hplc troubleshooting separations retention time...

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HPLC Troubleshooting Separations Retention Time, Efficiency and Peak Shape carlopez-humax-2012

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Page 1: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

HPLC Troubleshooting Separations Retention Time Efficiency and Peak Shape

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Rectangle

This essential guide examines the common causes of retention time drift loss of chromatographic efficiency and peak shape issues ndash such as splitting fronting tailing and shouldering Strategies for problem identification and calculation of critical chromatography performance indicators will be discussed

The degree to which these parameters are affected by mobile phase composition temperature sample solvent strength and many other variables will be investigated alongside strategies for isolating the precise cause of the problem

Corrective and preventative actions will be described for the major causes of the symptoms observed In Part II Julyrsquos Essential Guide we will focus on Selectivity Resolution and Baseline issues

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Every separation in HPLC produces a chromatogram (we hope) Broadly speaking every analyte is represented by a single peak eluting at a particular time according to its various physicochemical properties Under ideal conditions each peak should be narrow and symmetrical (Gaussian distribution)[1-8]

A visual inspection of the chromatogram is often enough to highlight problems with a separation however sometimes we need to be more quantitative to describe the extent of the issue To this end a set of parameters the most popular being retention time retention factor selectivity efficiency asymmetry and tailing factor have been developed and in this Essential Guide we shall be focusing on retention time retention factor and efficiency

Retention Time (tR)

The analyte retention time can be defined as the elapsed time between sample injection and the time of elution of the peak maximum of that analyte The non-retained mobile phase elutes at a time t0 which is known as the lsquohold up timersquo or ldquodead timerdquo

Chromatographic Parameters

There are several ways to determine t0 including

bull The time at the baseline disturbance seen due to differences in absorbance or refractive index as the injection solvent passes through the detectorbull Retention time of Uracil (reverse phase)bullRetention time of Hexane (normal phase)

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Retention or Capacity Factor

Isocratic Operation

The retention factor (krsquo) also known as capacity factor is a means of measuring the retention of an analyteon the chromatographic columnA high krsquo value indicates that the sample is highly retained and has spent a significant amount of time interacting with the stationary phase

The retention factor is equal to the ratio of retention time of the analyte on the column to the retention time of a non-retained compound The non-retained compound has no affinity for the stationary phase and elutes with the solvent front at a time t0

Retention factor is independent of some key variable factors including small flow rate variations and column dimensions Because of this it is a useful parameter when comparing retention of chromatographic peaks obtained using different HPLC systems and when converting conventional HPLC methods to UHPLC systems (and vice versa) Chromatographers typically like to keep krsquo values between 1 and 10 for good HPLC separations or 05 and 5 for methods developed on more modern UHPLC systems where highly efficient separations are the norm Please do note however that there is the risk of peak overlap (poor resolution) when separating a moderate number of analytes at low krsquo values

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Gradient Operation

One cannot assign a fixed krsquo value to a compound when gradient elution is applied krsquo is the retention coefficient and changes during gradient elution

The equation for the gradient retention factor (k) takes the form

Gradient retention factor (k) is difficult to visualize as it differs from its isocratic counterpart (krsquo) and resembles morethe profile of the gradient elution It is effectively defined as the retention factor for an analyte that has migrated half way down the HPLC column

The Retention Factor as a Means to Evaluate the State of an HPLC Separation

The retention factor of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention factors may result from changes in mobile phase flow rate mobile phase composition column stationary phase and temperature

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Efficiency

The efficiency of a chromatographic peak is a measure of the dispersion of the analyte band as it travelled through the HPLC system and column In an ideal world chromatographic peaks would be pencil thin lines ndashhowever due to dispersion effects the peaks take on their familiar ldquoGaussianrdquo shape

The plate number (N) is a measure of the peak dispersion on the HPLC column reflecting the column performance N is derived from an analogy of Martin and Synge who likened column efficiency to fractional distillation where the column is divided into Theoretical Plates

Each plate is the distance over which the sample components achieve one equilibration between the stationary and mobile phase in the column Therefore the more ldquotheoreticalrdquo plates available within a column the more equilibrations possible and the better quality the separation

Higher values for the Plate Number (N) are expected for subsequent peaks within a chromatogram Later eluting peaks that look broad in comparison to early eluters may have a higher plate count If this is not the case then your system contains a large extra-column dead volume which is dominating the diffusion process

For a fractionating tower of a given length (L) the higher the number of plates the lower will be the distance between each plate shown as plate height in the diagram Therefore for high efficiency separations the plate number (N) will be high and the plate height (H) low Note that plate height is often called ndash ldquoHeight Equivalent to a Theoretical Plate (HETP)rdquo Modern columns which employ are reduced particle size sub 2 microm enjoy an increased plate number (N) without increasing the length of the column (L) due to the reduction in distance between active sites plate height (H)

These two terms are related through the expression H = L N

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Figure 3 Factional distillation model of efficiency theorycarlo

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The number of theoretical plates is often used to establish the efficacy of a column for a given method The method developer may decide that a given method is no longer valid when the plate number falls below a predetermined value At that time the column would be replaced with a new one

Figure 4 Comparison of two chromatograms with the same selectivity and different efficiency (and resolution)carlo

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Efficiency as a Means to Evaluate the State of an HPLC Separation

Peak efficiency within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Reduced efficiency may result from incorrect or mobile phase deterioration incorrect or deteriorated column increased dead volume addition of a disproportionate length of tubing or wider ID tubing etc

Peak Asymmetry

In the ideal world all chromatographic peaks would be symmetrical (or Gaussian)

However due to the effects of instrument dead-volume adsorptive effects of the stationary phase and the quality of the column packing peaks may often show a tailing behavior Tailing describes a peak whose tail portion (distance lsquoBrsquo in the diagram) is wider than the front portion (distance lsquoArsquo in the diagram) Also if the sample concentration is too high or if the column is damaged and contains lsquochannelsrsquo then a fronting peak shape may occur

There are different ways of calculating the amount of peak tailing (or fronting) However one of them peak asymmetry (As) is probably the most commonly used and is defined as

Where A and B are measured at 10 of the peak heightcarlo

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Figure 5 Determination of Peak Asymmetry (AS) and examples of good and poor peak shape

The US Pharmacopeia (USP) recommends the use of a different measurement the tailing factor (Tf) which has been defined as

Where A and B are measured at 5 of the peak height

In general terms it doesnrsquot matter which measurement parameter is used (either As or Tf) as long as we are consistent in our measurement and are aware of the ldquoacceptablerdquo values for each measure The definition of lsquoacceptablersquo peak tailing or fronting for the two different measurements is roughly the same - see Table 1carlo

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Peak Distortion as a Means to Evaluate the State of an HPLC Separation

Peak asymmetry and tailing factor can aid in the identification of many problems that are associated with HPLC system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions Peak tailing and fronting are the most common forms of peak distortion in HPLC

Basically the primary cause of peak distortion is due to the occurrence of more than one mechanism of analyte retention however there are other reasons that account for this condition (mobile phase issues column degradation injection problems etc)

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Mobile phase and solvent Considerations

In order to effectively troubleshoot our separations for retention time efficiency and peak shape issues we need to understand the basics of retention and separation in HPLC This section presents a brief overview of the controlling variables and how they might be manipulated to change chromatographic behavior

Reversed Phase solvents

Reversed phase mobile phases usually consist of water and an organic solvent often called a ldquomodifierrdquo When ionisable compounds are analyzed buffers and other additives may be present in the aqueous phase to control retention and peak shape[9 10 11]

Chromatographically in reversed phase HPLC water is the ldquoweakestrdquo solvent As water is most polar it repels the hydrophobic analytes into the stationary phase more than any other solvent and hence retention times are long ndashthis makes it chromatographically lsquoweakrsquo The organic modifier is added (usually only one modifier type at a time for modern chromatography) and as these are less polar the (hydrophobic) analyte is no longer as strongly repelled into the stationary phase will spend less time in the stationary phase and therefore elute earlier This makes the modifier chromatographically ldquostrongrdquo as it speeds up elution

As progressively more organic modifier is added to the mobile phase the analyte retention time will continue to decrease

The values alongside the solvent chemical structures in Figure 6 represent the Snyder polarity index value The more polar a solvent the lsquoweakerrsquo it is in terms of eluotropic strength in reversed phase HPLC

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Figure 6 Solvents in reversed phase HPLC Figure 7 Properties for selected HPLC solvents

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Use Figure 7 to assess the relative physics-chemical properties of the various solvents one might use as organic modifiers to decide upon their suitability for a particular application

Changing the organic modifier within a mobile phase can alter the selectivity of the separation as well as the retention characteristics

So how would one chose the most appropriate solvent ndash what are the major considerations

First of all the chosen organic solvent must be miscible with water All of the solvents listed in Figure 6 are miscible with water Second a low viscosity mobile phase is favored to reduce dispersion and keep system back pressure low

The solvent must be stable for long periods of time This disfavors tetrahydrofuran which after exposure to air degrades rapidly often forming explosive peroxides

The two remaining mobile phase solvents are methanol and acetonitrile The use of both solvents usually gives excellent retention characteristics

Acetonitrile however has a lower viscosity and lower UV-cutoff (which is advantageous as the possibility detection interference is reduced) The UV-cutoff for methanol is 205 nm while the UV cut-off for acetonitrile is 190 nm For these reasons most analysts begin reversed-phase method development with acetonitrile

It is also important to note that whilst the polarity index values of methanol and acetonitrile are similar they have different Lewis acid base characteristics (proton donator acceptor) which allows them to act differently to alter separation characteristics We will study this in greater detail in Part II of this Essential Guide carlo

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Mobile Phase Strength and Retention

Increasing the percentage of organic modifier in the mobile phase has a profound effect on analyte retention due to the change in polarity of the mobile phase Use the slider below to investigate the effect of altering the mobile phase composition

Figure 8 Solvent strength

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Eluotropic Series

Solvent strength in Reverse Phase HPLC depends upon the solvent type and amount used in the mobile phase (percentage organic modifier (B))

It is possible to interrelate the relative ldquostrengthrdquo of each of the common solvents using an Eluotropic Series This is a table or listing of the various solvents and their relative strength on various media ndash for example Aluminium Oxide or Silica for Normal Phase HPLC and C18 for Reverse Phase HPLC

Commonly ndash a nomograph might be used to find equivalent eluotropic strength between mobile phases that use different organic modifiers The nomograph relates each solvent using a vertical line to indicate mobile phase composition (B) which give equivalent elution strength ndashknown as lsquoisoelutropicrsquo mobile phase compositions

Isoelutropic mobile phases produce separations in approximately the same time frame (measured using retention (capacity) factor of the last peak) ndash however they show altered selectivity This can be very useful when developing or optimising separations

You can use the slider bar below to find various isoelutropic compositions ndash note that the relationship yields results which are only approximately accurate to within about plusmn 5 B

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Figure 9 Solvent nomogram form reversed phase HPLC

Table 2 Eluotropic series of various reverse phase solventscarlo

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Matching Injection Volume with Sample Solvent Strength

Under ideal conditions the strength of the sample solvent used would make no difference to the chromatographic separation because the solvent would be diluted instantaneously to the same composition as the mobile phase However in practice it takes a finite period of time for the injected sample solvent to become diluted with the mobile phase

The following table provides guidelines for sample injection volumes based on the sample solvent strength with respect to that of mobile phase

100 Strong Solvent

In this demanding situation it is necessary to keep the injection volume as small as possible thereby minimising peak distortion The recommendation is for no more than 10μL

When the sample solvent is greater than 80 of the mobile phase it may be possible to inject larger volumes as the compositional difference between the sample solvent and mobile phase is correspondingly less

Stronger than Mobile Phase

When the sample solvent is no more than approximately 25 stronger than the mobile phase then injection volumes as high as 25μLshould be possible However always examine the chromatogram for possible peak distortion given the inter-relationship of this and the preceding rule

Generally analyte solubility issues are the primary reason for increasing the sample solvent strength utilised To minimise such solvent strength variations on injection dissolve the analyte at a high concentration in the strong solvent then dilute with the weaker solvent to progressively correct for the difference in eluotropic strength In a worst-case scenario dissolve in 100 strong solvent and inject as small a volume as possible

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Mobile Phase

The best injection scenario is when the sample solvent and the mobile phase are matched both in terms of eluotropicstrength and pH In this situation given the solvent homogeneity you donrsquot have to be concerned with dilution effects

However the injection volume is limited The contribution of the sample injection volume to the chromatographic peak width can be determined by the following equation

Weaker than Mobile Phase

To assess the effect of the mobile phase strength consider the ldquoRule of Threerdquo which states that a 10 change in the strong solvent will result in an approximate threefold change in analyte retention time Therefore if a chromatographic peak had a retention time of 5min in a mobile phase of 4060 wateracetonitrile then in a mobile phase of 6040 wateracetonitrile its retention time would increase to approximately 45min (3 times 3 times 5min = 45min)

Consequently if the sample were to be injected in 6040 wateracetonitrile instead then the analyte molecules would travel much more slowly through the column until the sample solvent was fully diluted with the mobile phase

Chromatographic bands travelling more slowly than the mobile phase tend to compress because as mobile phase reaches the analyte molecules at the peak ldquotailrdquo they will begin to move faster down the column catching those analyte molecules further down the column that are still effectively residing in a weaker mobile phase strength

As the difference between the solvent strength of the sample solvent and the mobile phase increases it is possible to use progressively larger and larger injection volumes This effectively allows analyte on-column sample ldquofocussingrdquo or concentration and is often employed in environmental analysis to assist in the detection of trace quantities of pesticides

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Exceptions to the above are whenever the mobile phase contains trace additives then it is always advisable to inject samples dissolved in the mobile phase eg ion-pair separations rely on the equilibrium between the ion pair reagent in the mobile phase and the stationary phase Injecting a sample in a solvent that doesnrsquot contain the ion pair shifts this equilibrium to the mobile phase with resulting peak distortion

Peak distortion can occur due to a mismatch between injection solvent and mobile phase strength In particular when large amounts of sample are injected in a solvent that is much ldquostrongerrdquo than the mobile phase The diagram below shows typical peak distortion problems

Figure 10 Peak shape abnormalities currently found when solvent strength is not considered when injecting

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Ionisable CompoundspH Considerations

The mobile phase pH can be used to influence the charge state of ionisable species in solution The extent of analyte ionisation can be used to affect retention and selectivity The pH of a solution will influence the charge state of an acidic or basic analyte

For example addition of an acid to an aqueous solution of a basic analyte will increase the concentration of charged analyte in solution as the hydrogen ion concentration increases Conversely raising the pH by addition of a base will increase the concentration of the neutral form of the basic analyte

Take the example of homovanillic acid shown below ndash equilibrium between the ionised and non-ionisedforms of the analyte will be established according to the solution pH

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Adding an acid to a buffered solution of homovanillic acid (ie lowering the mobile phase pH) will cause the equilibrium to shift to the left and the analyte will become less ionised (ion suppressed) as the analyterecombines to reduce the effect of the added hydrogen ions (protons) from the acidic species Adding a base to a buffered solution of homovanillic acid (ie increasing the mobile phase pH) will cause the analyte to become more ionised as the solution attempts to regain equilibrium by producing more hydrogen ions to neutralise the added base This principle was first described by Le Chetalier and the converse applies to acidic analyte species

The 2 pH rule

It can be shown that at 1 pH unit away from the analyte pKa the change in extent of ionisation is approximately 90 At 2 pH units away from the pKa the change in extent of ionisation is approximately 99 at 3 pH units 999 etc Therefore ndash a rule a thumb known as the ldquo2 pH rulerdquo is useful in predicting extent of ionisation

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Basic Analytes amp Ion Suppression

Unlike the dissociated (ionised) acids which when charged elute rapidly from the column protonated bases often have long retention times and poor peak shape This retention behaviour is due to the interaction with residual silanol species on the silica surface

Separations of basic compounds however are not usually carried out under ion suppression conditions The analyst would have to raise the pH to produce the neutral molecule High pH mobile phases can damage traditional silica columns unless specifically designed to cope with high pH

Traditionally the analysis of weak bases has been carried out at low pH ndash essentially because the surface silanol species are non-ionised (pKa approx 35 ndash 45) and peak tailing is improved somewhat

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically) highly surface active base such as Triethylamine (TEA) Piperazine NNNrsquoNrsquo-Tetramethylethylenediamine (TEMED) or Dimethyloctylamine (DMOA) These bases interact with the surface silanol species in preference to the analyte molecule and are called lsquosacrificial basesrsquo They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times

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Figure 13 Tailing factor versus concentration of TEAcarlo

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Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

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Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

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As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

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Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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012

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Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 2: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

This essential guide examines the common causes of retention time drift loss of chromatographic efficiency and peak shape issues ndash such as splitting fronting tailing and shouldering Strategies for problem identification and calculation of critical chromatography performance indicators will be discussed

The degree to which these parameters are affected by mobile phase composition temperature sample solvent strength and many other variables will be investigated alongside strategies for isolating the precise cause of the problem

Corrective and preventative actions will be described for the major causes of the symptoms observed In Part II Julyrsquos Essential Guide we will focus on Selectivity Resolution and Baseline issues

carlo

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Every separation in HPLC produces a chromatogram (we hope) Broadly speaking every analyte is represented by a single peak eluting at a particular time according to its various physicochemical properties Under ideal conditions each peak should be narrow and symmetrical (Gaussian distribution)[1-8]

A visual inspection of the chromatogram is often enough to highlight problems with a separation however sometimes we need to be more quantitative to describe the extent of the issue To this end a set of parameters the most popular being retention time retention factor selectivity efficiency asymmetry and tailing factor have been developed and in this Essential Guide we shall be focusing on retention time retention factor and efficiency

Retention Time (tR)

The analyte retention time can be defined as the elapsed time between sample injection and the time of elution of the peak maximum of that analyte The non-retained mobile phase elutes at a time t0 which is known as the lsquohold up timersquo or ldquodead timerdquo

Chromatographic Parameters

There are several ways to determine t0 including

bull The time at the baseline disturbance seen due to differences in absorbance or refractive index as the injection solvent passes through the detectorbull Retention time of Uracil (reverse phase)bullRetention time of Hexane (normal phase)

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Retention or Capacity Factor

Isocratic Operation

The retention factor (krsquo) also known as capacity factor is a means of measuring the retention of an analyteon the chromatographic columnA high krsquo value indicates that the sample is highly retained and has spent a significant amount of time interacting with the stationary phase

The retention factor is equal to the ratio of retention time of the analyte on the column to the retention time of a non-retained compound The non-retained compound has no affinity for the stationary phase and elutes with the solvent front at a time t0

Retention factor is independent of some key variable factors including small flow rate variations and column dimensions Because of this it is a useful parameter when comparing retention of chromatographic peaks obtained using different HPLC systems and when converting conventional HPLC methods to UHPLC systems (and vice versa) Chromatographers typically like to keep krsquo values between 1 and 10 for good HPLC separations or 05 and 5 for methods developed on more modern UHPLC systems where highly efficient separations are the norm Please do note however that there is the risk of peak overlap (poor resolution) when separating a moderate number of analytes at low krsquo values

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Gradient Operation

One cannot assign a fixed krsquo value to a compound when gradient elution is applied krsquo is the retention coefficient and changes during gradient elution

The equation for the gradient retention factor (k) takes the form

Gradient retention factor (k) is difficult to visualize as it differs from its isocratic counterpart (krsquo) and resembles morethe profile of the gradient elution It is effectively defined as the retention factor for an analyte that has migrated half way down the HPLC column

The Retention Factor as a Means to Evaluate the State of an HPLC Separation

The retention factor of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention factors may result from changes in mobile phase flow rate mobile phase composition column stationary phase and temperature

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Efficiency

The efficiency of a chromatographic peak is a measure of the dispersion of the analyte band as it travelled through the HPLC system and column In an ideal world chromatographic peaks would be pencil thin lines ndashhowever due to dispersion effects the peaks take on their familiar ldquoGaussianrdquo shape

The plate number (N) is a measure of the peak dispersion on the HPLC column reflecting the column performance N is derived from an analogy of Martin and Synge who likened column efficiency to fractional distillation where the column is divided into Theoretical Plates

Each plate is the distance over which the sample components achieve one equilibration between the stationary and mobile phase in the column Therefore the more ldquotheoreticalrdquo plates available within a column the more equilibrations possible and the better quality the separation

Higher values for the Plate Number (N) are expected for subsequent peaks within a chromatogram Later eluting peaks that look broad in comparison to early eluters may have a higher plate count If this is not the case then your system contains a large extra-column dead volume which is dominating the diffusion process

For a fractionating tower of a given length (L) the higher the number of plates the lower will be the distance between each plate shown as plate height in the diagram Therefore for high efficiency separations the plate number (N) will be high and the plate height (H) low Note that plate height is often called ndash ldquoHeight Equivalent to a Theoretical Plate (HETP)rdquo Modern columns which employ are reduced particle size sub 2 microm enjoy an increased plate number (N) without increasing the length of the column (L) due to the reduction in distance between active sites plate height (H)

These two terms are related through the expression H = L N

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Figure 3 Factional distillation model of efficiency theorycarlo

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012

The number of theoretical plates is often used to establish the efficacy of a column for a given method The method developer may decide that a given method is no longer valid when the plate number falls below a predetermined value At that time the column would be replaced with a new one

Figure 4 Comparison of two chromatograms with the same selectivity and different efficiency (and resolution)carlo

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Efficiency as a Means to Evaluate the State of an HPLC Separation

Peak efficiency within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Reduced efficiency may result from incorrect or mobile phase deterioration incorrect or deteriorated column increased dead volume addition of a disproportionate length of tubing or wider ID tubing etc

Peak Asymmetry

In the ideal world all chromatographic peaks would be symmetrical (or Gaussian)

However due to the effects of instrument dead-volume adsorptive effects of the stationary phase and the quality of the column packing peaks may often show a tailing behavior Tailing describes a peak whose tail portion (distance lsquoBrsquo in the diagram) is wider than the front portion (distance lsquoArsquo in the diagram) Also if the sample concentration is too high or if the column is damaged and contains lsquochannelsrsquo then a fronting peak shape may occur

There are different ways of calculating the amount of peak tailing (or fronting) However one of them peak asymmetry (As) is probably the most commonly used and is defined as

Where A and B are measured at 10 of the peak heightcarlo

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Figure 5 Determination of Peak Asymmetry (AS) and examples of good and poor peak shape

The US Pharmacopeia (USP) recommends the use of a different measurement the tailing factor (Tf) which has been defined as

Where A and B are measured at 5 of the peak height

In general terms it doesnrsquot matter which measurement parameter is used (either As or Tf) as long as we are consistent in our measurement and are aware of the ldquoacceptablerdquo values for each measure The definition of lsquoacceptablersquo peak tailing or fronting for the two different measurements is roughly the same - see Table 1carlo

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Peak Distortion as a Means to Evaluate the State of an HPLC Separation

Peak asymmetry and tailing factor can aid in the identification of many problems that are associated with HPLC system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions Peak tailing and fronting are the most common forms of peak distortion in HPLC

Basically the primary cause of peak distortion is due to the occurrence of more than one mechanism of analyte retention however there are other reasons that account for this condition (mobile phase issues column degradation injection problems etc)

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Mobile phase and solvent Considerations

In order to effectively troubleshoot our separations for retention time efficiency and peak shape issues we need to understand the basics of retention and separation in HPLC This section presents a brief overview of the controlling variables and how they might be manipulated to change chromatographic behavior

Reversed Phase solvents

Reversed phase mobile phases usually consist of water and an organic solvent often called a ldquomodifierrdquo When ionisable compounds are analyzed buffers and other additives may be present in the aqueous phase to control retention and peak shape[9 10 11]

Chromatographically in reversed phase HPLC water is the ldquoweakestrdquo solvent As water is most polar it repels the hydrophobic analytes into the stationary phase more than any other solvent and hence retention times are long ndashthis makes it chromatographically lsquoweakrsquo The organic modifier is added (usually only one modifier type at a time for modern chromatography) and as these are less polar the (hydrophobic) analyte is no longer as strongly repelled into the stationary phase will spend less time in the stationary phase and therefore elute earlier This makes the modifier chromatographically ldquostrongrdquo as it speeds up elution

As progressively more organic modifier is added to the mobile phase the analyte retention time will continue to decrease

The values alongside the solvent chemical structures in Figure 6 represent the Snyder polarity index value The more polar a solvent the lsquoweakerrsquo it is in terms of eluotropic strength in reversed phase HPLC

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Figure 6 Solvents in reversed phase HPLC Figure 7 Properties for selected HPLC solvents

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Use Figure 7 to assess the relative physics-chemical properties of the various solvents one might use as organic modifiers to decide upon their suitability for a particular application

Changing the organic modifier within a mobile phase can alter the selectivity of the separation as well as the retention characteristics

So how would one chose the most appropriate solvent ndash what are the major considerations

First of all the chosen organic solvent must be miscible with water All of the solvents listed in Figure 6 are miscible with water Second a low viscosity mobile phase is favored to reduce dispersion and keep system back pressure low

The solvent must be stable for long periods of time This disfavors tetrahydrofuran which after exposure to air degrades rapidly often forming explosive peroxides

The two remaining mobile phase solvents are methanol and acetonitrile The use of both solvents usually gives excellent retention characteristics

Acetonitrile however has a lower viscosity and lower UV-cutoff (which is advantageous as the possibility detection interference is reduced) The UV-cutoff for methanol is 205 nm while the UV cut-off for acetonitrile is 190 nm For these reasons most analysts begin reversed-phase method development with acetonitrile

It is also important to note that whilst the polarity index values of methanol and acetonitrile are similar they have different Lewis acid base characteristics (proton donator acceptor) which allows them to act differently to alter separation characteristics We will study this in greater detail in Part II of this Essential Guide carlo

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Mobile Phase Strength and Retention

Increasing the percentage of organic modifier in the mobile phase has a profound effect on analyte retention due to the change in polarity of the mobile phase Use the slider below to investigate the effect of altering the mobile phase composition

Figure 8 Solvent strength

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Eluotropic Series

Solvent strength in Reverse Phase HPLC depends upon the solvent type and amount used in the mobile phase (percentage organic modifier (B))

It is possible to interrelate the relative ldquostrengthrdquo of each of the common solvents using an Eluotropic Series This is a table or listing of the various solvents and their relative strength on various media ndash for example Aluminium Oxide or Silica for Normal Phase HPLC and C18 for Reverse Phase HPLC

Commonly ndash a nomograph might be used to find equivalent eluotropic strength between mobile phases that use different organic modifiers The nomograph relates each solvent using a vertical line to indicate mobile phase composition (B) which give equivalent elution strength ndashknown as lsquoisoelutropicrsquo mobile phase compositions

Isoelutropic mobile phases produce separations in approximately the same time frame (measured using retention (capacity) factor of the last peak) ndash however they show altered selectivity This can be very useful when developing or optimising separations

You can use the slider bar below to find various isoelutropic compositions ndash note that the relationship yields results which are only approximately accurate to within about plusmn 5 B

carlo

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Figure 9 Solvent nomogram form reversed phase HPLC

Table 2 Eluotropic series of various reverse phase solventscarlo

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Matching Injection Volume with Sample Solvent Strength

Under ideal conditions the strength of the sample solvent used would make no difference to the chromatographic separation because the solvent would be diluted instantaneously to the same composition as the mobile phase However in practice it takes a finite period of time for the injected sample solvent to become diluted with the mobile phase

The following table provides guidelines for sample injection volumes based on the sample solvent strength with respect to that of mobile phase

100 Strong Solvent

In this demanding situation it is necessary to keep the injection volume as small as possible thereby minimising peak distortion The recommendation is for no more than 10μL

When the sample solvent is greater than 80 of the mobile phase it may be possible to inject larger volumes as the compositional difference between the sample solvent and mobile phase is correspondingly less

Stronger than Mobile Phase

When the sample solvent is no more than approximately 25 stronger than the mobile phase then injection volumes as high as 25μLshould be possible However always examine the chromatogram for possible peak distortion given the inter-relationship of this and the preceding rule

Generally analyte solubility issues are the primary reason for increasing the sample solvent strength utilised To minimise such solvent strength variations on injection dissolve the analyte at a high concentration in the strong solvent then dilute with the weaker solvent to progressively correct for the difference in eluotropic strength In a worst-case scenario dissolve in 100 strong solvent and inject as small a volume as possible

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Mobile Phase

The best injection scenario is when the sample solvent and the mobile phase are matched both in terms of eluotropicstrength and pH In this situation given the solvent homogeneity you donrsquot have to be concerned with dilution effects

However the injection volume is limited The contribution of the sample injection volume to the chromatographic peak width can be determined by the following equation

Weaker than Mobile Phase

To assess the effect of the mobile phase strength consider the ldquoRule of Threerdquo which states that a 10 change in the strong solvent will result in an approximate threefold change in analyte retention time Therefore if a chromatographic peak had a retention time of 5min in a mobile phase of 4060 wateracetonitrile then in a mobile phase of 6040 wateracetonitrile its retention time would increase to approximately 45min (3 times 3 times 5min = 45min)

Consequently if the sample were to be injected in 6040 wateracetonitrile instead then the analyte molecules would travel much more slowly through the column until the sample solvent was fully diluted with the mobile phase

Chromatographic bands travelling more slowly than the mobile phase tend to compress because as mobile phase reaches the analyte molecules at the peak ldquotailrdquo they will begin to move faster down the column catching those analyte molecules further down the column that are still effectively residing in a weaker mobile phase strength

As the difference between the solvent strength of the sample solvent and the mobile phase increases it is possible to use progressively larger and larger injection volumes This effectively allows analyte on-column sample ldquofocussingrdquo or concentration and is often employed in environmental analysis to assist in the detection of trace quantities of pesticides

carlo

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Exceptions to the above are whenever the mobile phase contains trace additives then it is always advisable to inject samples dissolved in the mobile phase eg ion-pair separations rely on the equilibrium between the ion pair reagent in the mobile phase and the stationary phase Injecting a sample in a solvent that doesnrsquot contain the ion pair shifts this equilibrium to the mobile phase with resulting peak distortion

Peak distortion can occur due to a mismatch between injection solvent and mobile phase strength In particular when large amounts of sample are injected in a solvent that is much ldquostrongerrdquo than the mobile phase The diagram below shows typical peak distortion problems

Figure 10 Peak shape abnormalities currently found when solvent strength is not considered when injecting

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Ionisable CompoundspH Considerations

The mobile phase pH can be used to influence the charge state of ionisable species in solution The extent of analyte ionisation can be used to affect retention and selectivity The pH of a solution will influence the charge state of an acidic or basic analyte

For example addition of an acid to an aqueous solution of a basic analyte will increase the concentration of charged analyte in solution as the hydrogen ion concentration increases Conversely raising the pH by addition of a base will increase the concentration of the neutral form of the basic analyte

Take the example of homovanillic acid shown below ndash equilibrium between the ionised and non-ionisedforms of the analyte will be established according to the solution pH

carlo

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Adding an acid to a buffered solution of homovanillic acid (ie lowering the mobile phase pH) will cause the equilibrium to shift to the left and the analyte will become less ionised (ion suppressed) as the analyterecombines to reduce the effect of the added hydrogen ions (protons) from the acidic species Adding a base to a buffered solution of homovanillic acid (ie increasing the mobile phase pH) will cause the analyte to become more ionised as the solution attempts to regain equilibrium by producing more hydrogen ions to neutralise the added base This principle was first described by Le Chetalier and the converse applies to acidic analyte species

The 2 pH rule

It can be shown that at 1 pH unit away from the analyte pKa the change in extent of ionisation is approximately 90 At 2 pH units away from the pKa the change in extent of ionisation is approximately 99 at 3 pH units 999 etc Therefore ndash a rule a thumb known as the ldquo2 pH rulerdquo is useful in predicting extent of ionisation

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Basic Analytes amp Ion Suppression

Unlike the dissociated (ionised) acids which when charged elute rapidly from the column protonated bases often have long retention times and poor peak shape This retention behaviour is due to the interaction with residual silanol species on the silica surface

Separations of basic compounds however are not usually carried out under ion suppression conditions The analyst would have to raise the pH to produce the neutral molecule High pH mobile phases can damage traditional silica columns unless specifically designed to cope with high pH

Traditionally the analysis of weak bases has been carried out at low pH ndash essentially because the surface silanol species are non-ionised (pKa approx 35 ndash 45) and peak tailing is improved somewhat

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically) highly surface active base such as Triethylamine (TEA) Piperazine NNNrsquoNrsquo-Tetramethylethylenediamine (TEMED) or Dimethyloctylamine (DMOA) These bases interact with the surface silanol species in preference to the analyte molecule and are called lsquosacrificial basesrsquo They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times

carlo

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Figure 13 Tailing factor versus concentration of TEAcarlo

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Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

carlo

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Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

carlo

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

carlo

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

carlo

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

carlo

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012

As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

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2

Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

carlo

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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2

Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

carlo

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

carlo

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

carlo

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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2

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

carlo

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

carlo

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

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012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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012

carlo

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Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 3: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Every separation in HPLC produces a chromatogram (we hope) Broadly speaking every analyte is represented by a single peak eluting at a particular time according to its various physicochemical properties Under ideal conditions each peak should be narrow and symmetrical (Gaussian distribution)[1-8]

A visual inspection of the chromatogram is often enough to highlight problems with a separation however sometimes we need to be more quantitative to describe the extent of the issue To this end a set of parameters the most popular being retention time retention factor selectivity efficiency asymmetry and tailing factor have been developed and in this Essential Guide we shall be focusing on retention time retention factor and efficiency

Retention Time (tR)

The analyte retention time can be defined as the elapsed time between sample injection and the time of elution of the peak maximum of that analyte The non-retained mobile phase elutes at a time t0 which is known as the lsquohold up timersquo or ldquodead timerdquo

Chromatographic Parameters

There are several ways to determine t0 including

bull The time at the baseline disturbance seen due to differences in absorbance or refractive index as the injection solvent passes through the detectorbull Retention time of Uracil (reverse phase)bullRetention time of Hexane (normal phase)

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Retention or Capacity Factor

Isocratic Operation

The retention factor (krsquo) also known as capacity factor is a means of measuring the retention of an analyteon the chromatographic columnA high krsquo value indicates that the sample is highly retained and has spent a significant amount of time interacting with the stationary phase

The retention factor is equal to the ratio of retention time of the analyte on the column to the retention time of a non-retained compound The non-retained compound has no affinity for the stationary phase and elutes with the solvent front at a time t0

Retention factor is independent of some key variable factors including small flow rate variations and column dimensions Because of this it is a useful parameter when comparing retention of chromatographic peaks obtained using different HPLC systems and when converting conventional HPLC methods to UHPLC systems (and vice versa) Chromatographers typically like to keep krsquo values between 1 and 10 for good HPLC separations or 05 and 5 for methods developed on more modern UHPLC systems where highly efficient separations are the norm Please do note however that there is the risk of peak overlap (poor resolution) when separating a moderate number of analytes at low krsquo values

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Gradient Operation

One cannot assign a fixed krsquo value to a compound when gradient elution is applied krsquo is the retention coefficient and changes during gradient elution

The equation for the gradient retention factor (k) takes the form

Gradient retention factor (k) is difficult to visualize as it differs from its isocratic counterpart (krsquo) and resembles morethe profile of the gradient elution It is effectively defined as the retention factor for an analyte that has migrated half way down the HPLC column

The Retention Factor as a Means to Evaluate the State of an HPLC Separation

The retention factor of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention factors may result from changes in mobile phase flow rate mobile phase composition column stationary phase and temperature

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Efficiency

The efficiency of a chromatographic peak is a measure of the dispersion of the analyte band as it travelled through the HPLC system and column In an ideal world chromatographic peaks would be pencil thin lines ndashhowever due to dispersion effects the peaks take on their familiar ldquoGaussianrdquo shape

The plate number (N) is a measure of the peak dispersion on the HPLC column reflecting the column performance N is derived from an analogy of Martin and Synge who likened column efficiency to fractional distillation where the column is divided into Theoretical Plates

Each plate is the distance over which the sample components achieve one equilibration between the stationary and mobile phase in the column Therefore the more ldquotheoreticalrdquo plates available within a column the more equilibrations possible and the better quality the separation

Higher values for the Plate Number (N) are expected for subsequent peaks within a chromatogram Later eluting peaks that look broad in comparison to early eluters may have a higher plate count If this is not the case then your system contains a large extra-column dead volume which is dominating the diffusion process

For a fractionating tower of a given length (L) the higher the number of plates the lower will be the distance between each plate shown as plate height in the diagram Therefore for high efficiency separations the plate number (N) will be high and the plate height (H) low Note that plate height is often called ndash ldquoHeight Equivalent to a Theoretical Plate (HETP)rdquo Modern columns which employ are reduced particle size sub 2 microm enjoy an increased plate number (N) without increasing the length of the column (L) due to the reduction in distance between active sites plate height (H)

These two terms are related through the expression H = L N

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Figure 3 Factional distillation model of efficiency theorycarlo

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The number of theoretical plates is often used to establish the efficacy of a column for a given method The method developer may decide that a given method is no longer valid when the plate number falls below a predetermined value At that time the column would be replaced with a new one

Figure 4 Comparison of two chromatograms with the same selectivity and different efficiency (and resolution)carlo

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Efficiency as a Means to Evaluate the State of an HPLC Separation

Peak efficiency within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Reduced efficiency may result from incorrect or mobile phase deterioration incorrect or deteriorated column increased dead volume addition of a disproportionate length of tubing or wider ID tubing etc

Peak Asymmetry

In the ideal world all chromatographic peaks would be symmetrical (or Gaussian)

However due to the effects of instrument dead-volume adsorptive effects of the stationary phase and the quality of the column packing peaks may often show a tailing behavior Tailing describes a peak whose tail portion (distance lsquoBrsquo in the diagram) is wider than the front portion (distance lsquoArsquo in the diagram) Also if the sample concentration is too high or if the column is damaged and contains lsquochannelsrsquo then a fronting peak shape may occur

There are different ways of calculating the amount of peak tailing (or fronting) However one of them peak asymmetry (As) is probably the most commonly used and is defined as

Where A and B are measured at 10 of the peak heightcarlo

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Figure 5 Determination of Peak Asymmetry (AS) and examples of good and poor peak shape

The US Pharmacopeia (USP) recommends the use of a different measurement the tailing factor (Tf) which has been defined as

Where A and B are measured at 5 of the peak height

In general terms it doesnrsquot matter which measurement parameter is used (either As or Tf) as long as we are consistent in our measurement and are aware of the ldquoacceptablerdquo values for each measure The definition of lsquoacceptablersquo peak tailing or fronting for the two different measurements is roughly the same - see Table 1carlo

pez-

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ax-2

012

Peak Distortion as a Means to Evaluate the State of an HPLC Separation

Peak asymmetry and tailing factor can aid in the identification of many problems that are associated with HPLC system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions Peak tailing and fronting are the most common forms of peak distortion in HPLC

Basically the primary cause of peak distortion is due to the occurrence of more than one mechanism of analyte retention however there are other reasons that account for this condition (mobile phase issues column degradation injection problems etc)

carlo

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Mobile phase and solvent Considerations

In order to effectively troubleshoot our separations for retention time efficiency and peak shape issues we need to understand the basics of retention and separation in HPLC This section presents a brief overview of the controlling variables and how they might be manipulated to change chromatographic behavior

Reversed Phase solvents

Reversed phase mobile phases usually consist of water and an organic solvent often called a ldquomodifierrdquo When ionisable compounds are analyzed buffers and other additives may be present in the aqueous phase to control retention and peak shape[9 10 11]

Chromatographically in reversed phase HPLC water is the ldquoweakestrdquo solvent As water is most polar it repels the hydrophobic analytes into the stationary phase more than any other solvent and hence retention times are long ndashthis makes it chromatographically lsquoweakrsquo The organic modifier is added (usually only one modifier type at a time for modern chromatography) and as these are less polar the (hydrophobic) analyte is no longer as strongly repelled into the stationary phase will spend less time in the stationary phase and therefore elute earlier This makes the modifier chromatographically ldquostrongrdquo as it speeds up elution

As progressively more organic modifier is added to the mobile phase the analyte retention time will continue to decrease

The values alongside the solvent chemical structures in Figure 6 represent the Snyder polarity index value The more polar a solvent the lsquoweakerrsquo it is in terms of eluotropic strength in reversed phase HPLC

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Figure 6 Solvents in reversed phase HPLC Figure 7 Properties for selected HPLC solvents

carlo

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Use Figure 7 to assess the relative physics-chemical properties of the various solvents one might use as organic modifiers to decide upon their suitability for a particular application

Changing the organic modifier within a mobile phase can alter the selectivity of the separation as well as the retention characteristics

So how would one chose the most appropriate solvent ndash what are the major considerations

First of all the chosen organic solvent must be miscible with water All of the solvents listed in Figure 6 are miscible with water Second a low viscosity mobile phase is favored to reduce dispersion and keep system back pressure low

The solvent must be stable for long periods of time This disfavors tetrahydrofuran which after exposure to air degrades rapidly often forming explosive peroxides

The two remaining mobile phase solvents are methanol and acetonitrile The use of both solvents usually gives excellent retention characteristics

Acetonitrile however has a lower viscosity and lower UV-cutoff (which is advantageous as the possibility detection interference is reduced) The UV-cutoff for methanol is 205 nm while the UV cut-off for acetonitrile is 190 nm For these reasons most analysts begin reversed-phase method development with acetonitrile

It is also important to note that whilst the polarity index values of methanol and acetonitrile are similar they have different Lewis acid base characteristics (proton donator acceptor) which allows them to act differently to alter separation characteristics We will study this in greater detail in Part II of this Essential Guide carlo

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Mobile Phase Strength and Retention

Increasing the percentage of organic modifier in the mobile phase has a profound effect on analyte retention due to the change in polarity of the mobile phase Use the slider below to investigate the effect of altering the mobile phase composition

Figure 8 Solvent strength

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Eluotropic Series

Solvent strength in Reverse Phase HPLC depends upon the solvent type and amount used in the mobile phase (percentage organic modifier (B))

It is possible to interrelate the relative ldquostrengthrdquo of each of the common solvents using an Eluotropic Series This is a table or listing of the various solvents and their relative strength on various media ndash for example Aluminium Oxide or Silica for Normal Phase HPLC and C18 for Reverse Phase HPLC

Commonly ndash a nomograph might be used to find equivalent eluotropic strength between mobile phases that use different organic modifiers The nomograph relates each solvent using a vertical line to indicate mobile phase composition (B) which give equivalent elution strength ndashknown as lsquoisoelutropicrsquo mobile phase compositions

Isoelutropic mobile phases produce separations in approximately the same time frame (measured using retention (capacity) factor of the last peak) ndash however they show altered selectivity This can be very useful when developing or optimising separations

You can use the slider bar below to find various isoelutropic compositions ndash note that the relationship yields results which are only approximately accurate to within about plusmn 5 B

carlo

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Figure 9 Solvent nomogram form reversed phase HPLC

Table 2 Eluotropic series of various reverse phase solventscarlo

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Matching Injection Volume with Sample Solvent Strength

Under ideal conditions the strength of the sample solvent used would make no difference to the chromatographic separation because the solvent would be diluted instantaneously to the same composition as the mobile phase However in practice it takes a finite period of time for the injected sample solvent to become diluted with the mobile phase

The following table provides guidelines for sample injection volumes based on the sample solvent strength with respect to that of mobile phase

100 Strong Solvent

In this demanding situation it is necessary to keep the injection volume as small as possible thereby minimising peak distortion The recommendation is for no more than 10μL

When the sample solvent is greater than 80 of the mobile phase it may be possible to inject larger volumes as the compositional difference between the sample solvent and mobile phase is correspondingly less

Stronger than Mobile Phase

When the sample solvent is no more than approximately 25 stronger than the mobile phase then injection volumes as high as 25μLshould be possible However always examine the chromatogram for possible peak distortion given the inter-relationship of this and the preceding rule

Generally analyte solubility issues are the primary reason for increasing the sample solvent strength utilised To minimise such solvent strength variations on injection dissolve the analyte at a high concentration in the strong solvent then dilute with the weaker solvent to progressively correct for the difference in eluotropic strength In a worst-case scenario dissolve in 100 strong solvent and inject as small a volume as possible

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Mobile Phase

The best injection scenario is when the sample solvent and the mobile phase are matched both in terms of eluotropicstrength and pH In this situation given the solvent homogeneity you donrsquot have to be concerned with dilution effects

However the injection volume is limited The contribution of the sample injection volume to the chromatographic peak width can be determined by the following equation

Weaker than Mobile Phase

To assess the effect of the mobile phase strength consider the ldquoRule of Threerdquo which states that a 10 change in the strong solvent will result in an approximate threefold change in analyte retention time Therefore if a chromatographic peak had a retention time of 5min in a mobile phase of 4060 wateracetonitrile then in a mobile phase of 6040 wateracetonitrile its retention time would increase to approximately 45min (3 times 3 times 5min = 45min)

Consequently if the sample were to be injected in 6040 wateracetonitrile instead then the analyte molecules would travel much more slowly through the column until the sample solvent was fully diluted with the mobile phase

Chromatographic bands travelling more slowly than the mobile phase tend to compress because as mobile phase reaches the analyte molecules at the peak ldquotailrdquo they will begin to move faster down the column catching those analyte molecules further down the column that are still effectively residing in a weaker mobile phase strength

As the difference between the solvent strength of the sample solvent and the mobile phase increases it is possible to use progressively larger and larger injection volumes This effectively allows analyte on-column sample ldquofocussingrdquo or concentration and is often employed in environmental analysis to assist in the detection of trace quantities of pesticides

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Exceptions to the above are whenever the mobile phase contains trace additives then it is always advisable to inject samples dissolved in the mobile phase eg ion-pair separations rely on the equilibrium between the ion pair reagent in the mobile phase and the stationary phase Injecting a sample in a solvent that doesnrsquot contain the ion pair shifts this equilibrium to the mobile phase with resulting peak distortion

Peak distortion can occur due to a mismatch between injection solvent and mobile phase strength In particular when large amounts of sample are injected in a solvent that is much ldquostrongerrdquo than the mobile phase The diagram below shows typical peak distortion problems

Figure 10 Peak shape abnormalities currently found when solvent strength is not considered when injecting

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Ionisable CompoundspH Considerations

The mobile phase pH can be used to influence the charge state of ionisable species in solution The extent of analyte ionisation can be used to affect retention and selectivity The pH of a solution will influence the charge state of an acidic or basic analyte

For example addition of an acid to an aqueous solution of a basic analyte will increase the concentration of charged analyte in solution as the hydrogen ion concentration increases Conversely raising the pH by addition of a base will increase the concentration of the neutral form of the basic analyte

Take the example of homovanillic acid shown below ndash equilibrium between the ionised and non-ionisedforms of the analyte will be established according to the solution pH

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Adding an acid to a buffered solution of homovanillic acid (ie lowering the mobile phase pH) will cause the equilibrium to shift to the left and the analyte will become less ionised (ion suppressed) as the analyterecombines to reduce the effect of the added hydrogen ions (protons) from the acidic species Adding a base to a buffered solution of homovanillic acid (ie increasing the mobile phase pH) will cause the analyte to become more ionised as the solution attempts to regain equilibrium by producing more hydrogen ions to neutralise the added base This principle was first described by Le Chetalier and the converse applies to acidic analyte species

The 2 pH rule

It can be shown that at 1 pH unit away from the analyte pKa the change in extent of ionisation is approximately 90 At 2 pH units away from the pKa the change in extent of ionisation is approximately 99 at 3 pH units 999 etc Therefore ndash a rule a thumb known as the ldquo2 pH rulerdquo is useful in predicting extent of ionisation

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Basic Analytes amp Ion Suppression

Unlike the dissociated (ionised) acids which when charged elute rapidly from the column protonated bases often have long retention times and poor peak shape This retention behaviour is due to the interaction with residual silanol species on the silica surface

Separations of basic compounds however are not usually carried out under ion suppression conditions The analyst would have to raise the pH to produce the neutral molecule High pH mobile phases can damage traditional silica columns unless specifically designed to cope with high pH

Traditionally the analysis of weak bases has been carried out at low pH ndash essentially because the surface silanol species are non-ionised (pKa approx 35 ndash 45) and peak tailing is improved somewhat

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically) highly surface active base such as Triethylamine (TEA) Piperazine NNNrsquoNrsquo-Tetramethylethylenediamine (TEMED) or Dimethyloctylamine (DMOA) These bases interact with the surface silanol species in preference to the analyte molecule and are called lsquosacrificial basesrsquo They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times

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Figure 13 Tailing factor versus concentration of TEAcarlo

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Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

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Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

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As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

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Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

carlo

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

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012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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ax-2

012

carlo

pez-

hum

ax-2

012

carlo

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012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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012

carlo

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 4: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Retention or Capacity Factor

Isocratic Operation

The retention factor (krsquo) also known as capacity factor is a means of measuring the retention of an analyteon the chromatographic columnA high krsquo value indicates that the sample is highly retained and has spent a significant amount of time interacting with the stationary phase

The retention factor is equal to the ratio of retention time of the analyte on the column to the retention time of a non-retained compound The non-retained compound has no affinity for the stationary phase and elutes with the solvent front at a time t0

Retention factor is independent of some key variable factors including small flow rate variations and column dimensions Because of this it is a useful parameter when comparing retention of chromatographic peaks obtained using different HPLC systems and when converting conventional HPLC methods to UHPLC systems (and vice versa) Chromatographers typically like to keep krsquo values between 1 and 10 for good HPLC separations or 05 and 5 for methods developed on more modern UHPLC systems where highly efficient separations are the norm Please do note however that there is the risk of peak overlap (poor resolution) when separating a moderate number of analytes at low krsquo values

carlo

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Gradient Operation

One cannot assign a fixed krsquo value to a compound when gradient elution is applied krsquo is the retention coefficient and changes during gradient elution

The equation for the gradient retention factor (k) takes the form

Gradient retention factor (k) is difficult to visualize as it differs from its isocratic counterpart (krsquo) and resembles morethe profile of the gradient elution It is effectively defined as the retention factor for an analyte that has migrated half way down the HPLC column

The Retention Factor as a Means to Evaluate the State of an HPLC Separation

The retention factor of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention factors may result from changes in mobile phase flow rate mobile phase composition column stationary phase and temperature

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Efficiency

The efficiency of a chromatographic peak is a measure of the dispersion of the analyte band as it travelled through the HPLC system and column In an ideal world chromatographic peaks would be pencil thin lines ndashhowever due to dispersion effects the peaks take on their familiar ldquoGaussianrdquo shape

The plate number (N) is a measure of the peak dispersion on the HPLC column reflecting the column performance N is derived from an analogy of Martin and Synge who likened column efficiency to fractional distillation where the column is divided into Theoretical Plates

Each plate is the distance over which the sample components achieve one equilibration between the stationary and mobile phase in the column Therefore the more ldquotheoreticalrdquo plates available within a column the more equilibrations possible and the better quality the separation

Higher values for the Plate Number (N) are expected for subsequent peaks within a chromatogram Later eluting peaks that look broad in comparison to early eluters may have a higher plate count If this is not the case then your system contains a large extra-column dead volume which is dominating the diffusion process

For a fractionating tower of a given length (L) the higher the number of plates the lower will be the distance between each plate shown as plate height in the diagram Therefore for high efficiency separations the plate number (N) will be high and the plate height (H) low Note that plate height is often called ndash ldquoHeight Equivalent to a Theoretical Plate (HETP)rdquo Modern columns which employ are reduced particle size sub 2 microm enjoy an increased plate number (N) without increasing the length of the column (L) due to the reduction in distance between active sites plate height (H)

These two terms are related through the expression H = L N

carlo

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Figure 3 Factional distillation model of efficiency theorycarlo

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The number of theoretical plates is often used to establish the efficacy of a column for a given method The method developer may decide that a given method is no longer valid when the plate number falls below a predetermined value At that time the column would be replaced with a new one

Figure 4 Comparison of two chromatograms with the same selectivity and different efficiency (and resolution)carlo

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Efficiency as a Means to Evaluate the State of an HPLC Separation

Peak efficiency within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Reduced efficiency may result from incorrect or mobile phase deterioration incorrect or deteriorated column increased dead volume addition of a disproportionate length of tubing or wider ID tubing etc

Peak Asymmetry

In the ideal world all chromatographic peaks would be symmetrical (or Gaussian)

However due to the effects of instrument dead-volume adsorptive effects of the stationary phase and the quality of the column packing peaks may often show a tailing behavior Tailing describes a peak whose tail portion (distance lsquoBrsquo in the diagram) is wider than the front portion (distance lsquoArsquo in the diagram) Also if the sample concentration is too high or if the column is damaged and contains lsquochannelsrsquo then a fronting peak shape may occur

There are different ways of calculating the amount of peak tailing (or fronting) However one of them peak asymmetry (As) is probably the most commonly used and is defined as

Where A and B are measured at 10 of the peak heightcarlo

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Figure 5 Determination of Peak Asymmetry (AS) and examples of good and poor peak shape

The US Pharmacopeia (USP) recommends the use of a different measurement the tailing factor (Tf) which has been defined as

Where A and B are measured at 5 of the peak height

In general terms it doesnrsquot matter which measurement parameter is used (either As or Tf) as long as we are consistent in our measurement and are aware of the ldquoacceptablerdquo values for each measure The definition of lsquoacceptablersquo peak tailing or fronting for the two different measurements is roughly the same - see Table 1carlo

pez-

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ax-2

012

Peak Distortion as a Means to Evaluate the State of an HPLC Separation

Peak asymmetry and tailing factor can aid in the identification of many problems that are associated with HPLC system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions Peak tailing and fronting are the most common forms of peak distortion in HPLC

Basically the primary cause of peak distortion is due to the occurrence of more than one mechanism of analyte retention however there are other reasons that account for this condition (mobile phase issues column degradation injection problems etc)

carlo

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Mobile phase and solvent Considerations

In order to effectively troubleshoot our separations for retention time efficiency and peak shape issues we need to understand the basics of retention and separation in HPLC This section presents a brief overview of the controlling variables and how they might be manipulated to change chromatographic behavior

Reversed Phase solvents

Reversed phase mobile phases usually consist of water and an organic solvent often called a ldquomodifierrdquo When ionisable compounds are analyzed buffers and other additives may be present in the aqueous phase to control retention and peak shape[9 10 11]

Chromatographically in reversed phase HPLC water is the ldquoweakestrdquo solvent As water is most polar it repels the hydrophobic analytes into the stationary phase more than any other solvent and hence retention times are long ndashthis makes it chromatographically lsquoweakrsquo The organic modifier is added (usually only one modifier type at a time for modern chromatography) and as these are less polar the (hydrophobic) analyte is no longer as strongly repelled into the stationary phase will spend less time in the stationary phase and therefore elute earlier This makes the modifier chromatographically ldquostrongrdquo as it speeds up elution

As progressively more organic modifier is added to the mobile phase the analyte retention time will continue to decrease

The values alongside the solvent chemical structures in Figure 6 represent the Snyder polarity index value The more polar a solvent the lsquoweakerrsquo it is in terms of eluotropic strength in reversed phase HPLC

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Figure 6 Solvents in reversed phase HPLC Figure 7 Properties for selected HPLC solvents

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Use Figure 7 to assess the relative physics-chemical properties of the various solvents one might use as organic modifiers to decide upon their suitability for a particular application

Changing the organic modifier within a mobile phase can alter the selectivity of the separation as well as the retention characteristics

So how would one chose the most appropriate solvent ndash what are the major considerations

First of all the chosen organic solvent must be miscible with water All of the solvents listed in Figure 6 are miscible with water Second a low viscosity mobile phase is favored to reduce dispersion and keep system back pressure low

The solvent must be stable for long periods of time This disfavors tetrahydrofuran which after exposure to air degrades rapidly often forming explosive peroxides

The two remaining mobile phase solvents are methanol and acetonitrile The use of both solvents usually gives excellent retention characteristics

Acetonitrile however has a lower viscosity and lower UV-cutoff (which is advantageous as the possibility detection interference is reduced) The UV-cutoff for methanol is 205 nm while the UV cut-off for acetonitrile is 190 nm For these reasons most analysts begin reversed-phase method development with acetonitrile

It is also important to note that whilst the polarity index values of methanol and acetonitrile are similar they have different Lewis acid base characteristics (proton donator acceptor) which allows them to act differently to alter separation characteristics We will study this in greater detail in Part II of this Essential Guide carlo

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Mobile Phase Strength and Retention

Increasing the percentage of organic modifier in the mobile phase has a profound effect on analyte retention due to the change in polarity of the mobile phase Use the slider below to investigate the effect of altering the mobile phase composition

Figure 8 Solvent strength

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Eluotropic Series

Solvent strength in Reverse Phase HPLC depends upon the solvent type and amount used in the mobile phase (percentage organic modifier (B))

It is possible to interrelate the relative ldquostrengthrdquo of each of the common solvents using an Eluotropic Series This is a table or listing of the various solvents and their relative strength on various media ndash for example Aluminium Oxide or Silica for Normal Phase HPLC and C18 for Reverse Phase HPLC

Commonly ndash a nomograph might be used to find equivalent eluotropic strength between mobile phases that use different organic modifiers The nomograph relates each solvent using a vertical line to indicate mobile phase composition (B) which give equivalent elution strength ndashknown as lsquoisoelutropicrsquo mobile phase compositions

Isoelutropic mobile phases produce separations in approximately the same time frame (measured using retention (capacity) factor of the last peak) ndash however they show altered selectivity This can be very useful when developing or optimising separations

You can use the slider bar below to find various isoelutropic compositions ndash note that the relationship yields results which are only approximately accurate to within about plusmn 5 B

carlo

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Figure 9 Solvent nomogram form reversed phase HPLC

Table 2 Eluotropic series of various reverse phase solventscarlo

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Matching Injection Volume with Sample Solvent Strength

Under ideal conditions the strength of the sample solvent used would make no difference to the chromatographic separation because the solvent would be diluted instantaneously to the same composition as the mobile phase However in practice it takes a finite period of time for the injected sample solvent to become diluted with the mobile phase

The following table provides guidelines for sample injection volumes based on the sample solvent strength with respect to that of mobile phase

100 Strong Solvent

In this demanding situation it is necessary to keep the injection volume as small as possible thereby minimising peak distortion The recommendation is for no more than 10μL

When the sample solvent is greater than 80 of the mobile phase it may be possible to inject larger volumes as the compositional difference between the sample solvent and mobile phase is correspondingly less

Stronger than Mobile Phase

When the sample solvent is no more than approximately 25 stronger than the mobile phase then injection volumes as high as 25μLshould be possible However always examine the chromatogram for possible peak distortion given the inter-relationship of this and the preceding rule

Generally analyte solubility issues are the primary reason for increasing the sample solvent strength utilised To minimise such solvent strength variations on injection dissolve the analyte at a high concentration in the strong solvent then dilute with the weaker solvent to progressively correct for the difference in eluotropic strength In a worst-case scenario dissolve in 100 strong solvent and inject as small a volume as possible

carlo

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Mobile Phase

The best injection scenario is when the sample solvent and the mobile phase are matched both in terms of eluotropicstrength and pH In this situation given the solvent homogeneity you donrsquot have to be concerned with dilution effects

However the injection volume is limited The contribution of the sample injection volume to the chromatographic peak width can be determined by the following equation

Weaker than Mobile Phase

To assess the effect of the mobile phase strength consider the ldquoRule of Threerdquo which states that a 10 change in the strong solvent will result in an approximate threefold change in analyte retention time Therefore if a chromatographic peak had a retention time of 5min in a mobile phase of 4060 wateracetonitrile then in a mobile phase of 6040 wateracetonitrile its retention time would increase to approximately 45min (3 times 3 times 5min = 45min)

Consequently if the sample were to be injected in 6040 wateracetonitrile instead then the analyte molecules would travel much more slowly through the column until the sample solvent was fully diluted with the mobile phase

Chromatographic bands travelling more slowly than the mobile phase tend to compress because as mobile phase reaches the analyte molecules at the peak ldquotailrdquo they will begin to move faster down the column catching those analyte molecules further down the column that are still effectively residing in a weaker mobile phase strength

As the difference between the solvent strength of the sample solvent and the mobile phase increases it is possible to use progressively larger and larger injection volumes This effectively allows analyte on-column sample ldquofocussingrdquo or concentration and is often employed in environmental analysis to assist in the detection of trace quantities of pesticides

carlo

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012

Exceptions to the above are whenever the mobile phase contains trace additives then it is always advisable to inject samples dissolved in the mobile phase eg ion-pair separations rely on the equilibrium between the ion pair reagent in the mobile phase and the stationary phase Injecting a sample in a solvent that doesnrsquot contain the ion pair shifts this equilibrium to the mobile phase with resulting peak distortion

Peak distortion can occur due to a mismatch between injection solvent and mobile phase strength In particular when large amounts of sample are injected in a solvent that is much ldquostrongerrdquo than the mobile phase The diagram below shows typical peak distortion problems

Figure 10 Peak shape abnormalities currently found when solvent strength is not considered when injecting

carlo

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012

Ionisable CompoundspH Considerations

The mobile phase pH can be used to influence the charge state of ionisable species in solution The extent of analyte ionisation can be used to affect retention and selectivity The pH of a solution will influence the charge state of an acidic or basic analyte

For example addition of an acid to an aqueous solution of a basic analyte will increase the concentration of charged analyte in solution as the hydrogen ion concentration increases Conversely raising the pH by addition of a base will increase the concentration of the neutral form of the basic analyte

Take the example of homovanillic acid shown below ndash equilibrium between the ionised and non-ionisedforms of the analyte will be established according to the solution pH

carlo

pez-

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ax-2

012

Adding an acid to a buffered solution of homovanillic acid (ie lowering the mobile phase pH) will cause the equilibrium to shift to the left and the analyte will become less ionised (ion suppressed) as the analyterecombines to reduce the effect of the added hydrogen ions (protons) from the acidic species Adding a base to a buffered solution of homovanillic acid (ie increasing the mobile phase pH) will cause the analyte to become more ionised as the solution attempts to regain equilibrium by producing more hydrogen ions to neutralise the added base This principle was first described by Le Chetalier and the converse applies to acidic analyte species

The 2 pH rule

It can be shown that at 1 pH unit away from the analyte pKa the change in extent of ionisation is approximately 90 At 2 pH units away from the pKa the change in extent of ionisation is approximately 99 at 3 pH units 999 etc Therefore ndash a rule a thumb known as the ldquo2 pH rulerdquo is useful in predicting extent of ionisation

carlo

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012

Basic Analytes amp Ion Suppression

Unlike the dissociated (ionised) acids which when charged elute rapidly from the column protonated bases often have long retention times and poor peak shape This retention behaviour is due to the interaction with residual silanol species on the silica surface

Separations of basic compounds however are not usually carried out under ion suppression conditions The analyst would have to raise the pH to produce the neutral molecule High pH mobile phases can damage traditional silica columns unless specifically designed to cope with high pH

Traditionally the analysis of weak bases has been carried out at low pH ndash essentially because the surface silanol species are non-ionised (pKa approx 35 ndash 45) and peak tailing is improved somewhat

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically) highly surface active base such as Triethylamine (TEA) Piperazine NNNrsquoNrsquo-Tetramethylethylenediamine (TEMED) or Dimethyloctylamine (DMOA) These bases interact with the surface silanol species in preference to the analyte molecule and are called lsquosacrificial basesrsquo They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times

carlo

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ax-2

012

Figure 13 Tailing factor versus concentration of TEAcarlo

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ax-2

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Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

carlo

pez-

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ax-2

012

Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

carlo

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

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As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

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Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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2

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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012

carlo

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ax-2

012

carlo

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Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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carlo

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012

carlo

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carlo

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012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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012

Page 5: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Gradient Operation

One cannot assign a fixed krsquo value to a compound when gradient elution is applied krsquo is the retention coefficient and changes during gradient elution

The equation for the gradient retention factor (k) takes the form

Gradient retention factor (k) is difficult to visualize as it differs from its isocratic counterpart (krsquo) and resembles morethe profile of the gradient elution It is effectively defined as the retention factor for an analyte that has migrated half way down the HPLC column

The Retention Factor as a Means to Evaluate the State of an HPLC Separation

The retention factor of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention factors may result from changes in mobile phase flow rate mobile phase composition column stationary phase and temperature

carlo

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Efficiency

The efficiency of a chromatographic peak is a measure of the dispersion of the analyte band as it travelled through the HPLC system and column In an ideal world chromatographic peaks would be pencil thin lines ndashhowever due to dispersion effects the peaks take on their familiar ldquoGaussianrdquo shape

The plate number (N) is a measure of the peak dispersion on the HPLC column reflecting the column performance N is derived from an analogy of Martin and Synge who likened column efficiency to fractional distillation where the column is divided into Theoretical Plates

Each plate is the distance over which the sample components achieve one equilibration between the stationary and mobile phase in the column Therefore the more ldquotheoreticalrdquo plates available within a column the more equilibrations possible and the better quality the separation

Higher values for the Plate Number (N) are expected for subsequent peaks within a chromatogram Later eluting peaks that look broad in comparison to early eluters may have a higher plate count If this is not the case then your system contains a large extra-column dead volume which is dominating the diffusion process

For a fractionating tower of a given length (L) the higher the number of plates the lower will be the distance between each plate shown as plate height in the diagram Therefore for high efficiency separations the plate number (N) will be high and the plate height (H) low Note that plate height is often called ndash ldquoHeight Equivalent to a Theoretical Plate (HETP)rdquo Modern columns which employ are reduced particle size sub 2 microm enjoy an increased plate number (N) without increasing the length of the column (L) due to the reduction in distance between active sites plate height (H)

These two terms are related through the expression H = L N

carlo

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Figure 3 Factional distillation model of efficiency theorycarlo

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The number of theoretical plates is often used to establish the efficacy of a column for a given method The method developer may decide that a given method is no longer valid when the plate number falls below a predetermined value At that time the column would be replaced with a new one

Figure 4 Comparison of two chromatograms with the same selectivity and different efficiency (and resolution)carlo

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Efficiency as a Means to Evaluate the State of an HPLC Separation

Peak efficiency within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Reduced efficiency may result from incorrect or mobile phase deterioration incorrect or deteriorated column increased dead volume addition of a disproportionate length of tubing or wider ID tubing etc

Peak Asymmetry

In the ideal world all chromatographic peaks would be symmetrical (or Gaussian)

However due to the effects of instrument dead-volume adsorptive effects of the stationary phase and the quality of the column packing peaks may often show a tailing behavior Tailing describes a peak whose tail portion (distance lsquoBrsquo in the diagram) is wider than the front portion (distance lsquoArsquo in the diagram) Also if the sample concentration is too high or if the column is damaged and contains lsquochannelsrsquo then a fronting peak shape may occur

There are different ways of calculating the amount of peak tailing (or fronting) However one of them peak asymmetry (As) is probably the most commonly used and is defined as

Where A and B are measured at 10 of the peak heightcarlo

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Figure 5 Determination of Peak Asymmetry (AS) and examples of good and poor peak shape

The US Pharmacopeia (USP) recommends the use of a different measurement the tailing factor (Tf) which has been defined as

Where A and B are measured at 5 of the peak height

In general terms it doesnrsquot matter which measurement parameter is used (either As or Tf) as long as we are consistent in our measurement and are aware of the ldquoacceptablerdquo values for each measure The definition of lsquoacceptablersquo peak tailing or fronting for the two different measurements is roughly the same - see Table 1carlo

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ax-2

012

Peak Distortion as a Means to Evaluate the State of an HPLC Separation

Peak asymmetry and tailing factor can aid in the identification of many problems that are associated with HPLC system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions Peak tailing and fronting are the most common forms of peak distortion in HPLC

Basically the primary cause of peak distortion is due to the occurrence of more than one mechanism of analyte retention however there are other reasons that account for this condition (mobile phase issues column degradation injection problems etc)

carlo

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Mobile phase and solvent Considerations

In order to effectively troubleshoot our separations for retention time efficiency and peak shape issues we need to understand the basics of retention and separation in HPLC This section presents a brief overview of the controlling variables and how they might be manipulated to change chromatographic behavior

Reversed Phase solvents

Reversed phase mobile phases usually consist of water and an organic solvent often called a ldquomodifierrdquo When ionisable compounds are analyzed buffers and other additives may be present in the aqueous phase to control retention and peak shape[9 10 11]

Chromatographically in reversed phase HPLC water is the ldquoweakestrdquo solvent As water is most polar it repels the hydrophobic analytes into the stationary phase more than any other solvent and hence retention times are long ndashthis makes it chromatographically lsquoweakrsquo The organic modifier is added (usually only one modifier type at a time for modern chromatography) and as these are less polar the (hydrophobic) analyte is no longer as strongly repelled into the stationary phase will spend less time in the stationary phase and therefore elute earlier This makes the modifier chromatographically ldquostrongrdquo as it speeds up elution

As progressively more organic modifier is added to the mobile phase the analyte retention time will continue to decrease

The values alongside the solvent chemical structures in Figure 6 represent the Snyder polarity index value The more polar a solvent the lsquoweakerrsquo it is in terms of eluotropic strength in reversed phase HPLC

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Figure 6 Solvents in reversed phase HPLC Figure 7 Properties for selected HPLC solvents

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Use Figure 7 to assess the relative physics-chemical properties of the various solvents one might use as organic modifiers to decide upon their suitability for a particular application

Changing the organic modifier within a mobile phase can alter the selectivity of the separation as well as the retention characteristics

So how would one chose the most appropriate solvent ndash what are the major considerations

First of all the chosen organic solvent must be miscible with water All of the solvents listed in Figure 6 are miscible with water Second a low viscosity mobile phase is favored to reduce dispersion and keep system back pressure low

The solvent must be stable for long periods of time This disfavors tetrahydrofuran which after exposure to air degrades rapidly often forming explosive peroxides

The two remaining mobile phase solvents are methanol and acetonitrile The use of both solvents usually gives excellent retention characteristics

Acetonitrile however has a lower viscosity and lower UV-cutoff (which is advantageous as the possibility detection interference is reduced) The UV-cutoff for methanol is 205 nm while the UV cut-off for acetonitrile is 190 nm For these reasons most analysts begin reversed-phase method development with acetonitrile

It is also important to note that whilst the polarity index values of methanol and acetonitrile are similar they have different Lewis acid base characteristics (proton donator acceptor) which allows them to act differently to alter separation characteristics We will study this in greater detail in Part II of this Essential Guide carlo

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Mobile Phase Strength and Retention

Increasing the percentage of organic modifier in the mobile phase has a profound effect on analyte retention due to the change in polarity of the mobile phase Use the slider below to investigate the effect of altering the mobile phase composition

Figure 8 Solvent strength

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Eluotropic Series

Solvent strength in Reverse Phase HPLC depends upon the solvent type and amount used in the mobile phase (percentage organic modifier (B))

It is possible to interrelate the relative ldquostrengthrdquo of each of the common solvents using an Eluotropic Series This is a table or listing of the various solvents and their relative strength on various media ndash for example Aluminium Oxide or Silica for Normal Phase HPLC and C18 for Reverse Phase HPLC

Commonly ndash a nomograph might be used to find equivalent eluotropic strength between mobile phases that use different organic modifiers The nomograph relates each solvent using a vertical line to indicate mobile phase composition (B) which give equivalent elution strength ndashknown as lsquoisoelutropicrsquo mobile phase compositions

Isoelutropic mobile phases produce separations in approximately the same time frame (measured using retention (capacity) factor of the last peak) ndash however they show altered selectivity This can be very useful when developing or optimising separations

You can use the slider bar below to find various isoelutropic compositions ndash note that the relationship yields results which are only approximately accurate to within about plusmn 5 B

carlo

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Figure 9 Solvent nomogram form reversed phase HPLC

Table 2 Eluotropic series of various reverse phase solventscarlo

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Matching Injection Volume with Sample Solvent Strength

Under ideal conditions the strength of the sample solvent used would make no difference to the chromatographic separation because the solvent would be diluted instantaneously to the same composition as the mobile phase However in practice it takes a finite period of time for the injected sample solvent to become diluted with the mobile phase

The following table provides guidelines for sample injection volumes based on the sample solvent strength with respect to that of mobile phase

100 Strong Solvent

In this demanding situation it is necessary to keep the injection volume as small as possible thereby minimising peak distortion The recommendation is for no more than 10μL

When the sample solvent is greater than 80 of the mobile phase it may be possible to inject larger volumes as the compositional difference between the sample solvent and mobile phase is correspondingly less

Stronger than Mobile Phase

When the sample solvent is no more than approximately 25 stronger than the mobile phase then injection volumes as high as 25μLshould be possible However always examine the chromatogram for possible peak distortion given the inter-relationship of this and the preceding rule

Generally analyte solubility issues are the primary reason for increasing the sample solvent strength utilised To minimise such solvent strength variations on injection dissolve the analyte at a high concentration in the strong solvent then dilute with the weaker solvent to progressively correct for the difference in eluotropic strength In a worst-case scenario dissolve in 100 strong solvent and inject as small a volume as possible

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Mobile Phase

The best injection scenario is when the sample solvent and the mobile phase are matched both in terms of eluotropicstrength and pH In this situation given the solvent homogeneity you donrsquot have to be concerned with dilution effects

However the injection volume is limited The contribution of the sample injection volume to the chromatographic peak width can be determined by the following equation

Weaker than Mobile Phase

To assess the effect of the mobile phase strength consider the ldquoRule of Threerdquo which states that a 10 change in the strong solvent will result in an approximate threefold change in analyte retention time Therefore if a chromatographic peak had a retention time of 5min in a mobile phase of 4060 wateracetonitrile then in a mobile phase of 6040 wateracetonitrile its retention time would increase to approximately 45min (3 times 3 times 5min = 45min)

Consequently if the sample were to be injected in 6040 wateracetonitrile instead then the analyte molecules would travel much more slowly through the column until the sample solvent was fully diluted with the mobile phase

Chromatographic bands travelling more slowly than the mobile phase tend to compress because as mobile phase reaches the analyte molecules at the peak ldquotailrdquo they will begin to move faster down the column catching those analyte molecules further down the column that are still effectively residing in a weaker mobile phase strength

As the difference between the solvent strength of the sample solvent and the mobile phase increases it is possible to use progressively larger and larger injection volumes This effectively allows analyte on-column sample ldquofocussingrdquo or concentration and is often employed in environmental analysis to assist in the detection of trace quantities of pesticides

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012

Exceptions to the above are whenever the mobile phase contains trace additives then it is always advisable to inject samples dissolved in the mobile phase eg ion-pair separations rely on the equilibrium between the ion pair reagent in the mobile phase and the stationary phase Injecting a sample in a solvent that doesnrsquot contain the ion pair shifts this equilibrium to the mobile phase with resulting peak distortion

Peak distortion can occur due to a mismatch between injection solvent and mobile phase strength In particular when large amounts of sample are injected in a solvent that is much ldquostrongerrdquo than the mobile phase The diagram below shows typical peak distortion problems

Figure 10 Peak shape abnormalities currently found when solvent strength is not considered when injecting

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Ionisable CompoundspH Considerations

The mobile phase pH can be used to influence the charge state of ionisable species in solution The extent of analyte ionisation can be used to affect retention and selectivity The pH of a solution will influence the charge state of an acidic or basic analyte

For example addition of an acid to an aqueous solution of a basic analyte will increase the concentration of charged analyte in solution as the hydrogen ion concentration increases Conversely raising the pH by addition of a base will increase the concentration of the neutral form of the basic analyte

Take the example of homovanillic acid shown below ndash equilibrium between the ionised and non-ionisedforms of the analyte will be established according to the solution pH

carlo

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012

Adding an acid to a buffered solution of homovanillic acid (ie lowering the mobile phase pH) will cause the equilibrium to shift to the left and the analyte will become less ionised (ion suppressed) as the analyterecombines to reduce the effect of the added hydrogen ions (protons) from the acidic species Adding a base to a buffered solution of homovanillic acid (ie increasing the mobile phase pH) will cause the analyte to become more ionised as the solution attempts to regain equilibrium by producing more hydrogen ions to neutralise the added base This principle was first described by Le Chetalier and the converse applies to acidic analyte species

The 2 pH rule

It can be shown that at 1 pH unit away from the analyte pKa the change in extent of ionisation is approximately 90 At 2 pH units away from the pKa the change in extent of ionisation is approximately 99 at 3 pH units 999 etc Therefore ndash a rule a thumb known as the ldquo2 pH rulerdquo is useful in predicting extent of ionisation

carlo

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Basic Analytes amp Ion Suppression

Unlike the dissociated (ionised) acids which when charged elute rapidly from the column protonated bases often have long retention times and poor peak shape This retention behaviour is due to the interaction with residual silanol species on the silica surface

Separations of basic compounds however are not usually carried out under ion suppression conditions The analyst would have to raise the pH to produce the neutral molecule High pH mobile phases can damage traditional silica columns unless specifically designed to cope with high pH

Traditionally the analysis of weak bases has been carried out at low pH ndash essentially because the surface silanol species are non-ionised (pKa approx 35 ndash 45) and peak tailing is improved somewhat

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically) highly surface active base such as Triethylamine (TEA) Piperazine NNNrsquoNrsquo-Tetramethylethylenediamine (TEMED) or Dimethyloctylamine (DMOA) These bases interact with the surface silanol species in preference to the analyte molecule and are called lsquosacrificial basesrsquo They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times

carlo

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Figure 13 Tailing factor versus concentration of TEAcarlo

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Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

carlo

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Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

carlo

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

carlo

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

carlo

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

carlo

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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012

Figure 16 Basic polar analytes analysed using Type I and Type II silica

carlo

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012

Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

carlo

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012

As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

carlo

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012

End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

rlope

z-hu

max

-201

2

Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

carlo

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

carlo

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012

In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

rlope

z-hu

max

-201

2

Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

carlo

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012

Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

carlo

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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2

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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2

carlo

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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ax-2

012

carlo

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012

carlo

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012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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012

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012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 6: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Efficiency

The efficiency of a chromatographic peak is a measure of the dispersion of the analyte band as it travelled through the HPLC system and column In an ideal world chromatographic peaks would be pencil thin lines ndashhowever due to dispersion effects the peaks take on their familiar ldquoGaussianrdquo shape

The plate number (N) is a measure of the peak dispersion on the HPLC column reflecting the column performance N is derived from an analogy of Martin and Synge who likened column efficiency to fractional distillation where the column is divided into Theoretical Plates

Each plate is the distance over which the sample components achieve one equilibration between the stationary and mobile phase in the column Therefore the more ldquotheoreticalrdquo plates available within a column the more equilibrations possible and the better quality the separation

Higher values for the Plate Number (N) are expected for subsequent peaks within a chromatogram Later eluting peaks that look broad in comparison to early eluters may have a higher plate count If this is not the case then your system contains a large extra-column dead volume which is dominating the diffusion process

For a fractionating tower of a given length (L) the higher the number of plates the lower will be the distance between each plate shown as plate height in the diagram Therefore for high efficiency separations the plate number (N) will be high and the plate height (H) low Note that plate height is often called ndash ldquoHeight Equivalent to a Theoretical Plate (HETP)rdquo Modern columns which employ are reduced particle size sub 2 microm enjoy an increased plate number (N) without increasing the length of the column (L) due to the reduction in distance between active sites plate height (H)

These two terms are related through the expression H = L N

carlo

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Figure 3 Factional distillation model of efficiency theorycarlo

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The number of theoretical plates is often used to establish the efficacy of a column for a given method The method developer may decide that a given method is no longer valid when the plate number falls below a predetermined value At that time the column would be replaced with a new one

Figure 4 Comparison of two chromatograms with the same selectivity and different efficiency (and resolution)carlo

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Efficiency as a Means to Evaluate the State of an HPLC Separation

Peak efficiency within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Reduced efficiency may result from incorrect or mobile phase deterioration incorrect or deteriorated column increased dead volume addition of a disproportionate length of tubing or wider ID tubing etc

Peak Asymmetry

In the ideal world all chromatographic peaks would be symmetrical (or Gaussian)

However due to the effects of instrument dead-volume adsorptive effects of the stationary phase and the quality of the column packing peaks may often show a tailing behavior Tailing describes a peak whose tail portion (distance lsquoBrsquo in the diagram) is wider than the front portion (distance lsquoArsquo in the diagram) Also if the sample concentration is too high or if the column is damaged and contains lsquochannelsrsquo then a fronting peak shape may occur

There are different ways of calculating the amount of peak tailing (or fronting) However one of them peak asymmetry (As) is probably the most commonly used and is defined as

Where A and B are measured at 10 of the peak heightcarlo

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Figure 5 Determination of Peak Asymmetry (AS) and examples of good and poor peak shape

The US Pharmacopeia (USP) recommends the use of a different measurement the tailing factor (Tf) which has been defined as

Where A and B are measured at 5 of the peak height

In general terms it doesnrsquot matter which measurement parameter is used (either As or Tf) as long as we are consistent in our measurement and are aware of the ldquoacceptablerdquo values for each measure The definition of lsquoacceptablersquo peak tailing or fronting for the two different measurements is roughly the same - see Table 1carlo

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Peak Distortion as a Means to Evaluate the State of an HPLC Separation

Peak asymmetry and tailing factor can aid in the identification of many problems that are associated with HPLC system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions Peak tailing and fronting are the most common forms of peak distortion in HPLC

Basically the primary cause of peak distortion is due to the occurrence of more than one mechanism of analyte retention however there are other reasons that account for this condition (mobile phase issues column degradation injection problems etc)

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Mobile phase and solvent Considerations

In order to effectively troubleshoot our separations for retention time efficiency and peak shape issues we need to understand the basics of retention and separation in HPLC This section presents a brief overview of the controlling variables and how they might be manipulated to change chromatographic behavior

Reversed Phase solvents

Reversed phase mobile phases usually consist of water and an organic solvent often called a ldquomodifierrdquo When ionisable compounds are analyzed buffers and other additives may be present in the aqueous phase to control retention and peak shape[9 10 11]

Chromatographically in reversed phase HPLC water is the ldquoweakestrdquo solvent As water is most polar it repels the hydrophobic analytes into the stationary phase more than any other solvent and hence retention times are long ndashthis makes it chromatographically lsquoweakrsquo The organic modifier is added (usually only one modifier type at a time for modern chromatography) and as these are less polar the (hydrophobic) analyte is no longer as strongly repelled into the stationary phase will spend less time in the stationary phase and therefore elute earlier This makes the modifier chromatographically ldquostrongrdquo as it speeds up elution

As progressively more organic modifier is added to the mobile phase the analyte retention time will continue to decrease

The values alongside the solvent chemical structures in Figure 6 represent the Snyder polarity index value The more polar a solvent the lsquoweakerrsquo it is in terms of eluotropic strength in reversed phase HPLC

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Figure 6 Solvents in reversed phase HPLC Figure 7 Properties for selected HPLC solvents

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Use Figure 7 to assess the relative physics-chemical properties of the various solvents one might use as organic modifiers to decide upon their suitability for a particular application

Changing the organic modifier within a mobile phase can alter the selectivity of the separation as well as the retention characteristics

So how would one chose the most appropriate solvent ndash what are the major considerations

First of all the chosen organic solvent must be miscible with water All of the solvents listed in Figure 6 are miscible with water Second a low viscosity mobile phase is favored to reduce dispersion and keep system back pressure low

The solvent must be stable for long periods of time This disfavors tetrahydrofuran which after exposure to air degrades rapidly often forming explosive peroxides

The two remaining mobile phase solvents are methanol and acetonitrile The use of both solvents usually gives excellent retention characteristics

Acetonitrile however has a lower viscosity and lower UV-cutoff (which is advantageous as the possibility detection interference is reduced) The UV-cutoff for methanol is 205 nm while the UV cut-off for acetonitrile is 190 nm For these reasons most analysts begin reversed-phase method development with acetonitrile

It is also important to note that whilst the polarity index values of methanol and acetonitrile are similar they have different Lewis acid base characteristics (proton donator acceptor) which allows them to act differently to alter separation characteristics We will study this in greater detail in Part II of this Essential Guide carlo

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Mobile Phase Strength and Retention

Increasing the percentage of organic modifier in the mobile phase has a profound effect on analyte retention due to the change in polarity of the mobile phase Use the slider below to investigate the effect of altering the mobile phase composition

Figure 8 Solvent strength

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Eluotropic Series

Solvent strength in Reverse Phase HPLC depends upon the solvent type and amount used in the mobile phase (percentage organic modifier (B))

It is possible to interrelate the relative ldquostrengthrdquo of each of the common solvents using an Eluotropic Series This is a table or listing of the various solvents and their relative strength on various media ndash for example Aluminium Oxide or Silica for Normal Phase HPLC and C18 for Reverse Phase HPLC

Commonly ndash a nomograph might be used to find equivalent eluotropic strength between mobile phases that use different organic modifiers The nomograph relates each solvent using a vertical line to indicate mobile phase composition (B) which give equivalent elution strength ndashknown as lsquoisoelutropicrsquo mobile phase compositions

Isoelutropic mobile phases produce separations in approximately the same time frame (measured using retention (capacity) factor of the last peak) ndash however they show altered selectivity This can be very useful when developing or optimising separations

You can use the slider bar below to find various isoelutropic compositions ndash note that the relationship yields results which are only approximately accurate to within about plusmn 5 B

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Figure 9 Solvent nomogram form reversed phase HPLC

Table 2 Eluotropic series of various reverse phase solventscarlo

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Matching Injection Volume with Sample Solvent Strength

Under ideal conditions the strength of the sample solvent used would make no difference to the chromatographic separation because the solvent would be diluted instantaneously to the same composition as the mobile phase However in practice it takes a finite period of time for the injected sample solvent to become diluted with the mobile phase

The following table provides guidelines for sample injection volumes based on the sample solvent strength with respect to that of mobile phase

100 Strong Solvent

In this demanding situation it is necessary to keep the injection volume as small as possible thereby minimising peak distortion The recommendation is for no more than 10μL

When the sample solvent is greater than 80 of the mobile phase it may be possible to inject larger volumes as the compositional difference between the sample solvent and mobile phase is correspondingly less

Stronger than Mobile Phase

When the sample solvent is no more than approximately 25 stronger than the mobile phase then injection volumes as high as 25μLshould be possible However always examine the chromatogram for possible peak distortion given the inter-relationship of this and the preceding rule

Generally analyte solubility issues are the primary reason for increasing the sample solvent strength utilised To minimise such solvent strength variations on injection dissolve the analyte at a high concentration in the strong solvent then dilute with the weaker solvent to progressively correct for the difference in eluotropic strength In a worst-case scenario dissolve in 100 strong solvent and inject as small a volume as possible

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Mobile Phase

The best injection scenario is when the sample solvent and the mobile phase are matched both in terms of eluotropicstrength and pH In this situation given the solvent homogeneity you donrsquot have to be concerned with dilution effects

However the injection volume is limited The contribution of the sample injection volume to the chromatographic peak width can be determined by the following equation

Weaker than Mobile Phase

To assess the effect of the mobile phase strength consider the ldquoRule of Threerdquo which states that a 10 change in the strong solvent will result in an approximate threefold change in analyte retention time Therefore if a chromatographic peak had a retention time of 5min in a mobile phase of 4060 wateracetonitrile then in a mobile phase of 6040 wateracetonitrile its retention time would increase to approximately 45min (3 times 3 times 5min = 45min)

Consequently if the sample were to be injected in 6040 wateracetonitrile instead then the analyte molecules would travel much more slowly through the column until the sample solvent was fully diluted with the mobile phase

Chromatographic bands travelling more slowly than the mobile phase tend to compress because as mobile phase reaches the analyte molecules at the peak ldquotailrdquo they will begin to move faster down the column catching those analyte molecules further down the column that are still effectively residing in a weaker mobile phase strength

As the difference between the solvent strength of the sample solvent and the mobile phase increases it is possible to use progressively larger and larger injection volumes This effectively allows analyte on-column sample ldquofocussingrdquo or concentration and is often employed in environmental analysis to assist in the detection of trace quantities of pesticides

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Exceptions to the above are whenever the mobile phase contains trace additives then it is always advisable to inject samples dissolved in the mobile phase eg ion-pair separations rely on the equilibrium between the ion pair reagent in the mobile phase and the stationary phase Injecting a sample in a solvent that doesnrsquot contain the ion pair shifts this equilibrium to the mobile phase with resulting peak distortion

Peak distortion can occur due to a mismatch between injection solvent and mobile phase strength In particular when large amounts of sample are injected in a solvent that is much ldquostrongerrdquo than the mobile phase The diagram below shows typical peak distortion problems

Figure 10 Peak shape abnormalities currently found when solvent strength is not considered when injecting

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Ionisable CompoundspH Considerations

The mobile phase pH can be used to influence the charge state of ionisable species in solution The extent of analyte ionisation can be used to affect retention and selectivity The pH of a solution will influence the charge state of an acidic or basic analyte

For example addition of an acid to an aqueous solution of a basic analyte will increase the concentration of charged analyte in solution as the hydrogen ion concentration increases Conversely raising the pH by addition of a base will increase the concentration of the neutral form of the basic analyte

Take the example of homovanillic acid shown below ndash equilibrium between the ionised and non-ionisedforms of the analyte will be established according to the solution pH

carlo

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Adding an acid to a buffered solution of homovanillic acid (ie lowering the mobile phase pH) will cause the equilibrium to shift to the left and the analyte will become less ionised (ion suppressed) as the analyterecombines to reduce the effect of the added hydrogen ions (protons) from the acidic species Adding a base to a buffered solution of homovanillic acid (ie increasing the mobile phase pH) will cause the analyte to become more ionised as the solution attempts to regain equilibrium by producing more hydrogen ions to neutralise the added base This principle was first described by Le Chetalier and the converse applies to acidic analyte species

The 2 pH rule

It can be shown that at 1 pH unit away from the analyte pKa the change in extent of ionisation is approximately 90 At 2 pH units away from the pKa the change in extent of ionisation is approximately 99 at 3 pH units 999 etc Therefore ndash a rule a thumb known as the ldquo2 pH rulerdquo is useful in predicting extent of ionisation

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Basic Analytes amp Ion Suppression

Unlike the dissociated (ionised) acids which when charged elute rapidly from the column protonated bases often have long retention times and poor peak shape This retention behaviour is due to the interaction with residual silanol species on the silica surface

Separations of basic compounds however are not usually carried out under ion suppression conditions The analyst would have to raise the pH to produce the neutral molecule High pH mobile phases can damage traditional silica columns unless specifically designed to cope with high pH

Traditionally the analysis of weak bases has been carried out at low pH ndash essentially because the surface silanol species are non-ionised (pKa approx 35 ndash 45) and peak tailing is improved somewhat

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically) highly surface active base such as Triethylamine (TEA) Piperazine NNNrsquoNrsquo-Tetramethylethylenediamine (TEMED) or Dimethyloctylamine (DMOA) These bases interact with the surface silanol species in preference to the analyte molecule and are called lsquosacrificial basesrsquo They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times

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Figure 13 Tailing factor versus concentration of TEAcarlo

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Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

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Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

carlo

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

carlo

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

carlo

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012

As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

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-201

2

Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

carlo

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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2

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

carlo

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

carlo

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

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012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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ax-2

012

carlo

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012

carlo

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Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 7: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Figure 3 Factional distillation model of efficiency theorycarlo

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The number of theoretical plates is often used to establish the efficacy of a column for a given method The method developer may decide that a given method is no longer valid when the plate number falls below a predetermined value At that time the column would be replaced with a new one

Figure 4 Comparison of two chromatograms with the same selectivity and different efficiency (and resolution)carlo

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Efficiency as a Means to Evaluate the State of an HPLC Separation

Peak efficiency within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Reduced efficiency may result from incorrect or mobile phase deterioration incorrect or deteriorated column increased dead volume addition of a disproportionate length of tubing or wider ID tubing etc

Peak Asymmetry

In the ideal world all chromatographic peaks would be symmetrical (or Gaussian)

However due to the effects of instrument dead-volume adsorptive effects of the stationary phase and the quality of the column packing peaks may often show a tailing behavior Tailing describes a peak whose tail portion (distance lsquoBrsquo in the diagram) is wider than the front portion (distance lsquoArsquo in the diagram) Also if the sample concentration is too high or if the column is damaged and contains lsquochannelsrsquo then a fronting peak shape may occur

There are different ways of calculating the amount of peak tailing (or fronting) However one of them peak asymmetry (As) is probably the most commonly used and is defined as

Where A and B are measured at 10 of the peak heightcarlo

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Figure 5 Determination of Peak Asymmetry (AS) and examples of good and poor peak shape

The US Pharmacopeia (USP) recommends the use of a different measurement the tailing factor (Tf) which has been defined as

Where A and B are measured at 5 of the peak height

In general terms it doesnrsquot matter which measurement parameter is used (either As or Tf) as long as we are consistent in our measurement and are aware of the ldquoacceptablerdquo values for each measure The definition of lsquoacceptablersquo peak tailing or fronting for the two different measurements is roughly the same - see Table 1carlo

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ax-2

012

Peak Distortion as a Means to Evaluate the State of an HPLC Separation

Peak asymmetry and tailing factor can aid in the identification of many problems that are associated with HPLC system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions Peak tailing and fronting are the most common forms of peak distortion in HPLC

Basically the primary cause of peak distortion is due to the occurrence of more than one mechanism of analyte retention however there are other reasons that account for this condition (mobile phase issues column degradation injection problems etc)

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Mobile phase and solvent Considerations

In order to effectively troubleshoot our separations for retention time efficiency and peak shape issues we need to understand the basics of retention and separation in HPLC This section presents a brief overview of the controlling variables and how they might be manipulated to change chromatographic behavior

Reversed Phase solvents

Reversed phase mobile phases usually consist of water and an organic solvent often called a ldquomodifierrdquo When ionisable compounds are analyzed buffers and other additives may be present in the aqueous phase to control retention and peak shape[9 10 11]

Chromatographically in reversed phase HPLC water is the ldquoweakestrdquo solvent As water is most polar it repels the hydrophobic analytes into the stationary phase more than any other solvent and hence retention times are long ndashthis makes it chromatographically lsquoweakrsquo The organic modifier is added (usually only one modifier type at a time for modern chromatography) and as these are less polar the (hydrophobic) analyte is no longer as strongly repelled into the stationary phase will spend less time in the stationary phase and therefore elute earlier This makes the modifier chromatographically ldquostrongrdquo as it speeds up elution

As progressively more organic modifier is added to the mobile phase the analyte retention time will continue to decrease

The values alongside the solvent chemical structures in Figure 6 represent the Snyder polarity index value The more polar a solvent the lsquoweakerrsquo it is in terms of eluotropic strength in reversed phase HPLC

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Figure 6 Solvents in reversed phase HPLC Figure 7 Properties for selected HPLC solvents

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Use Figure 7 to assess the relative physics-chemical properties of the various solvents one might use as organic modifiers to decide upon their suitability for a particular application

Changing the organic modifier within a mobile phase can alter the selectivity of the separation as well as the retention characteristics

So how would one chose the most appropriate solvent ndash what are the major considerations

First of all the chosen organic solvent must be miscible with water All of the solvents listed in Figure 6 are miscible with water Second a low viscosity mobile phase is favored to reduce dispersion and keep system back pressure low

The solvent must be stable for long periods of time This disfavors tetrahydrofuran which after exposure to air degrades rapidly often forming explosive peroxides

The two remaining mobile phase solvents are methanol and acetonitrile The use of both solvents usually gives excellent retention characteristics

Acetonitrile however has a lower viscosity and lower UV-cutoff (which is advantageous as the possibility detection interference is reduced) The UV-cutoff for methanol is 205 nm while the UV cut-off for acetonitrile is 190 nm For these reasons most analysts begin reversed-phase method development with acetonitrile

It is also important to note that whilst the polarity index values of methanol and acetonitrile are similar they have different Lewis acid base characteristics (proton donator acceptor) which allows them to act differently to alter separation characteristics We will study this in greater detail in Part II of this Essential Guide carlo

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Mobile Phase Strength and Retention

Increasing the percentage of organic modifier in the mobile phase has a profound effect on analyte retention due to the change in polarity of the mobile phase Use the slider below to investigate the effect of altering the mobile phase composition

Figure 8 Solvent strength

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Eluotropic Series

Solvent strength in Reverse Phase HPLC depends upon the solvent type and amount used in the mobile phase (percentage organic modifier (B))

It is possible to interrelate the relative ldquostrengthrdquo of each of the common solvents using an Eluotropic Series This is a table or listing of the various solvents and their relative strength on various media ndash for example Aluminium Oxide or Silica for Normal Phase HPLC and C18 for Reverse Phase HPLC

Commonly ndash a nomograph might be used to find equivalent eluotropic strength between mobile phases that use different organic modifiers The nomograph relates each solvent using a vertical line to indicate mobile phase composition (B) which give equivalent elution strength ndashknown as lsquoisoelutropicrsquo mobile phase compositions

Isoelutropic mobile phases produce separations in approximately the same time frame (measured using retention (capacity) factor of the last peak) ndash however they show altered selectivity This can be very useful when developing or optimising separations

You can use the slider bar below to find various isoelutropic compositions ndash note that the relationship yields results which are only approximately accurate to within about plusmn 5 B

carlo

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Figure 9 Solvent nomogram form reversed phase HPLC

Table 2 Eluotropic series of various reverse phase solventscarlo

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Matching Injection Volume with Sample Solvent Strength

Under ideal conditions the strength of the sample solvent used would make no difference to the chromatographic separation because the solvent would be diluted instantaneously to the same composition as the mobile phase However in practice it takes a finite period of time for the injected sample solvent to become diluted with the mobile phase

The following table provides guidelines for sample injection volumes based on the sample solvent strength with respect to that of mobile phase

100 Strong Solvent

In this demanding situation it is necessary to keep the injection volume as small as possible thereby minimising peak distortion The recommendation is for no more than 10μL

When the sample solvent is greater than 80 of the mobile phase it may be possible to inject larger volumes as the compositional difference between the sample solvent and mobile phase is correspondingly less

Stronger than Mobile Phase

When the sample solvent is no more than approximately 25 stronger than the mobile phase then injection volumes as high as 25μLshould be possible However always examine the chromatogram for possible peak distortion given the inter-relationship of this and the preceding rule

Generally analyte solubility issues are the primary reason for increasing the sample solvent strength utilised To minimise such solvent strength variations on injection dissolve the analyte at a high concentration in the strong solvent then dilute with the weaker solvent to progressively correct for the difference in eluotropic strength In a worst-case scenario dissolve in 100 strong solvent and inject as small a volume as possible

carlo

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Mobile Phase

The best injection scenario is when the sample solvent and the mobile phase are matched both in terms of eluotropicstrength and pH In this situation given the solvent homogeneity you donrsquot have to be concerned with dilution effects

However the injection volume is limited The contribution of the sample injection volume to the chromatographic peak width can be determined by the following equation

Weaker than Mobile Phase

To assess the effect of the mobile phase strength consider the ldquoRule of Threerdquo which states that a 10 change in the strong solvent will result in an approximate threefold change in analyte retention time Therefore if a chromatographic peak had a retention time of 5min in a mobile phase of 4060 wateracetonitrile then in a mobile phase of 6040 wateracetonitrile its retention time would increase to approximately 45min (3 times 3 times 5min = 45min)

Consequently if the sample were to be injected in 6040 wateracetonitrile instead then the analyte molecules would travel much more slowly through the column until the sample solvent was fully diluted with the mobile phase

Chromatographic bands travelling more slowly than the mobile phase tend to compress because as mobile phase reaches the analyte molecules at the peak ldquotailrdquo they will begin to move faster down the column catching those analyte molecules further down the column that are still effectively residing in a weaker mobile phase strength

As the difference between the solvent strength of the sample solvent and the mobile phase increases it is possible to use progressively larger and larger injection volumes This effectively allows analyte on-column sample ldquofocussingrdquo or concentration and is often employed in environmental analysis to assist in the detection of trace quantities of pesticides

carlo

pez-

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012

Exceptions to the above are whenever the mobile phase contains trace additives then it is always advisable to inject samples dissolved in the mobile phase eg ion-pair separations rely on the equilibrium between the ion pair reagent in the mobile phase and the stationary phase Injecting a sample in a solvent that doesnrsquot contain the ion pair shifts this equilibrium to the mobile phase with resulting peak distortion

Peak distortion can occur due to a mismatch between injection solvent and mobile phase strength In particular when large amounts of sample are injected in a solvent that is much ldquostrongerrdquo than the mobile phase The diagram below shows typical peak distortion problems

Figure 10 Peak shape abnormalities currently found when solvent strength is not considered when injecting

carlo

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Ionisable CompoundspH Considerations

The mobile phase pH can be used to influence the charge state of ionisable species in solution The extent of analyte ionisation can be used to affect retention and selectivity The pH of a solution will influence the charge state of an acidic or basic analyte

For example addition of an acid to an aqueous solution of a basic analyte will increase the concentration of charged analyte in solution as the hydrogen ion concentration increases Conversely raising the pH by addition of a base will increase the concentration of the neutral form of the basic analyte

Take the example of homovanillic acid shown below ndash equilibrium between the ionised and non-ionisedforms of the analyte will be established according to the solution pH

carlo

pez-

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ax-2

012

Adding an acid to a buffered solution of homovanillic acid (ie lowering the mobile phase pH) will cause the equilibrium to shift to the left and the analyte will become less ionised (ion suppressed) as the analyterecombines to reduce the effect of the added hydrogen ions (protons) from the acidic species Adding a base to a buffered solution of homovanillic acid (ie increasing the mobile phase pH) will cause the analyte to become more ionised as the solution attempts to regain equilibrium by producing more hydrogen ions to neutralise the added base This principle was first described by Le Chetalier and the converse applies to acidic analyte species

The 2 pH rule

It can be shown that at 1 pH unit away from the analyte pKa the change in extent of ionisation is approximately 90 At 2 pH units away from the pKa the change in extent of ionisation is approximately 99 at 3 pH units 999 etc Therefore ndash a rule a thumb known as the ldquo2 pH rulerdquo is useful in predicting extent of ionisation

carlo

pez-

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012

Basic Analytes amp Ion Suppression

Unlike the dissociated (ionised) acids which when charged elute rapidly from the column protonated bases often have long retention times and poor peak shape This retention behaviour is due to the interaction with residual silanol species on the silica surface

Separations of basic compounds however are not usually carried out under ion suppression conditions The analyst would have to raise the pH to produce the neutral molecule High pH mobile phases can damage traditional silica columns unless specifically designed to cope with high pH

Traditionally the analysis of weak bases has been carried out at low pH ndash essentially because the surface silanol species are non-ionised (pKa approx 35 ndash 45) and peak tailing is improved somewhat

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically) highly surface active base such as Triethylamine (TEA) Piperazine NNNrsquoNrsquo-Tetramethylethylenediamine (TEMED) or Dimethyloctylamine (DMOA) These bases interact with the surface silanol species in preference to the analyte molecule and are called lsquosacrificial basesrsquo They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times

carlo

pez-

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Figure 13 Tailing factor versus concentration of TEAcarlo

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Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

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Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

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As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

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Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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2

carlo

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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012

carlo

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012

carlo

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012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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012

carlo

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012

carlo

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Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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012

carlo

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012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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012

Page 8: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

The number of theoretical plates is often used to establish the efficacy of a column for a given method The method developer may decide that a given method is no longer valid when the plate number falls below a predetermined value At that time the column would be replaced with a new one

Figure 4 Comparison of two chromatograms with the same selectivity and different efficiency (and resolution)carlo

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Efficiency as a Means to Evaluate the State of an HPLC Separation

Peak efficiency within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Reduced efficiency may result from incorrect or mobile phase deterioration incorrect or deteriorated column increased dead volume addition of a disproportionate length of tubing or wider ID tubing etc

Peak Asymmetry

In the ideal world all chromatographic peaks would be symmetrical (or Gaussian)

However due to the effects of instrument dead-volume adsorptive effects of the stationary phase and the quality of the column packing peaks may often show a tailing behavior Tailing describes a peak whose tail portion (distance lsquoBrsquo in the diagram) is wider than the front portion (distance lsquoArsquo in the diagram) Also if the sample concentration is too high or if the column is damaged and contains lsquochannelsrsquo then a fronting peak shape may occur

There are different ways of calculating the amount of peak tailing (or fronting) However one of them peak asymmetry (As) is probably the most commonly used and is defined as

Where A and B are measured at 10 of the peak heightcarlo

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Figure 5 Determination of Peak Asymmetry (AS) and examples of good and poor peak shape

The US Pharmacopeia (USP) recommends the use of a different measurement the tailing factor (Tf) which has been defined as

Where A and B are measured at 5 of the peak height

In general terms it doesnrsquot matter which measurement parameter is used (either As or Tf) as long as we are consistent in our measurement and are aware of the ldquoacceptablerdquo values for each measure The definition of lsquoacceptablersquo peak tailing or fronting for the two different measurements is roughly the same - see Table 1carlo

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ax-2

012

Peak Distortion as a Means to Evaluate the State of an HPLC Separation

Peak asymmetry and tailing factor can aid in the identification of many problems that are associated with HPLC system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions Peak tailing and fronting are the most common forms of peak distortion in HPLC

Basically the primary cause of peak distortion is due to the occurrence of more than one mechanism of analyte retention however there are other reasons that account for this condition (mobile phase issues column degradation injection problems etc)

carlo

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Mobile phase and solvent Considerations

In order to effectively troubleshoot our separations for retention time efficiency and peak shape issues we need to understand the basics of retention and separation in HPLC This section presents a brief overview of the controlling variables and how they might be manipulated to change chromatographic behavior

Reversed Phase solvents

Reversed phase mobile phases usually consist of water and an organic solvent often called a ldquomodifierrdquo When ionisable compounds are analyzed buffers and other additives may be present in the aqueous phase to control retention and peak shape[9 10 11]

Chromatographically in reversed phase HPLC water is the ldquoweakestrdquo solvent As water is most polar it repels the hydrophobic analytes into the stationary phase more than any other solvent and hence retention times are long ndashthis makes it chromatographically lsquoweakrsquo The organic modifier is added (usually only one modifier type at a time for modern chromatography) and as these are less polar the (hydrophobic) analyte is no longer as strongly repelled into the stationary phase will spend less time in the stationary phase and therefore elute earlier This makes the modifier chromatographically ldquostrongrdquo as it speeds up elution

As progressively more organic modifier is added to the mobile phase the analyte retention time will continue to decrease

The values alongside the solvent chemical structures in Figure 6 represent the Snyder polarity index value The more polar a solvent the lsquoweakerrsquo it is in terms of eluotropic strength in reversed phase HPLC

carlo

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012

Figure 6 Solvents in reversed phase HPLC Figure 7 Properties for selected HPLC solvents

carlo

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Use Figure 7 to assess the relative physics-chemical properties of the various solvents one might use as organic modifiers to decide upon their suitability for a particular application

Changing the organic modifier within a mobile phase can alter the selectivity of the separation as well as the retention characteristics

So how would one chose the most appropriate solvent ndash what are the major considerations

First of all the chosen organic solvent must be miscible with water All of the solvents listed in Figure 6 are miscible with water Second a low viscosity mobile phase is favored to reduce dispersion and keep system back pressure low

The solvent must be stable for long periods of time This disfavors tetrahydrofuran which after exposure to air degrades rapidly often forming explosive peroxides

The two remaining mobile phase solvents are methanol and acetonitrile The use of both solvents usually gives excellent retention characteristics

Acetonitrile however has a lower viscosity and lower UV-cutoff (which is advantageous as the possibility detection interference is reduced) The UV-cutoff for methanol is 205 nm while the UV cut-off for acetonitrile is 190 nm For these reasons most analysts begin reversed-phase method development with acetonitrile

It is also important to note that whilst the polarity index values of methanol and acetonitrile are similar they have different Lewis acid base characteristics (proton donator acceptor) which allows them to act differently to alter separation characteristics We will study this in greater detail in Part II of this Essential Guide carlo

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Mobile Phase Strength and Retention

Increasing the percentage of organic modifier in the mobile phase has a profound effect on analyte retention due to the change in polarity of the mobile phase Use the slider below to investigate the effect of altering the mobile phase composition

Figure 8 Solvent strength

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Eluotropic Series

Solvent strength in Reverse Phase HPLC depends upon the solvent type and amount used in the mobile phase (percentage organic modifier (B))

It is possible to interrelate the relative ldquostrengthrdquo of each of the common solvents using an Eluotropic Series This is a table or listing of the various solvents and their relative strength on various media ndash for example Aluminium Oxide or Silica for Normal Phase HPLC and C18 for Reverse Phase HPLC

Commonly ndash a nomograph might be used to find equivalent eluotropic strength between mobile phases that use different organic modifiers The nomograph relates each solvent using a vertical line to indicate mobile phase composition (B) which give equivalent elution strength ndashknown as lsquoisoelutropicrsquo mobile phase compositions

Isoelutropic mobile phases produce separations in approximately the same time frame (measured using retention (capacity) factor of the last peak) ndash however they show altered selectivity This can be very useful when developing or optimising separations

You can use the slider bar below to find various isoelutropic compositions ndash note that the relationship yields results which are only approximately accurate to within about plusmn 5 B

carlo

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Figure 9 Solvent nomogram form reversed phase HPLC

Table 2 Eluotropic series of various reverse phase solventscarlo

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Matching Injection Volume with Sample Solvent Strength

Under ideal conditions the strength of the sample solvent used would make no difference to the chromatographic separation because the solvent would be diluted instantaneously to the same composition as the mobile phase However in practice it takes a finite period of time for the injected sample solvent to become diluted with the mobile phase

The following table provides guidelines for sample injection volumes based on the sample solvent strength with respect to that of mobile phase

100 Strong Solvent

In this demanding situation it is necessary to keep the injection volume as small as possible thereby minimising peak distortion The recommendation is for no more than 10μL

When the sample solvent is greater than 80 of the mobile phase it may be possible to inject larger volumes as the compositional difference between the sample solvent and mobile phase is correspondingly less

Stronger than Mobile Phase

When the sample solvent is no more than approximately 25 stronger than the mobile phase then injection volumes as high as 25μLshould be possible However always examine the chromatogram for possible peak distortion given the inter-relationship of this and the preceding rule

Generally analyte solubility issues are the primary reason for increasing the sample solvent strength utilised To minimise such solvent strength variations on injection dissolve the analyte at a high concentration in the strong solvent then dilute with the weaker solvent to progressively correct for the difference in eluotropic strength In a worst-case scenario dissolve in 100 strong solvent and inject as small a volume as possible

carlo

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Mobile Phase

The best injection scenario is when the sample solvent and the mobile phase are matched both in terms of eluotropicstrength and pH In this situation given the solvent homogeneity you donrsquot have to be concerned with dilution effects

However the injection volume is limited The contribution of the sample injection volume to the chromatographic peak width can be determined by the following equation

Weaker than Mobile Phase

To assess the effect of the mobile phase strength consider the ldquoRule of Threerdquo which states that a 10 change in the strong solvent will result in an approximate threefold change in analyte retention time Therefore if a chromatographic peak had a retention time of 5min in a mobile phase of 4060 wateracetonitrile then in a mobile phase of 6040 wateracetonitrile its retention time would increase to approximately 45min (3 times 3 times 5min = 45min)

Consequently if the sample were to be injected in 6040 wateracetonitrile instead then the analyte molecules would travel much more slowly through the column until the sample solvent was fully diluted with the mobile phase

Chromatographic bands travelling more slowly than the mobile phase tend to compress because as mobile phase reaches the analyte molecules at the peak ldquotailrdquo they will begin to move faster down the column catching those analyte molecules further down the column that are still effectively residing in a weaker mobile phase strength

As the difference between the solvent strength of the sample solvent and the mobile phase increases it is possible to use progressively larger and larger injection volumes This effectively allows analyte on-column sample ldquofocussingrdquo or concentration and is often employed in environmental analysis to assist in the detection of trace quantities of pesticides

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012

Exceptions to the above are whenever the mobile phase contains trace additives then it is always advisable to inject samples dissolved in the mobile phase eg ion-pair separations rely on the equilibrium between the ion pair reagent in the mobile phase and the stationary phase Injecting a sample in a solvent that doesnrsquot contain the ion pair shifts this equilibrium to the mobile phase with resulting peak distortion

Peak distortion can occur due to a mismatch between injection solvent and mobile phase strength In particular when large amounts of sample are injected in a solvent that is much ldquostrongerrdquo than the mobile phase The diagram below shows typical peak distortion problems

Figure 10 Peak shape abnormalities currently found when solvent strength is not considered when injecting

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Ionisable CompoundspH Considerations

The mobile phase pH can be used to influence the charge state of ionisable species in solution The extent of analyte ionisation can be used to affect retention and selectivity The pH of a solution will influence the charge state of an acidic or basic analyte

For example addition of an acid to an aqueous solution of a basic analyte will increase the concentration of charged analyte in solution as the hydrogen ion concentration increases Conversely raising the pH by addition of a base will increase the concentration of the neutral form of the basic analyte

Take the example of homovanillic acid shown below ndash equilibrium between the ionised and non-ionisedforms of the analyte will be established according to the solution pH

carlo

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012

Adding an acid to a buffered solution of homovanillic acid (ie lowering the mobile phase pH) will cause the equilibrium to shift to the left and the analyte will become less ionised (ion suppressed) as the analyterecombines to reduce the effect of the added hydrogen ions (protons) from the acidic species Adding a base to a buffered solution of homovanillic acid (ie increasing the mobile phase pH) will cause the analyte to become more ionised as the solution attempts to regain equilibrium by producing more hydrogen ions to neutralise the added base This principle was first described by Le Chetalier and the converse applies to acidic analyte species

The 2 pH rule

It can be shown that at 1 pH unit away from the analyte pKa the change in extent of ionisation is approximately 90 At 2 pH units away from the pKa the change in extent of ionisation is approximately 99 at 3 pH units 999 etc Therefore ndash a rule a thumb known as the ldquo2 pH rulerdquo is useful in predicting extent of ionisation

carlo

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Basic Analytes amp Ion Suppression

Unlike the dissociated (ionised) acids which when charged elute rapidly from the column protonated bases often have long retention times and poor peak shape This retention behaviour is due to the interaction with residual silanol species on the silica surface

Separations of basic compounds however are not usually carried out under ion suppression conditions The analyst would have to raise the pH to produce the neutral molecule High pH mobile phases can damage traditional silica columns unless specifically designed to cope with high pH

Traditionally the analysis of weak bases has been carried out at low pH ndash essentially because the surface silanol species are non-ionised (pKa approx 35 ndash 45) and peak tailing is improved somewhat

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically) highly surface active base such as Triethylamine (TEA) Piperazine NNNrsquoNrsquo-Tetramethylethylenediamine (TEMED) or Dimethyloctylamine (DMOA) These bases interact with the surface silanol species in preference to the analyte molecule and are called lsquosacrificial basesrsquo They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times

carlo

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Figure 13 Tailing factor versus concentration of TEAcarlo

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Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

carlo

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Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

carlo

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

carlo

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

carlo

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

carlo

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012

This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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012

Figure 16 Basic polar analytes analysed using Type I and Type II silica

carlo

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

carlo

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ax-2

012

As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

carlo

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012

End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

rlope

z-hu

max

-201

2

Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

carlo

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

carlo

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012

In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

rlope

z-hu

max

-201

2

Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

carlo

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ax-2

012

Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

carlo

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ax-2

012

Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

carlo

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ax-2

012

By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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-201

2

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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2

carlo

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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ax-2

012

carlo

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ax-2

012

carlo

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012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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012

carlo

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012

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012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 9: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Efficiency as a Means to Evaluate the State of an HPLC Separation

Peak efficiency within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Reduced efficiency may result from incorrect or mobile phase deterioration incorrect or deteriorated column increased dead volume addition of a disproportionate length of tubing or wider ID tubing etc

Peak Asymmetry

In the ideal world all chromatographic peaks would be symmetrical (or Gaussian)

However due to the effects of instrument dead-volume adsorptive effects of the stationary phase and the quality of the column packing peaks may often show a tailing behavior Tailing describes a peak whose tail portion (distance lsquoBrsquo in the diagram) is wider than the front portion (distance lsquoArsquo in the diagram) Also if the sample concentration is too high or if the column is damaged and contains lsquochannelsrsquo then a fronting peak shape may occur

There are different ways of calculating the amount of peak tailing (or fronting) However one of them peak asymmetry (As) is probably the most commonly used and is defined as

Where A and B are measured at 10 of the peak heightcarlo

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012

Figure 5 Determination of Peak Asymmetry (AS) and examples of good and poor peak shape

The US Pharmacopeia (USP) recommends the use of a different measurement the tailing factor (Tf) which has been defined as

Where A and B are measured at 5 of the peak height

In general terms it doesnrsquot matter which measurement parameter is used (either As or Tf) as long as we are consistent in our measurement and are aware of the ldquoacceptablerdquo values for each measure The definition of lsquoacceptablersquo peak tailing or fronting for the two different measurements is roughly the same - see Table 1carlo

pez-

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ax-2

012

Peak Distortion as a Means to Evaluate the State of an HPLC Separation

Peak asymmetry and tailing factor can aid in the identification of many problems that are associated with HPLC system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions Peak tailing and fronting are the most common forms of peak distortion in HPLC

Basically the primary cause of peak distortion is due to the occurrence of more than one mechanism of analyte retention however there are other reasons that account for this condition (mobile phase issues column degradation injection problems etc)

carlo

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Mobile phase and solvent Considerations

In order to effectively troubleshoot our separations for retention time efficiency and peak shape issues we need to understand the basics of retention and separation in HPLC This section presents a brief overview of the controlling variables and how they might be manipulated to change chromatographic behavior

Reversed Phase solvents

Reversed phase mobile phases usually consist of water and an organic solvent often called a ldquomodifierrdquo When ionisable compounds are analyzed buffers and other additives may be present in the aqueous phase to control retention and peak shape[9 10 11]

Chromatographically in reversed phase HPLC water is the ldquoweakestrdquo solvent As water is most polar it repels the hydrophobic analytes into the stationary phase more than any other solvent and hence retention times are long ndashthis makes it chromatographically lsquoweakrsquo The organic modifier is added (usually only one modifier type at a time for modern chromatography) and as these are less polar the (hydrophobic) analyte is no longer as strongly repelled into the stationary phase will spend less time in the stationary phase and therefore elute earlier This makes the modifier chromatographically ldquostrongrdquo as it speeds up elution

As progressively more organic modifier is added to the mobile phase the analyte retention time will continue to decrease

The values alongside the solvent chemical structures in Figure 6 represent the Snyder polarity index value The more polar a solvent the lsquoweakerrsquo it is in terms of eluotropic strength in reversed phase HPLC

carlo

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012

Figure 6 Solvents in reversed phase HPLC Figure 7 Properties for selected HPLC solvents

carlo

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012

Use Figure 7 to assess the relative physics-chemical properties of the various solvents one might use as organic modifiers to decide upon their suitability for a particular application

Changing the organic modifier within a mobile phase can alter the selectivity of the separation as well as the retention characteristics

So how would one chose the most appropriate solvent ndash what are the major considerations

First of all the chosen organic solvent must be miscible with water All of the solvents listed in Figure 6 are miscible with water Second a low viscosity mobile phase is favored to reduce dispersion and keep system back pressure low

The solvent must be stable for long periods of time This disfavors tetrahydrofuran which after exposure to air degrades rapidly often forming explosive peroxides

The two remaining mobile phase solvents are methanol and acetonitrile The use of both solvents usually gives excellent retention characteristics

Acetonitrile however has a lower viscosity and lower UV-cutoff (which is advantageous as the possibility detection interference is reduced) The UV-cutoff for methanol is 205 nm while the UV cut-off for acetonitrile is 190 nm For these reasons most analysts begin reversed-phase method development with acetonitrile

It is also important to note that whilst the polarity index values of methanol and acetonitrile are similar they have different Lewis acid base characteristics (proton donator acceptor) which allows them to act differently to alter separation characteristics We will study this in greater detail in Part II of this Essential Guide carlo

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Mobile Phase Strength and Retention

Increasing the percentage of organic modifier in the mobile phase has a profound effect on analyte retention due to the change in polarity of the mobile phase Use the slider below to investigate the effect of altering the mobile phase composition

Figure 8 Solvent strength

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Eluotropic Series

Solvent strength in Reverse Phase HPLC depends upon the solvent type and amount used in the mobile phase (percentage organic modifier (B))

It is possible to interrelate the relative ldquostrengthrdquo of each of the common solvents using an Eluotropic Series This is a table or listing of the various solvents and their relative strength on various media ndash for example Aluminium Oxide or Silica for Normal Phase HPLC and C18 for Reverse Phase HPLC

Commonly ndash a nomograph might be used to find equivalent eluotropic strength between mobile phases that use different organic modifiers The nomograph relates each solvent using a vertical line to indicate mobile phase composition (B) which give equivalent elution strength ndashknown as lsquoisoelutropicrsquo mobile phase compositions

Isoelutropic mobile phases produce separations in approximately the same time frame (measured using retention (capacity) factor of the last peak) ndash however they show altered selectivity This can be very useful when developing or optimising separations

You can use the slider bar below to find various isoelutropic compositions ndash note that the relationship yields results which are only approximately accurate to within about plusmn 5 B

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Figure 9 Solvent nomogram form reversed phase HPLC

Table 2 Eluotropic series of various reverse phase solventscarlo

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Matching Injection Volume with Sample Solvent Strength

Under ideal conditions the strength of the sample solvent used would make no difference to the chromatographic separation because the solvent would be diluted instantaneously to the same composition as the mobile phase However in practice it takes a finite period of time for the injected sample solvent to become diluted with the mobile phase

The following table provides guidelines for sample injection volumes based on the sample solvent strength with respect to that of mobile phase

100 Strong Solvent

In this demanding situation it is necessary to keep the injection volume as small as possible thereby minimising peak distortion The recommendation is for no more than 10μL

When the sample solvent is greater than 80 of the mobile phase it may be possible to inject larger volumes as the compositional difference between the sample solvent and mobile phase is correspondingly less

Stronger than Mobile Phase

When the sample solvent is no more than approximately 25 stronger than the mobile phase then injection volumes as high as 25μLshould be possible However always examine the chromatogram for possible peak distortion given the inter-relationship of this and the preceding rule

Generally analyte solubility issues are the primary reason for increasing the sample solvent strength utilised To minimise such solvent strength variations on injection dissolve the analyte at a high concentration in the strong solvent then dilute with the weaker solvent to progressively correct for the difference in eluotropic strength In a worst-case scenario dissolve in 100 strong solvent and inject as small a volume as possible

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Mobile Phase

The best injection scenario is when the sample solvent and the mobile phase are matched both in terms of eluotropicstrength and pH In this situation given the solvent homogeneity you donrsquot have to be concerned with dilution effects

However the injection volume is limited The contribution of the sample injection volume to the chromatographic peak width can be determined by the following equation

Weaker than Mobile Phase

To assess the effect of the mobile phase strength consider the ldquoRule of Threerdquo which states that a 10 change in the strong solvent will result in an approximate threefold change in analyte retention time Therefore if a chromatographic peak had a retention time of 5min in a mobile phase of 4060 wateracetonitrile then in a mobile phase of 6040 wateracetonitrile its retention time would increase to approximately 45min (3 times 3 times 5min = 45min)

Consequently if the sample were to be injected in 6040 wateracetonitrile instead then the analyte molecules would travel much more slowly through the column until the sample solvent was fully diluted with the mobile phase

Chromatographic bands travelling more slowly than the mobile phase tend to compress because as mobile phase reaches the analyte molecules at the peak ldquotailrdquo they will begin to move faster down the column catching those analyte molecules further down the column that are still effectively residing in a weaker mobile phase strength

As the difference between the solvent strength of the sample solvent and the mobile phase increases it is possible to use progressively larger and larger injection volumes This effectively allows analyte on-column sample ldquofocussingrdquo or concentration and is often employed in environmental analysis to assist in the detection of trace quantities of pesticides

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Exceptions to the above are whenever the mobile phase contains trace additives then it is always advisable to inject samples dissolved in the mobile phase eg ion-pair separations rely on the equilibrium between the ion pair reagent in the mobile phase and the stationary phase Injecting a sample in a solvent that doesnrsquot contain the ion pair shifts this equilibrium to the mobile phase with resulting peak distortion

Peak distortion can occur due to a mismatch between injection solvent and mobile phase strength In particular when large amounts of sample are injected in a solvent that is much ldquostrongerrdquo than the mobile phase The diagram below shows typical peak distortion problems

Figure 10 Peak shape abnormalities currently found when solvent strength is not considered when injecting

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Ionisable CompoundspH Considerations

The mobile phase pH can be used to influence the charge state of ionisable species in solution The extent of analyte ionisation can be used to affect retention and selectivity The pH of a solution will influence the charge state of an acidic or basic analyte

For example addition of an acid to an aqueous solution of a basic analyte will increase the concentration of charged analyte in solution as the hydrogen ion concentration increases Conversely raising the pH by addition of a base will increase the concentration of the neutral form of the basic analyte

Take the example of homovanillic acid shown below ndash equilibrium between the ionised and non-ionisedforms of the analyte will be established according to the solution pH

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Adding an acid to a buffered solution of homovanillic acid (ie lowering the mobile phase pH) will cause the equilibrium to shift to the left and the analyte will become less ionised (ion suppressed) as the analyterecombines to reduce the effect of the added hydrogen ions (protons) from the acidic species Adding a base to a buffered solution of homovanillic acid (ie increasing the mobile phase pH) will cause the analyte to become more ionised as the solution attempts to regain equilibrium by producing more hydrogen ions to neutralise the added base This principle was first described by Le Chetalier and the converse applies to acidic analyte species

The 2 pH rule

It can be shown that at 1 pH unit away from the analyte pKa the change in extent of ionisation is approximately 90 At 2 pH units away from the pKa the change in extent of ionisation is approximately 99 at 3 pH units 999 etc Therefore ndash a rule a thumb known as the ldquo2 pH rulerdquo is useful in predicting extent of ionisation

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Basic Analytes amp Ion Suppression

Unlike the dissociated (ionised) acids which when charged elute rapidly from the column protonated bases often have long retention times and poor peak shape This retention behaviour is due to the interaction with residual silanol species on the silica surface

Separations of basic compounds however are not usually carried out under ion suppression conditions The analyst would have to raise the pH to produce the neutral molecule High pH mobile phases can damage traditional silica columns unless specifically designed to cope with high pH

Traditionally the analysis of weak bases has been carried out at low pH ndash essentially because the surface silanol species are non-ionised (pKa approx 35 ndash 45) and peak tailing is improved somewhat

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically) highly surface active base such as Triethylamine (TEA) Piperazine NNNrsquoNrsquo-Tetramethylethylenediamine (TEMED) or Dimethyloctylamine (DMOA) These bases interact with the surface silanol species in preference to the analyte molecule and are called lsquosacrificial basesrsquo They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times

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Figure 13 Tailing factor versus concentration of TEAcarlo

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Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

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Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

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As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

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Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

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012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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ax-2

012

carlo

pez-

hum

ax-2

012

carlo

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012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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carlo

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012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 10: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Figure 5 Determination of Peak Asymmetry (AS) and examples of good and poor peak shape

The US Pharmacopeia (USP) recommends the use of a different measurement the tailing factor (Tf) which has been defined as

Where A and B are measured at 5 of the peak height

In general terms it doesnrsquot matter which measurement parameter is used (either As or Tf) as long as we are consistent in our measurement and are aware of the ldquoacceptablerdquo values for each measure The definition of lsquoacceptablersquo peak tailing or fronting for the two different measurements is roughly the same - see Table 1carlo

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012

Peak Distortion as a Means to Evaluate the State of an HPLC Separation

Peak asymmetry and tailing factor can aid in the identification of many problems that are associated with HPLC system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions Peak tailing and fronting are the most common forms of peak distortion in HPLC

Basically the primary cause of peak distortion is due to the occurrence of more than one mechanism of analyte retention however there are other reasons that account for this condition (mobile phase issues column degradation injection problems etc)

carlo

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Mobile phase and solvent Considerations

In order to effectively troubleshoot our separations for retention time efficiency and peak shape issues we need to understand the basics of retention and separation in HPLC This section presents a brief overview of the controlling variables and how they might be manipulated to change chromatographic behavior

Reversed Phase solvents

Reversed phase mobile phases usually consist of water and an organic solvent often called a ldquomodifierrdquo When ionisable compounds are analyzed buffers and other additives may be present in the aqueous phase to control retention and peak shape[9 10 11]

Chromatographically in reversed phase HPLC water is the ldquoweakestrdquo solvent As water is most polar it repels the hydrophobic analytes into the stationary phase more than any other solvent and hence retention times are long ndashthis makes it chromatographically lsquoweakrsquo The organic modifier is added (usually only one modifier type at a time for modern chromatography) and as these are less polar the (hydrophobic) analyte is no longer as strongly repelled into the stationary phase will spend less time in the stationary phase and therefore elute earlier This makes the modifier chromatographically ldquostrongrdquo as it speeds up elution

As progressively more organic modifier is added to the mobile phase the analyte retention time will continue to decrease

The values alongside the solvent chemical structures in Figure 6 represent the Snyder polarity index value The more polar a solvent the lsquoweakerrsquo it is in terms of eluotropic strength in reversed phase HPLC

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Figure 6 Solvents in reversed phase HPLC Figure 7 Properties for selected HPLC solvents

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Use Figure 7 to assess the relative physics-chemical properties of the various solvents one might use as organic modifiers to decide upon their suitability for a particular application

Changing the organic modifier within a mobile phase can alter the selectivity of the separation as well as the retention characteristics

So how would one chose the most appropriate solvent ndash what are the major considerations

First of all the chosen organic solvent must be miscible with water All of the solvents listed in Figure 6 are miscible with water Second a low viscosity mobile phase is favored to reduce dispersion and keep system back pressure low

The solvent must be stable for long periods of time This disfavors tetrahydrofuran which after exposure to air degrades rapidly often forming explosive peroxides

The two remaining mobile phase solvents are methanol and acetonitrile The use of both solvents usually gives excellent retention characteristics

Acetonitrile however has a lower viscosity and lower UV-cutoff (which is advantageous as the possibility detection interference is reduced) The UV-cutoff for methanol is 205 nm while the UV cut-off for acetonitrile is 190 nm For these reasons most analysts begin reversed-phase method development with acetonitrile

It is also important to note that whilst the polarity index values of methanol and acetonitrile are similar they have different Lewis acid base characteristics (proton donator acceptor) which allows them to act differently to alter separation characteristics We will study this in greater detail in Part II of this Essential Guide carlo

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Mobile Phase Strength and Retention

Increasing the percentage of organic modifier in the mobile phase has a profound effect on analyte retention due to the change in polarity of the mobile phase Use the slider below to investigate the effect of altering the mobile phase composition

Figure 8 Solvent strength

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Eluotropic Series

Solvent strength in Reverse Phase HPLC depends upon the solvent type and amount used in the mobile phase (percentage organic modifier (B))

It is possible to interrelate the relative ldquostrengthrdquo of each of the common solvents using an Eluotropic Series This is a table or listing of the various solvents and their relative strength on various media ndash for example Aluminium Oxide or Silica for Normal Phase HPLC and C18 for Reverse Phase HPLC

Commonly ndash a nomograph might be used to find equivalent eluotropic strength between mobile phases that use different organic modifiers The nomograph relates each solvent using a vertical line to indicate mobile phase composition (B) which give equivalent elution strength ndashknown as lsquoisoelutropicrsquo mobile phase compositions

Isoelutropic mobile phases produce separations in approximately the same time frame (measured using retention (capacity) factor of the last peak) ndash however they show altered selectivity This can be very useful when developing or optimising separations

You can use the slider bar below to find various isoelutropic compositions ndash note that the relationship yields results which are only approximately accurate to within about plusmn 5 B

carlo

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Figure 9 Solvent nomogram form reversed phase HPLC

Table 2 Eluotropic series of various reverse phase solventscarlo

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Matching Injection Volume with Sample Solvent Strength

Under ideal conditions the strength of the sample solvent used would make no difference to the chromatographic separation because the solvent would be diluted instantaneously to the same composition as the mobile phase However in practice it takes a finite period of time for the injected sample solvent to become diluted with the mobile phase

The following table provides guidelines for sample injection volumes based on the sample solvent strength with respect to that of mobile phase

100 Strong Solvent

In this demanding situation it is necessary to keep the injection volume as small as possible thereby minimising peak distortion The recommendation is for no more than 10μL

When the sample solvent is greater than 80 of the mobile phase it may be possible to inject larger volumes as the compositional difference between the sample solvent and mobile phase is correspondingly less

Stronger than Mobile Phase

When the sample solvent is no more than approximately 25 stronger than the mobile phase then injection volumes as high as 25μLshould be possible However always examine the chromatogram for possible peak distortion given the inter-relationship of this and the preceding rule

Generally analyte solubility issues are the primary reason for increasing the sample solvent strength utilised To minimise such solvent strength variations on injection dissolve the analyte at a high concentration in the strong solvent then dilute with the weaker solvent to progressively correct for the difference in eluotropic strength In a worst-case scenario dissolve in 100 strong solvent and inject as small a volume as possible

carlo

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Mobile Phase

The best injection scenario is when the sample solvent and the mobile phase are matched both in terms of eluotropicstrength and pH In this situation given the solvent homogeneity you donrsquot have to be concerned with dilution effects

However the injection volume is limited The contribution of the sample injection volume to the chromatographic peak width can be determined by the following equation

Weaker than Mobile Phase

To assess the effect of the mobile phase strength consider the ldquoRule of Threerdquo which states that a 10 change in the strong solvent will result in an approximate threefold change in analyte retention time Therefore if a chromatographic peak had a retention time of 5min in a mobile phase of 4060 wateracetonitrile then in a mobile phase of 6040 wateracetonitrile its retention time would increase to approximately 45min (3 times 3 times 5min = 45min)

Consequently if the sample were to be injected in 6040 wateracetonitrile instead then the analyte molecules would travel much more slowly through the column until the sample solvent was fully diluted with the mobile phase

Chromatographic bands travelling more slowly than the mobile phase tend to compress because as mobile phase reaches the analyte molecules at the peak ldquotailrdquo they will begin to move faster down the column catching those analyte molecules further down the column that are still effectively residing in a weaker mobile phase strength

As the difference between the solvent strength of the sample solvent and the mobile phase increases it is possible to use progressively larger and larger injection volumes This effectively allows analyte on-column sample ldquofocussingrdquo or concentration and is often employed in environmental analysis to assist in the detection of trace quantities of pesticides

carlo

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012

Exceptions to the above are whenever the mobile phase contains trace additives then it is always advisable to inject samples dissolved in the mobile phase eg ion-pair separations rely on the equilibrium between the ion pair reagent in the mobile phase and the stationary phase Injecting a sample in a solvent that doesnrsquot contain the ion pair shifts this equilibrium to the mobile phase with resulting peak distortion

Peak distortion can occur due to a mismatch between injection solvent and mobile phase strength In particular when large amounts of sample are injected in a solvent that is much ldquostrongerrdquo than the mobile phase The diagram below shows typical peak distortion problems

Figure 10 Peak shape abnormalities currently found when solvent strength is not considered when injecting

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Ionisable CompoundspH Considerations

The mobile phase pH can be used to influence the charge state of ionisable species in solution The extent of analyte ionisation can be used to affect retention and selectivity The pH of a solution will influence the charge state of an acidic or basic analyte

For example addition of an acid to an aqueous solution of a basic analyte will increase the concentration of charged analyte in solution as the hydrogen ion concentration increases Conversely raising the pH by addition of a base will increase the concentration of the neutral form of the basic analyte

Take the example of homovanillic acid shown below ndash equilibrium between the ionised and non-ionisedforms of the analyte will be established according to the solution pH

carlo

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012

Adding an acid to a buffered solution of homovanillic acid (ie lowering the mobile phase pH) will cause the equilibrium to shift to the left and the analyte will become less ionised (ion suppressed) as the analyterecombines to reduce the effect of the added hydrogen ions (protons) from the acidic species Adding a base to a buffered solution of homovanillic acid (ie increasing the mobile phase pH) will cause the analyte to become more ionised as the solution attempts to regain equilibrium by producing more hydrogen ions to neutralise the added base This principle was first described by Le Chetalier and the converse applies to acidic analyte species

The 2 pH rule

It can be shown that at 1 pH unit away from the analyte pKa the change in extent of ionisation is approximately 90 At 2 pH units away from the pKa the change in extent of ionisation is approximately 99 at 3 pH units 999 etc Therefore ndash a rule a thumb known as the ldquo2 pH rulerdquo is useful in predicting extent of ionisation

carlo

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012

Basic Analytes amp Ion Suppression

Unlike the dissociated (ionised) acids which when charged elute rapidly from the column protonated bases often have long retention times and poor peak shape This retention behaviour is due to the interaction with residual silanol species on the silica surface

Separations of basic compounds however are not usually carried out under ion suppression conditions The analyst would have to raise the pH to produce the neutral molecule High pH mobile phases can damage traditional silica columns unless specifically designed to cope with high pH

Traditionally the analysis of weak bases has been carried out at low pH ndash essentially because the surface silanol species are non-ionised (pKa approx 35 ndash 45) and peak tailing is improved somewhat

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically) highly surface active base such as Triethylamine (TEA) Piperazine NNNrsquoNrsquo-Tetramethylethylenediamine (TEMED) or Dimethyloctylamine (DMOA) These bases interact with the surface silanol species in preference to the analyte molecule and are called lsquosacrificial basesrsquo They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times

carlo

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012

Figure 13 Tailing factor versus concentration of TEAcarlo

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Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

carlo

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012

Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

carlo

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

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As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

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Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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2

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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012

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Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 11: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Peak Distortion as a Means to Evaluate the State of an HPLC Separation

Peak asymmetry and tailing factor can aid in the identification of many problems that are associated with HPLC system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions Peak tailing and fronting are the most common forms of peak distortion in HPLC

Basically the primary cause of peak distortion is due to the occurrence of more than one mechanism of analyte retention however there are other reasons that account for this condition (mobile phase issues column degradation injection problems etc)

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Mobile phase and solvent Considerations

In order to effectively troubleshoot our separations for retention time efficiency and peak shape issues we need to understand the basics of retention and separation in HPLC This section presents a brief overview of the controlling variables and how they might be manipulated to change chromatographic behavior

Reversed Phase solvents

Reversed phase mobile phases usually consist of water and an organic solvent often called a ldquomodifierrdquo When ionisable compounds are analyzed buffers and other additives may be present in the aqueous phase to control retention and peak shape[9 10 11]

Chromatographically in reversed phase HPLC water is the ldquoweakestrdquo solvent As water is most polar it repels the hydrophobic analytes into the stationary phase more than any other solvent and hence retention times are long ndashthis makes it chromatographically lsquoweakrsquo The organic modifier is added (usually only one modifier type at a time for modern chromatography) and as these are less polar the (hydrophobic) analyte is no longer as strongly repelled into the stationary phase will spend less time in the stationary phase and therefore elute earlier This makes the modifier chromatographically ldquostrongrdquo as it speeds up elution

As progressively more organic modifier is added to the mobile phase the analyte retention time will continue to decrease

The values alongside the solvent chemical structures in Figure 6 represent the Snyder polarity index value The more polar a solvent the lsquoweakerrsquo it is in terms of eluotropic strength in reversed phase HPLC

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Figure 6 Solvents in reversed phase HPLC Figure 7 Properties for selected HPLC solvents

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Use Figure 7 to assess the relative physics-chemical properties of the various solvents one might use as organic modifiers to decide upon their suitability for a particular application

Changing the organic modifier within a mobile phase can alter the selectivity of the separation as well as the retention characteristics

So how would one chose the most appropriate solvent ndash what are the major considerations

First of all the chosen organic solvent must be miscible with water All of the solvents listed in Figure 6 are miscible with water Second a low viscosity mobile phase is favored to reduce dispersion and keep system back pressure low

The solvent must be stable for long periods of time This disfavors tetrahydrofuran which after exposure to air degrades rapidly often forming explosive peroxides

The two remaining mobile phase solvents are methanol and acetonitrile The use of both solvents usually gives excellent retention characteristics

Acetonitrile however has a lower viscosity and lower UV-cutoff (which is advantageous as the possibility detection interference is reduced) The UV-cutoff for methanol is 205 nm while the UV cut-off for acetonitrile is 190 nm For these reasons most analysts begin reversed-phase method development with acetonitrile

It is also important to note that whilst the polarity index values of methanol and acetonitrile are similar they have different Lewis acid base characteristics (proton donator acceptor) which allows them to act differently to alter separation characteristics We will study this in greater detail in Part II of this Essential Guide carlo

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Mobile Phase Strength and Retention

Increasing the percentage of organic modifier in the mobile phase has a profound effect on analyte retention due to the change in polarity of the mobile phase Use the slider below to investigate the effect of altering the mobile phase composition

Figure 8 Solvent strength

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Eluotropic Series

Solvent strength in Reverse Phase HPLC depends upon the solvent type and amount used in the mobile phase (percentage organic modifier (B))

It is possible to interrelate the relative ldquostrengthrdquo of each of the common solvents using an Eluotropic Series This is a table or listing of the various solvents and their relative strength on various media ndash for example Aluminium Oxide or Silica for Normal Phase HPLC and C18 for Reverse Phase HPLC

Commonly ndash a nomograph might be used to find equivalent eluotropic strength between mobile phases that use different organic modifiers The nomograph relates each solvent using a vertical line to indicate mobile phase composition (B) which give equivalent elution strength ndashknown as lsquoisoelutropicrsquo mobile phase compositions

Isoelutropic mobile phases produce separations in approximately the same time frame (measured using retention (capacity) factor of the last peak) ndash however they show altered selectivity This can be very useful when developing or optimising separations

You can use the slider bar below to find various isoelutropic compositions ndash note that the relationship yields results which are only approximately accurate to within about plusmn 5 B

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Figure 9 Solvent nomogram form reversed phase HPLC

Table 2 Eluotropic series of various reverse phase solventscarlo

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Matching Injection Volume with Sample Solvent Strength

Under ideal conditions the strength of the sample solvent used would make no difference to the chromatographic separation because the solvent would be diluted instantaneously to the same composition as the mobile phase However in practice it takes a finite period of time for the injected sample solvent to become diluted with the mobile phase

The following table provides guidelines for sample injection volumes based on the sample solvent strength with respect to that of mobile phase

100 Strong Solvent

In this demanding situation it is necessary to keep the injection volume as small as possible thereby minimising peak distortion The recommendation is for no more than 10μL

When the sample solvent is greater than 80 of the mobile phase it may be possible to inject larger volumes as the compositional difference between the sample solvent and mobile phase is correspondingly less

Stronger than Mobile Phase

When the sample solvent is no more than approximately 25 stronger than the mobile phase then injection volumes as high as 25μLshould be possible However always examine the chromatogram for possible peak distortion given the inter-relationship of this and the preceding rule

Generally analyte solubility issues are the primary reason for increasing the sample solvent strength utilised To minimise such solvent strength variations on injection dissolve the analyte at a high concentration in the strong solvent then dilute with the weaker solvent to progressively correct for the difference in eluotropic strength In a worst-case scenario dissolve in 100 strong solvent and inject as small a volume as possible

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Mobile Phase

The best injection scenario is when the sample solvent and the mobile phase are matched both in terms of eluotropicstrength and pH In this situation given the solvent homogeneity you donrsquot have to be concerned with dilution effects

However the injection volume is limited The contribution of the sample injection volume to the chromatographic peak width can be determined by the following equation

Weaker than Mobile Phase

To assess the effect of the mobile phase strength consider the ldquoRule of Threerdquo which states that a 10 change in the strong solvent will result in an approximate threefold change in analyte retention time Therefore if a chromatographic peak had a retention time of 5min in a mobile phase of 4060 wateracetonitrile then in a mobile phase of 6040 wateracetonitrile its retention time would increase to approximately 45min (3 times 3 times 5min = 45min)

Consequently if the sample were to be injected in 6040 wateracetonitrile instead then the analyte molecules would travel much more slowly through the column until the sample solvent was fully diluted with the mobile phase

Chromatographic bands travelling more slowly than the mobile phase tend to compress because as mobile phase reaches the analyte molecules at the peak ldquotailrdquo they will begin to move faster down the column catching those analyte molecules further down the column that are still effectively residing in a weaker mobile phase strength

As the difference between the solvent strength of the sample solvent and the mobile phase increases it is possible to use progressively larger and larger injection volumes This effectively allows analyte on-column sample ldquofocussingrdquo or concentration and is often employed in environmental analysis to assist in the detection of trace quantities of pesticides

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Exceptions to the above are whenever the mobile phase contains trace additives then it is always advisable to inject samples dissolved in the mobile phase eg ion-pair separations rely on the equilibrium between the ion pair reagent in the mobile phase and the stationary phase Injecting a sample in a solvent that doesnrsquot contain the ion pair shifts this equilibrium to the mobile phase with resulting peak distortion

Peak distortion can occur due to a mismatch between injection solvent and mobile phase strength In particular when large amounts of sample are injected in a solvent that is much ldquostrongerrdquo than the mobile phase The diagram below shows typical peak distortion problems

Figure 10 Peak shape abnormalities currently found when solvent strength is not considered when injecting

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Ionisable CompoundspH Considerations

The mobile phase pH can be used to influence the charge state of ionisable species in solution The extent of analyte ionisation can be used to affect retention and selectivity The pH of a solution will influence the charge state of an acidic or basic analyte

For example addition of an acid to an aqueous solution of a basic analyte will increase the concentration of charged analyte in solution as the hydrogen ion concentration increases Conversely raising the pH by addition of a base will increase the concentration of the neutral form of the basic analyte

Take the example of homovanillic acid shown below ndash equilibrium between the ionised and non-ionisedforms of the analyte will be established according to the solution pH

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Adding an acid to a buffered solution of homovanillic acid (ie lowering the mobile phase pH) will cause the equilibrium to shift to the left and the analyte will become less ionised (ion suppressed) as the analyterecombines to reduce the effect of the added hydrogen ions (protons) from the acidic species Adding a base to a buffered solution of homovanillic acid (ie increasing the mobile phase pH) will cause the analyte to become more ionised as the solution attempts to regain equilibrium by producing more hydrogen ions to neutralise the added base This principle was first described by Le Chetalier and the converse applies to acidic analyte species

The 2 pH rule

It can be shown that at 1 pH unit away from the analyte pKa the change in extent of ionisation is approximately 90 At 2 pH units away from the pKa the change in extent of ionisation is approximately 99 at 3 pH units 999 etc Therefore ndash a rule a thumb known as the ldquo2 pH rulerdquo is useful in predicting extent of ionisation

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Basic Analytes amp Ion Suppression

Unlike the dissociated (ionised) acids which when charged elute rapidly from the column protonated bases often have long retention times and poor peak shape This retention behaviour is due to the interaction with residual silanol species on the silica surface

Separations of basic compounds however are not usually carried out under ion suppression conditions The analyst would have to raise the pH to produce the neutral molecule High pH mobile phases can damage traditional silica columns unless specifically designed to cope with high pH

Traditionally the analysis of weak bases has been carried out at low pH ndash essentially because the surface silanol species are non-ionised (pKa approx 35 ndash 45) and peak tailing is improved somewhat

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically) highly surface active base such as Triethylamine (TEA) Piperazine NNNrsquoNrsquo-Tetramethylethylenediamine (TEMED) or Dimethyloctylamine (DMOA) These bases interact with the surface silanol species in preference to the analyte molecule and are called lsquosacrificial basesrsquo They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times

carlo

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Figure 13 Tailing factor versus concentration of TEAcarlo

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Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

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Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

carlo

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

carlo

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

carlo

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012

As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

carlo

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

rlope

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-201

2

Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

carlo

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

carlo

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

rlope

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-201

2

Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

carlo

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

carlo

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

carlo

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

rlope

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2

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

carlo

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012

Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

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012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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ax-2

012

carlo

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012

carlo

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012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 12: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Mobile phase and solvent Considerations

In order to effectively troubleshoot our separations for retention time efficiency and peak shape issues we need to understand the basics of retention and separation in HPLC This section presents a brief overview of the controlling variables and how they might be manipulated to change chromatographic behavior

Reversed Phase solvents

Reversed phase mobile phases usually consist of water and an organic solvent often called a ldquomodifierrdquo When ionisable compounds are analyzed buffers and other additives may be present in the aqueous phase to control retention and peak shape[9 10 11]

Chromatographically in reversed phase HPLC water is the ldquoweakestrdquo solvent As water is most polar it repels the hydrophobic analytes into the stationary phase more than any other solvent and hence retention times are long ndashthis makes it chromatographically lsquoweakrsquo The organic modifier is added (usually only one modifier type at a time for modern chromatography) and as these are less polar the (hydrophobic) analyte is no longer as strongly repelled into the stationary phase will spend less time in the stationary phase and therefore elute earlier This makes the modifier chromatographically ldquostrongrdquo as it speeds up elution

As progressively more organic modifier is added to the mobile phase the analyte retention time will continue to decrease

The values alongside the solvent chemical structures in Figure 6 represent the Snyder polarity index value The more polar a solvent the lsquoweakerrsquo it is in terms of eluotropic strength in reversed phase HPLC

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Figure 6 Solvents in reversed phase HPLC Figure 7 Properties for selected HPLC solvents

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Use Figure 7 to assess the relative physics-chemical properties of the various solvents one might use as organic modifiers to decide upon their suitability for a particular application

Changing the organic modifier within a mobile phase can alter the selectivity of the separation as well as the retention characteristics

So how would one chose the most appropriate solvent ndash what are the major considerations

First of all the chosen organic solvent must be miscible with water All of the solvents listed in Figure 6 are miscible with water Second a low viscosity mobile phase is favored to reduce dispersion and keep system back pressure low

The solvent must be stable for long periods of time This disfavors tetrahydrofuran which after exposure to air degrades rapidly often forming explosive peroxides

The two remaining mobile phase solvents are methanol and acetonitrile The use of both solvents usually gives excellent retention characteristics

Acetonitrile however has a lower viscosity and lower UV-cutoff (which is advantageous as the possibility detection interference is reduced) The UV-cutoff for methanol is 205 nm while the UV cut-off for acetonitrile is 190 nm For these reasons most analysts begin reversed-phase method development with acetonitrile

It is also important to note that whilst the polarity index values of methanol and acetonitrile are similar they have different Lewis acid base characteristics (proton donator acceptor) which allows them to act differently to alter separation characteristics We will study this in greater detail in Part II of this Essential Guide carlo

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Mobile Phase Strength and Retention

Increasing the percentage of organic modifier in the mobile phase has a profound effect on analyte retention due to the change in polarity of the mobile phase Use the slider below to investigate the effect of altering the mobile phase composition

Figure 8 Solvent strength

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Eluotropic Series

Solvent strength in Reverse Phase HPLC depends upon the solvent type and amount used in the mobile phase (percentage organic modifier (B))

It is possible to interrelate the relative ldquostrengthrdquo of each of the common solvents using an Eluotropic Series This is a table or listing of the various solvents and their relative strength on various media ndash for example Aluminium Oxide or Silica for Normal Phase HPLC and C18 for Reverse Phase HPLC

Commonly ndash a nomograph might be used to find equivalent eluotropic strength between mobile phases that use different organic modifiers The nomograph relates each solvent using a vertical line to indicate mobile phase composition (B) which give equivalent elution strength ndashknown as lsquoisoelutropicrsquo mobile phase compositions

Isoelutropic mobile phases produce separations in approximately the same time frame (measured using retention (capacity) factor of the last peak) ndash however they show altered selectivity This can be very useful when developing or optimising separations

You can use the slider bar below to find various isoelutropic compositions ndash note that the relationship yields results which are only approximately accurate to within about plusmn 5 B

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Figure 9 Solvent nomogram form reversed phase HPLC

Table 2 Eluotropic series of various reverse phase solventscarlo

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Matching Injection Volume with Sample Solvent Strength

Under ideal conditions the strength of the sample solvent used would make no difference to the chromatographic separation because the solvent would be diluted instantaneously to the same composition as the mobile phase However in practice it takes a finite period of time for the injected sample solvent to become diluted with the mobile phase

The following table provides guidelines for sample injection volumes based on the sample solvent strength with respect to that of mobile phase

100 Strong Solvent

In this demanding situation it is necessary to keep the injection volume as small as possible thereby minimising peak distortion The recommendation is for no more than 10μL

When the sample solvent is greater than 80 of the mobile phase it may be possible to inject larger volumes as the compositional difference between the sample solvent and mobile phase is correspondingly less

Stronger than Mobile Phase

When the sample solvent is no more than approximately 25 stronger than the mobile phase then injection volumes as high as 25μLshould be possible However always examine the chromatogram for possible peak distortion given the inter-relationship of this and the preceding rule

Generally analyte solubility issues are the primary reason for increasing the sample solvent strength utilised To minimise such solvent strength variations on injection dissolve the analyte at a high concentration in the strong solvent then dilute with the weaker solvent to progressively correct for the difference in eluotropic strength In a worst-case scenario dissolve in 100 strong solvent and inject as small a volume as possible

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Mobile Phase

The best injection scenario is when the sample solvent and the mobile phase are matched both in terms of eluotropicstrength and pH In this situation given the solvent homogeneity you donrsquot have to be concerned with dilution effects

However the injection volume is limited The contribution of the sample injection volume to the chromatographic peak width can be determined by the following equation

Weaker than Mobile Phase

To assess the effect of the mobile phase strength consider the ldquoRule of Threerdquo which states that a 10 change in the strong solvent will result in an approximate threefold change in analyte retention time Therefore if a chromatographic peak had a retention time of 5min in a mobile phase of 4060 wateracetonitrile then in a mobile phase of 6040 wateracetonitrile its retention time would increase to approximately 45min (3 times 3 times 5min = 45min)

Consequently if the sample were to be injected in 6040 wateracetonitrile instead then the analyte molecules would travel much more slowly through the column until the sample solvent was fully diluted with the mobile phase

Chromatographic bands travelling more slowly than the mobile phase tend to compress because as mobile phase reaches the analyte molecules at the peak ldquotailrdquo they will begin to move faster down the column catching those analyte molecules further down the column that are still effectively residing in a weaker mobile phase strength

As the difference between the solvent strength of the sample solvent and the mobile phase increases it is possible to use progressively larger and larger injection volumes This effectively allows analyte on-column sample ldquofocussingrdquo or concentration and is often employed in environmental analysis to assist in the detection of trace quantities of pesticides

carlo

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012

Exceptions to the above are whenever the mobile phase contains trace additives then it is always advisable to inject samples dissolved in the mobile phase eg ion-pair separations rely on the equilibrium between the ion pair reagent in the mobile phase and the stationary phase Injecting a sample in a solvent that doesnrsquot contain the ion pair shifts this equilibrium to the mobile phase with resulting peak distortion

Peak distortion can occur due to a mismatch between injection solvent and mobile phase strength In particular when large amounts of sample are injected in a solvent that is much ldquostrongerrdquo than the mobile phase The diagram below shows typical peak distortion problems

Figure 10 Peak shape abnormalities currently found when solvent strength is not considered when injecting

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Ionisable CompoundspH Considerations

The mobile phase pH can be used to influence the charge state of ionisable species in solution The extent of analyte ionisation can be used to affect retention and selectivity The pH of a solution will influence the charge state of an acidic or basic analyte

For example addition of an acid to an aqueous solution of a basic analyte will increase the concentration of charged analyte in solution as the hydrogen ion concentration increases Conversely raising the pH by addition of a base will increase the concentration of the neutral form of the basic analyte

Take the example of homovanillic acid shown below ndash equilibrium between the ionised and non-ionisedforms of the analyte will be established according to the solution pH

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Adding an acid to a buffered solution of homovanillic acid (ie lowering the mobile phase pH) will cause the equilibrium to shift to the left and the analyte will become less ionised (ion suppressed) as the analyterecombines to reduce the effect of the added hydrogen ions (protons) from the acidic species Adding a base to a buffered solution of homovanillic acid (ie increasing the mobile phase pH) will cause the analyte to become more ionised as the solution attempts to regain equilibrium by producing more hydrogen ions to neutralise the added base This principle was first described by Le Chetalier and the converse applies to acidic analyte species

The 2 pH rule

It can be shown that at 1 pH unit away from the analyte pKa the change in extent of ionisation is approximately 90 At 2 pH units away from the pKa the change in extent of ionisation is approximately 99 at 3 pH units 999 etc Therefore ndash a rule a thumb known as the ldquo2 pH rulerdquo is useful in predicting extent of ionisation

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Basic Analytes amp Ion Suppression

Unlike the dissociated (ionised) acids which when charged elute rapidly from the column protonated bases often have long retention times and poor peak shape This retention behaviour is due to the interaction with residual silanol species on the silica surface

Separations of basic compounds however are not usually carried out under ion suppression conditions The analyst would have to raise the pH to produce the neutral molecule High pH mobile phases can damage traditional silica columns unless specifically designed to cope with high pH

Traditionally the analysis of weak bases has been carried out at low pH ndash essentially because the surface silanol species are non-ionised (pKa approx 35 ndash 45) and peak tailing is improved somewhat

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically) highly surface active base such as Triethylamine (TEA) Piperazine NNNrsquoNrsquo-Tetramethylethylenediamine (TEMED) or Dimethyloctylamine (DMOA) These bases interact with the surface silanol species in preference to the analyte molecule and are called lsquosacrificial basesrsquo They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times

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Figure 13 Tailing factor versus concentration of TEAcarlo

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Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

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Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

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As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

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Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

pez-

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012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

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-201

2

carlo

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

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012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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ax-2

012

carlo

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012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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ax-2

012

carlo

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ax-2

012

carlo

pez-

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Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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carlo

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 13: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Figure 6 Solvents in reversed phase HPLC Figure 7 Properties for selected HPLC solvents

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Use Figure 7 to assess the relative physics-chemical properties of the various solvents one might use as organic modifiers to decide upon their suitability for a particular application

Changing the organic modifier within a mobile phase can alter the selectivity of the separation as well as the retention characteristics

So how would one chose the most appropriate solvent ndash what are the major considerations

First of all the chosen organic solvent must be miscible with water All of the solvents listed in Figure 6 are miscible with water Second a low viscosity mobile phase is favored to reduce dispersion and keep system back pressure low

The solvent must be stable for long periods of time This disfavors tetrahydrofuran which after exposure to air degrades rapidly often forming explosive peroxides

The two remaining mobile phase solvents are methanol and acetonitrile The use of both solvents usually gives excellent retention characteristics

Acetonitrile however has a lower viscosity and lower UV-cutoff (which is advantageous as the possibility detection interference is reduced) The UV-cutoff for methanol is 205 nm while the UV cut-off for acetonitrile is 190 nm For these reasons most analysts begin reversed-phase method development with acetonitrile

It is also important to note that whilst the polarity index values of methanol and acetonitrile are similar they have different Lewis acid base characteristics (proton donator acceptor) which allows them to act differently to alter separation characteristics We will study this in greater detail in Part II of this Essential Guide carlo

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Mobile Phase Strength and Retention

Increasing the percentage of organic modifier in the mobile phase has a profound effect on analyte retention due to the change in polarity of the mobile phase Use the slider below to investigate the effect of altering the mobile phase composition

Figure 8 Solvent strength

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Eluotropic Series

Solvent strength in Reverse Phase HPLC depends upon the solvent type and amount used in the mobile phase (percentage organic modifier (B))

It is possible to interrelate the relative ldquostrengthrdquo of each of the common solvents using an Eluotropic Series This is a table or listing of the various solvents and their relative strength on various media ndash for example Aluminium Oxide or Silica for Normal Phase HPLC and C18 for Reverse Phase HPLC

Commonly ndash a nomograph might be used to find equivalent eluotropic strength between mobile phases that use different organic modifiers The nomograph relates each solvent using a vertical line to indicate mobile phase composition (B) which give equivalent elution strength ndashknown as lsquoisoelutropicrsquo mobile phase compositions

Isoelutropic mobile phases produce separations in approximately the same time frame (measured using retention (capacity) factor of the last peak) ndash however they show altered selectivity This can be very useful when developing or optimising separations

You can use the slider bar below to find various isoelutropic compositions ndash note that the relationship yields results which are only approximately accurate to within about plusmn 5 B

carlo

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Figure 9 Solvent nomogram form reversed phase HPLC

Table 2 Eluotropic series of various reverse phase solventscarlo

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Matching Injection Volume with Sample Solvent Strength

Under ideal conditions the strength of the sample solvent used would make no difference to the chromatographic separation because the solvent would be diluted instantaneously to the same composition as the mobile phase However in practice it takes a finite period of time for the injected sample solvent to become diluted with the mobile phase

The following table provides guidelines for sample injection volumes based on the sample solvent strength with respect to that of mobile phase

100 Strong Solvent

In this demanding situation it is necessary to keep the injection volume as small as possible thereby minimising peak distortion The recommendation is for no more than 10μL

When the sample solvent is greater than 80 of the mobile phase it may be possible to inject larger volumes as the compositional difference between the sample solvent and mobile phase is correspondingly less

Stronger than Mobile Phase

When the sample solvent is no more than approximately 25 stronger than the mobile phase then injection volumes as high as 25μLshould be possible However always examine the chromatogram for possible peak distortion given the inter-relationship of this and the preceding rule

Generally analyte solubility issues are the primary reason for increasing the sample solvent strength utilised To minimise such solvent strength variations on injection dissolve the analyte at a high concentration in the strong solvent then dilute with the weaker solvent to progressively correct for the difference in eluotropic strength In a worst-case scenario dissolve in 100 strong solvent and inject as small a volume as possible

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Mobile Phase

The best injection scenario is when the sample solvent and the mobile phase are matched both in terms of eluotropicstrength and pH In this situation given the solvent homogeneity you donrsquot have to be concerned with dilution effects

However the injection volume is limited The contribution of the sample injection volume to the chromatographic peak width can be determined by the following equation

Weaker than Mobile Phase

To assess the effect of the mobile phase strength consider the ldquoRule of Threerdquo which states that a 10 change in the strong solvent will result in an approximate threefold change in analyte retention time Therefore if a chromatographic peak had a retention time of 5min in a mobile phase of 4060 wateracetonitrile then in a mobile phase of 6040 wateracetonitrile its retention time would increase to approximately 45min (3 times 3 times 5min = 45min)

Consequently if the sample were to be injected in 6040 wateracetonitrile instead then the analyte molecules would travel much more slowly through the column until the sample solvent was fully diluted with the mobile phase

Chromatographic bands travelling more slowly than the mobile phase tend to compress because as mobile phase reaches the analyte molecules at the peak ldquotailrdquo they will begin to move faster down the column catching those analyte molecules further down the column that are still effectively residing in a weaker mobile phase strength

As the difference between the solvent strength of the sample solvent and the mobile phase increases it is possible to use progressively larger and larger injection volumes This effectively allows analyte on-column sample ldquofocussingrdquo or concentration and is often employed in environmental analysis to assist in the detection of trace quantities of pesticides

carlo

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Exceptions to the above are whenever the mobile phase contains trace additives then it is always advisable to inject samples dissolved in the mobile phase eg ion-pair separations rely on the equilibrium between the ion pair reagent in the mobile phase and the stationary phase Injecting a sample in a solvent that doesnrsquot contain the ion pair shifts this equilibrium to the mobile phase with resulting peak distortion

Peak distortion can occur due to a mismatch between injection solvent and mobile phase strength In particular when large amounts of sample are injected in a solvent that is much ldquostrongerrdquo than the mobile phase The diagram below shows typical peak distortion problems

Figure 10 Peak shape abnormalities currently found when solvent strength is not considered when injecting

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Ionisable CompoundspH Considerations

The mobile phase pH can be used to influence the charge state of ionisable species in solution The extent of analyte ionisation can be used to affect retention and selectivity The pH of a solution will influence the charge state of an acidic or basic analyte

For example addition of an acid to an aqueous solution of a basic analyte will increase the concentration of charged analyte in solution as the hydrogen ion concentration increases Conversely raising the pH by addition of a base will increase the concentration of the neutral form of the basic analyte

Take the example of homovanillic acid shown below ndash equilibrium between the ionised and non-ionisedforms of the analyte will be established according to the solution pH

carlo

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Adding an acid to a buffered solution of homovanillic acid (ie lowering the mobile phase pH) will cause the equilibrium to shift to the left and the analyte will become less ionised (ion suppressed) as the analyterecombines to reduce the effect of the added hydrogen ions (protons) from the acidic species Adding a base to a buffered solution of homovanillic acid (ie increasing the mobile phase pH) will cause the analyte to become more ionised as the solution attempts to regain equilibrium by producing more hydrogen ions to neutralise the added base This principle was first described by Le Chetalier and the converse applies to acidic analyte species

The 2 pH rule

It can be shown that at 1 pH unit away from the analyte pKa the change in extent of ionisation is approximately 90 At 2 pH units away from the pKa the change in extent of ionisation is approximately 99 at 3 pH units 999 etc Therefore ndash a rule a thumb known as the ldquo2 pH rulerdquo is useful in predicting extent of ionisation

carlo

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Basic Analytes amp Ion Suppression

Unlike the dissociated (ionised) acids which when charged elute rapidly from the column protonated bases often have long retention times and poor peak shape This retention behaviour is due to the interaction with residual silanol species on the silica surface

Separations of basic compounds however are not usually carried out under ion suppression conditions The analyst would have to raise the pH to produce the neutral molecule High pH mobile phases can damage traditional silica columns unless specifically designed to cope with high pH

Traditionally the analysis of weak bases has been carried out at low pH ndash essentially because the surface silanol species are non-ionised (pKa approx 35 ndash 45) and peak tailing is improved somewhat

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically) highly surface active base such as Triethylamine (TEA) Piperazine NNNrsquoNrsquo-Tetramethylethylenediamine (TEMED) or Dimethyloctylamine (DMOA) These bases interact with the surface silanol species in preference to the analyte molecule and are called lsquosacrificial basesrsquo They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times

carlo

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Figure 13 Tailing factor versus concentration of TEAcarlo

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Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

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Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

carlo

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

carlo

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

carlo

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

carlo

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012

Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

carlo

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012

As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

carlo

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

rlope

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-201

2

Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

carlo

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

carlo

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

rlope

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-201

2

Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

carlo

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012

Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

carlo

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

carlo

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012

By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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012

Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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2

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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2

carlo

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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012

carlo

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012

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Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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012

Page 14: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Use Figure 7 to assess the relative physics-chemical properties of the various solvents one might use as organic modifiers to decide upon their suitability for a particular application

Changing the organic modifier within a mobile phase can alter the selectivity of the separation as well as the retention characteristics

So how would one chose the most appropriate solvent ndash what are the major considerations

First of all the chosen organic solvent must be miscible with water All of the solvents listed in Figure 6 are miscible with water Second a low viscosity mobile phase is favored to reduce dispersion and keep system back pressure low

The solvent must be stable for long periods of time This disfavors tetrahydrofuran which after exposure to air degrades rapidly often forming explosive peroxides

The two remaining mobile phase solvents are methanol and acetonitrile The use of both solvents usually gives excellent retention characteristics

Acetonitrile however has a lower viscosity and lower UV-cutoff (which is advantageous as the possibility detection interference is reduced) The UV-cutoff for methanol is 205 nm while the UV cut-off for acetonitrile is 190 nm For these reasons most analysts begin reversed-phase method development with acetonitrile

It is also important to note that whilst the polarity index values of methanol and acetonitrile are similar they have different Lewis acid base characteristics (proton donator acceptor) which allows them to act differently to alter separation characteristics We will study this in greater detail in Part II of this Essential Guide carlo

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Mobile Phase Strength and Retention

Increasing the percentage of organic modifier in the mobile phase has a profound effect on analyte retention due to the change in polarity of the mobile phase Use the slider below to investigate the effect of altering the mobile phase composition

Figure 8 Solvent strength

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Eluotropic Series

Solvent strength in Reverse Phase HPLC depends upon the solvent type and amount used in the mobile phase (percentage organic modifier (B))

It is possible to interrelate the relative ldquostrengthrdquo of each of the common solvents using an Eluotropic Series This is a table or listing of the various solvents and their relative strength on various media ndash for example Aluminium Oxide or Silica for Normal Phase HPLC and C18 for Reverse Phase HPLC

Commonly ndash a nomograph might be used to find equivalent eluotropic strength between mobile phases that use different organic modifiers The nomograph relates each solvent using a vertical line to indicate mobile phase composition (B) which give equivalent elution strength ndashknown as lsquoisoelutropicrsquo mobile phase compositions

Isoelutropic mobile phases produce separations in approximately the same time frame (measured using retention (capacity) factor of the last peak) ndash however they show altered selectivity This can be very useful when developing or optimising separations

You can use the slider bar below to find various isoelutropic compositions ndash note that the relationship yields results which are only approximately accurate to within about plusmn 5 B

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Figure 9 Solvent nomogram form reversed phase HPLC

Table 2 Eluotropic series of various reverse phase solventscarlo

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Matching Injection Volume with Sample Solvent Strength

Under ideal conditions the strength of the sample solvent used would make no difference to the chromatographic separation because the solvent would be diluted instantaneously to the same composition as the mobile phase However in practice it takes a finite period of time for the injected sample solvent to become diluted with the mobile phase

The following table provides guidelines for sample injection volumes based on the sample solvent strength with respect to that of mobile phase

100 Strong Solvent

In this demanding situation it is necessary to keep the injection volume as small as possible thereby minimising peak distortion The recommendation is for no more than 10μL

When the sample solvent is greater than 80 of the mobile phase it may be possible to inject larger volumes as the compositional difference between the sample solvent and mobile phase is correspondingly less

Stronger than Mobile Phase

When the sample solvent is no more than approximately 25 stronger than the mobile phase then injection volumes as high as 25μLshould be possible However always examine the chromatogram for possible peak distortion given the inter-relationship of this and the preceding rule

Generally analyte solubility issues are the primary reason for increasing the sample solvent strength utilised To minimise such solvent strength variations on injection dissolve the analyte at a high concentration in the strong solvent then dilute with the weaker solvent to progressively correct for the difference in eluotropic strength In a worst-case scenario dissolve in 100 strong solvent and inject as small a volume as possible

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Mobile Phase

The best injection scenario is when the sample solvent and the mobile phase are matched both in terms of eluotropicstrength and pH In this situation given the solvent homogeneity you donrsquot have to be concerned with dilution effects

However the injection volume is limited The contribution of the sample injection volume to the chromatographic peak width can be determined by the following equation

Weaker than Mobile Phase

To assess the effect of the mobile phase strength consider the ldquoRule of Threerdquo which states that a 10 change in the strong solvent will result in an approximate threefold change in analyte retention time Therefore if a chromatographic peak had a retention time of 5min in a mobile phase of 4060 wateracetonitrile then in a mobile phase of 6040 wateracetonitrile its retention time would increase to approximately 45min (3 times 3 times 5min = 45min)

Consequently if the sample were to be injected in 6040 wateracetonitrile instead then the analyte molecules would travel much more slowly through the column until the sample solvent was fully diluted with the mobile phase

Chromatographic bands travelling more slowly than the mobile phase tend to compress because as mobile phase reaches the analyte molecules at the peak ldquotailrdquo they will begin to move faster down the column catching those analyte molecules further down the column that are still effectively residing in a weaker mobile phase strength

As the difference between the solvent strength of the sample solvent and the mobile phase increases it is possible to use progressively larger and larger injection volumes This effectively allows analyte on-column sample ldquofocussingrdquo or concentration and is often employed in environmental analysis to assist in the detection of trace quantities of pesticides

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Exceptions to the above are whenever the mobile phase contains trace additives then it is always advisable to inject samples dissolved in the mobile phase eg ion-pair separations rely on the equilibrium between the ion pair reagent in the mobile phase and the stationary phase Injecting a sample in a solvent that doesnrsquot contain the ion pair shifts this equilibrium to the mobile phase with resulting peak distortion

Peak distortion can occur due to a mismatch between injection solvent and mobile phase strength In particular when large amounts of sample are injected in a solvent that is much ldquostrongerrdquo than the mobile phase The diagram below shows typical peak distortion problems

Figure 10 Peak shape abnormalities currently found when solvent strength is not considered when injecting

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Ionisable CompoundspH Considerations

The mobile phase pH can be used to influence the charge state of ionisable species in solution The extent of analyte ionisation can be used to affect retention and selectivity The pH of a solution will influence the charge state of an acidic or basic analyte

For example addition of an acid to an aqueous solution of a basic analyte will increase the concentration of charged analyte in solution as the hydrogen ion concentration increases Conversely raising the pH by addition of a base will increase the concentration of the neutral form of the basic analyte

Take the example of homovanillic acid shown below ndash equilibrium between the ionised and non-ionisedforms of the analyte will be established according to the solution pH

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Adding an acid to a buffered solution of homovanillic acid (ie lowering the mobile phase pH) will cause the equilibrium to shift to the left and the analyte will become less ionised (ion suppressed) as the analyterecombines to reduce the effect of the added hydrogen ions (protons) from the acidic species Adding a base to a buffered solution of homovanillic acid (ie increasing the mobile phase pH) will cause the analyte to become more ionised as the solution attempts to regain equilibrium by producing more hydrogen ions to neutralise the added base This principle was first described by Le Chetalier and the converse applies to acidic analyte species

The 2 pH rule

It can be shown that at 1 pH unit away from the analyte pKa the change in extent of ionisation is approximately 90 At 2 pH units away from the pKa the change in extent of ionisation is approximately 99 at 3 pH units 999 etc Therefore ndash a rule a thumb known as the ldquo2 pH rulerdquo is useful in predicting extent of ionisation

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Basic Analytes amp Ion Suppression

Unlike the dissociated (ionised) acids which when charged elute rapidly from the column protonated bases often have long retention times and poor peak shape This retention behaviour is due to the interaction with residual silanol species on the silica surface

Separations of basic compounds however are not usually carried out under ion suppression conditions The analyst would have to raise the pH to produce the neutral molecule High pH mobile phases can damage traditional silica columns unless specifically designed to cope with high pH

Traditionally the analysis of weak bases has been carried out at low pH ndash essentially because the surface silanol species are non-ionised (pKa approx 35 ndash 45) and peak tailing is improved somewhat

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically) highly surface active base such as Triethylamine (TEA) Piperazine NNNrsquoNrsquo-Tetramethylethylenediamine (TEMED) or Dimethyloctylamine (DMOA) These bases interact with the surface silanol species in preference to the analyte molecule and are called lsquosacrificial basesrsquo They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times

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Figure 13 Tailing factor versus concentration of TEAcarlo

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Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

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Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

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As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

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Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

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012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

pez-

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ax-2

012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

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-201

2

carlo

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012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

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012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

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012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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ax-2

012

carlo

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012

carlo

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012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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carlo

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012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 15: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Mobile Phase Strength and Retention

Increasing the percentage of organic modifier in the mobile phase has a profound effect on analyte retention due to the change in polarity of the mobile phase Use the slider below to investigate the effect of altering the mobile phase composition

Figure 8 Solvent strength

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Eluotropic Series

Solvent strength in Reverse Phase HPLC depends upon the solvent type and amount used in the mobile phase (percentage organic modifier (B))

It is possible to interrelate the relative ldquostrengthrdquo of each of the common solvents using an Eluotropic Series This is a table or listing of the various solvents and their relative strength on various media ndash for example Aluminium Oxide or Silica for Normal Phase HPLC and C18 for Reverse Phase HPLC

Commonly ndash a nomograph might be used to find equivalent eluotropic strength between mobile phases that use different organic modifiers The nomograph relates each solvent using a vertical line to indicate mobile phase composition (B) which give equivalent elution strength ndashknown as lsquoisoelutropicrsquo mobile phase compositions

Isoelutropic mobile phases produce separations in approximately the same time frame (measured using retention (capacity) factor of the last peak) ndash however they show altered selectivity This can be very useful when developing or optimising separations

You can use the slider bar below to find various isoelutropic compositions ndash note that the relationship yields results which are only approximately accurate to within about plusmn 5 B

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Figure 9 Solvent nomogram form reversed phase HPLC

Table 2 Eluotropic series of various reverse phase solventscarlo

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Matching Injection Volume with Sample Solvent Strength

Under ideal conditions the strength of the sample solvent used would make no difference to the chromatographic separation because the solvent would be diluted instantaneously to the same composition as the mobile phase However in practice it takes a finite period of time for the injected sample solvent to become diluted with the mobile phase

The following table provides guidelines for sample injection volumes based on the sample solvent strength with respect to that of mobile phase

100 Strong Solvent

In this demanding situation it is necessary to keep the injection volume as small as possible thereby minimising peak distortion The recommendation is for no more than 10μL

When the sample solvent is greater than 80 of the mobile phase it may be possible to inject larger volumes as the compositional difference between the sample solvent and mobile phase is correspondingly less

Stronger than Mobile Phase

When the sample solvent is no more than approximately 25 stronger than the mobile phase then injection volumes as high as 25μLshould be possible However always examine the chromatogram for possible peak distortion given the inter-relationship of this and the preceding rule

Generally analyte solubility issues are the primary reason for increasing the sample solvent strength utilised To minimise such solvent strength variations on injection dissolve the analyte at a high concentration in the strong solvent then dilute with the weaker solvent to progressively correct for the difference in eluotropic strength In a worst-case scenario dissolve in 100 strong solvent and inject as small a volume as possible

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Mobile Phase

The best injection scenario is when the sample solvent and the mobile phase are matched both in terms of eluotropicstrength and pH In this situation given the solvent homogeneity you donrsquot have to be concerned with dilution effects

However the injection volume is limited The contribution of the sample injection volume to the chromatographic peak width can be determined by the following equation

Weaker than Mobile Phase

To assess the effect of the mobile phase strength consider the ldquoRule of Threerdquo which states that a 10 change in the strong solvent will result in an approximate threefold change in analyte retention time Therefore if a chromatographic peak had a retention time of 5min in a mobile phase of 4060 wateracetonitrile then in a mobile phase of 6040 wateracetonitrile its retention time would increase to approximately 45min (3 times 3 times 5min = 45min)

Consequently if the sample were to be injected in 6040 wateracetonitrile instead then the analyte molecules would travel much more slowly through the column until the sample solvent was fully diluted with the mobile phase

Chromatographic bands travelling more slowly than the mobile phase tend to compress because as mobile phase reaches the analyte molecules at the peak ldquotailrdquo they will begin to move faster down the column catching those analyte molecules further down the column that are still effectively residing in a weaker mobile phase strength

As the difference between the solvent strength of the sample solvent and the mobile phase increases it is possible to use progressively larger and larger injection volumes This effectively allows analyte on-column sample ldquofocussingrdquo or concentration and is often employed in environmental analysis to assist in the detection of trace quantities of pesticides

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012

Exceptions to the above are whenever the mobile phase contains trace additives then it is always advisable to inject samples dissolved in the mobile phase eg ion-pair separations rely on the equilibrium between the ion pair reagent in the mobile phase and the stationary phase Injecting a sample in a solvent that doesnrsquot contain the ion pair shifts this equilibrium to the mobile phase with resulting peak distortion

Peak distortion can occur due to a mismatch between injection solvent and mobile phase strength In particular when large amounts of sample are injected in a solvent that is much ldquostrongerrdquo than the mobile phase The diagram below shows typical peak distortion problems

Figure 10 Peak shape abnormalities currently found when solvent strength is not considered when injecting

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Ionisable CompoundspH Considerations

The mobile phase pH can be used to influence the charge state of ionisable species in solution The extent of analyte ionisation can be used to affect retention and selectivity The pH of a solution will influence the charge state of an acidic or basic analyte

For example addition of an acid to an aqueous solution of a basic analyte will increase the concentration of charged analyte in solution as the hydrogen ion concentration increases Conversely raising the pH by addition of a base will increase the concentration of the neutral form of the basic analyte

Take the example of homovanillic acid shown below ndash equilibrium between the ionised and non-ionisedforms of the analyte will be established according to the solution pH

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Adding an acid to a buffered solution of homovanillic acid (ie lowering the mobile phase pH) will cause the equilibrium to shift to the left and the analyte will become less ionised (ion suppressed) as the analyterecombines to reduce the effect of the added hydrogen ions (protons) from the acidic species Adding a base to a buffered solution of homovanillic acid (ie increasing the mobile phase pH) will cause the analyte to become more ionised as the solution attempts to regain equilibrium by producing more hydrogen ions to neutralise the added base This principle was first described by Le Chetalier and the converse applies to acidic analyte species

The 2 pH rule

It can be shown that at 1 pH unit away from the analyte pKa the change in extent of ionisation is approximately 90 At 2 pH units away from the pKa the change in extent of ionisation is approximately 99 at 3 pH units 999 etc Therefore ndash a rule a thumb known as the ldquo2 pH rulerdquo is useful in predicting extent of ionisation

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Basic Analytes amp Ion Suppression

Unlike the dissociated (ionised) acids which when charged elute rapidly from the column protonated bases often have long retention times and poor peak shape This retention behaviour is due to the interaction with residual silanol species on the silica surface

Separations of basic compounds however are not usually carried out under ion suppression conditions The analyst would have to raise the pH to produce the neutral molecule High pH mobile phases can damage traditional silica columns unless specifically designed to cope with high pH

Traditionally the analysis of weak bases has been carried out at low pH ndash essentially because the surface silanol species are non-ionised (pKa approx 35 ndash 45) and peak tailing is improved somewhat

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically) highly surface active base such as Triethylamine (TEA) Piperazine NNNrsquoNrsquo-Tetramethylethylenediamine (TEMED) or Dimethyloctylamine (DMOA) These bases interact with the surface silanol species in preference to the analyte molecule and are called lsquosacrificial basesrsquo They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times

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Figure 13 Tailing factor versus concentration of TEAcarlo

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Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

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Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

carlo

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

carlo

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012

As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

carlo

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

rlope

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2

Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

carlo

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

carlo

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

rlope

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-201

2

Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

carlo

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012

Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

carlo

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

carlo

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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2

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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2

carlo

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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ax-2

012

carlo

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012

carlo

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012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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carlo

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012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 16: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Eluotropic Series

Solvent strength in Reverse Phase HPLC depends upon the solvent type and amount used in the mobile phase (percentage organic modifier (B))

It is possible to interrelate the relative ldquostrengthrdquo of each of the common solvents using an Eluotropic Series This is a table or listing of the various solvents and their relative strength on various media ndash for example Aluminium Oxide or Silica for Normal Phase HPLC and C18 for Reverse Phase HPLC

Commonly ndash a nomograph might be used to find equivalent eluotropic strength between mobile phases that use different organic modifiers The nomograph relates each solvent using a vertical line to indicate mobile phase composition (B) which give equivalent elution strength ndashknown as lsquoisoelutropicrsquo mobile phase compositions

Isoelutropic mobile phases produce separations in approximately the same time frame (measured using retention (capacity) factor of the last peak) ndash however they show altered selectivity This can be very useful when developing or optimising separations

You can use the slider bar below to find various isoelutropic compositions ndash note that the relationship yields results which are only approximately accurate to within about plusmn 5 B

carlo

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Figure 9 Solvent nomogram form reversed phase HPLC

Table 2 Eluotropic series of various reverse phase solventscarlo

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Matching Injection Volume with Sample Solvent Strength

Under ideal conditions the strength of the sample solvent used would make no difference to the chromatographic separation because the solvent would be diluted instantaneously to the same composition as the mobile phase However in practice it takes a finite period of time for the injected sample solvent to become diluted with the mobile phase

The following table provides guidelines for sample injection volumes based on the sample solvent strength with respect to that of mobile phase

100 Strong Solvent

In this demanding situation it is necessary to keep the injection volume as small as possible thereby minimising peak distortion The recommendation is for no more than 10μL

When the sample solvent is greater than 80 of the mobile phase it may be possible to inject larger volumes as the compositional difference between the sample solvent and mobile phase is correspondingly less

Stronger than Mobile Phase

When the sample solvent is no more than approximately 25 stronger than the mobile phase then injection volumes as high as 25μLshould be possible However always examine the chromatogram for possible peak distortion given the inter-relationship of this and the preceding rule

Generally analyte solubility issues are the primary reason for increasing the sample solvent strength utilised To minimise such solvent strength variations on injection dissolve the analyte at a high concentration in the strong solvent then dilute with the weaker solvent to progressively correct for the difference in eluotropic strength In a worst-case scenario dissolve in 100 strong solvent and inject as small a volume as possible

carlo

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Mobile Phase

The best injection scenario is when the sample solvent and the mobile phase are matched both in terms of eluotropicstrength and pH In this situation given the solvent homogeneity you donrsquot have to be concerned with dilution effects

However the injection volume is limited The contribution of the sample injection volume to the chromatographic peak width can be determined by the following equation

Weaker than Mobile Phase

To assess the effect of the mobile phase strength consider the ldquoRule of Threerdquo which states that a 10 change in the strong solvent will result in an approximate threefold change in analyte retention time Therefore if a chromatographic peak had a retention time of 5min in a mobile phase of 4060 wateracetonitrile then in a mobile phase of 6040 wateracetonitrile its retention time would increase to approximately 45min (3 times 3 times 5min = 45min)

Consequently if the sample were to be injected in 6040 wateracetonitrile instead then the analyte molecules would travel much more slowly through the column until the sample solvent was fully diluted with the mobile phase

Chromatographic bands travelling more slowly than the mobile phase tend to compress because as mobile phase reaches the analyte molecules at the peak ldquotailrdquo they will begin to move faster down the column catching those analyte molecules further down the column that are still effectively residing in a weaker mobile phase strength

As the difference between the solvent strength of the sample solvent and the mobile phase increases it is possible to use progressively larger and larger injection volumes This effectively allows analyte on-column sample ldquofocussingrdquo or concentration and is often employed in environmental analysis to assist in the detection of trace quantities of pesticides

carlo

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Exceptions to the above are whenever the mobile phase contains trace additives then it is always advisable to inject samples dissolved in the mobile phase eg ion-pair separations rely on the equilibrium between the ion pair reagent in the mobile phase and the stationary phase Injecting a sample in a solvent that doesnrsquot contain the ion pair shifts this equilibrium to the mobile phase with resulting peak distortion

Peak distortion can occur due to a mismatch between injection solvent and mobile phase strength In particular when large amounts of sample are injected in a solvent that is much ldquostrongerrdquo than the mobile phase The diagram below shows typical peak distortion problems

Figure 10 Peak shape abnormalities currently found when solvent strength is not considered when injecting

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Ionisable CompoundspH Considerations

The mobile phase pH can be used to influence the charge state of ionisable species in solution The extent of analyte ionisation can be used to affect retention and selectivity The pH of a solution will influence the charge state of an acidic or basic analyte

For example addition of an acid to an aqueous solution of a basic analyte will increase the concentration of charged analyte in solution as the hydrogen ion concentration increases Conversely raising the pH by addition of a base will increase the concentration of the neutral form of the basic analyte

Take the example of homovanillic acid shown below ndash equilibrium between the ionised and non-ionisedforms of the analyte will be established according to the solution pH

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Adding an acid to a buffered solution of homovanillic acid (ie lowering the mobile phase pH) will cause the equilibrium to shift to the left and the analyte will become less ionised (ion suppressed) as the analyterecombines to reduce the effect of the added hydrogen ions (protons) from the acidic species Adding a base to a buffered solution of homovanillic acid (ie increasing the mobile phase pH) will cause the analyte to become more ionised as the solution attempts to regain equilibrium by producing more hydrogen ions to neutralise the added base This principle was first described by Le Chetalier and the converse applies to acidic analyte species

The 2 pH rule

It can be shown that at 1 pH unit away from the analyte pKa the change in extent of ionisation is approximately 90 At 2 pH units away from the pKa the change in extent of ionisation is approximately 99 at 3 pH units 999 etc Therefore ndash a rule a thumb known as the ldquo2 pH rulerdquo is useful in predicting extent of ionisation

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Basic Analytes amp Ion Suppression

Unlike the dissociated (ionised) acids which when charged elute rapidly from the column protonated bases often have long retention times and poor peak shape This retention behaviour is due to the interaction with residual silanol species on the silica surface

Separations of basic compounds however are not usually carried out under ion suppression conditions The analyst would have to raise the pH to produce the neutral molecule High pH mobile phases can damage traditional silica columns unless specifically designed to cope with high pH

Traditionally the analysis of weak bases has been carried out at low pH ndash essentially because the surface silanol species are non-ionised (pKa approx 35 ndash 45) and peak tailing is improved somewhat

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically) highly surface active base such as Triethylamine (TEA) Piperazine NNNrsquoNrsquo-Tetramethylethylenediamine (TEMED) or Dimethyloctylamine (DMOA) These bases interact with the surface silanol species in preference to the analyte molecule and are called lsquosacrificial basesrsquo They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times

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Figure 13 Tailing factor versus concentration of TEAcarlo

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Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

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Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

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As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

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Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

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012

First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

pez-

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ax-2

012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

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-201

2

carlo

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012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

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012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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012

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012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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ax-2

012

carlo

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ax-2

012

carlo

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Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 17: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Figure 9 Solvent nomogram form reversed phase HPLC

Table 2 Eluotropic series of various reverse phase solventscarlo

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Matching Injection Volume with Sample Solvent Strength

Under ideal conditions the strength of the sample solvent used would make no difference to the chromatographic separation because the solvent would be diluted instantaneously to the same composition as the mobile phase However in practice it takes a finite period of time for the injected sample solvent to become diluted with the mobile phase

The following table provides guidelines for sample injection volumes based on the sample solvent strength with respect to that of mobile phase

100 Strong Solvent

In this demanding situation it is necessary to keep the injection volume as small as possible thereby minimising peak distortion The recommendation is for no more than 10μL

When the sample solvent is greater than 80 of the mobile phase it may be possible to inject larger volumes as the compositional difference between the sample solvent and mobile phase is correspondingly less

Stronger than Mobile Phase

When the sample solvent is no more than approximately 25 stronger than the mobile phase then injection volumes as high as 25μLshould be possible However always examine the chromatogram for possible peak distortion given the inter-relationship of this and the preceding rule

Generally analyte solubility issues are the primary reason for increasing the sample solvent strength utilised To minimise such solvent strength variations on injection dissolve the analyte at a high concentration in the strong solvent then dilute with the weaker solvent to progressively correct for the difference in eluotropic strength In a worst-case scenario dissolve in 100 strong solvent and inject as small a volume as possible

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Mobile Phase

The best injection scenario is when the sample solvent and the mobile phase are matched both in terms of eluotropicstrength and pH In this situation given the solvent homogeneity you donrsquot have to be concerned with dilution effects

However the injection volume is limited The contribution of the sample injection volume to the chromatographic peak width can be determined by the following equation

Weaker than Mobile Phase

To assess the effect of the mobile phase strength consider the ldquoRule of Threerdquo which states that a 10 change in the strong solvent will result in an approximate threefold change in analyte retention time Therefore if a chromatographic peak had a retention time of 5min in a mobile phase of 4060 wateracetonitrile then in a mobile phase of 6040 wateracetonitrile its retention time would increase to approximately 45min (3 times 3 times 5min = 45min)

Consequently if the sample were to be injected in 6040 wateracetonitrile instead then the analyte molecules would travel much more slowly through the column until the sample solvent was fully diluted with the mobile phase

Chromatographic bands travelling more slowly than the mobile phase tend to compress because as mobile phase reaches the analyte molecules at the peak ldquotailrdquo they will begin to move faster down the column catching those analyte molecules further down the column that are still effectively residing in a weaker mobile phase strength

As the difference between the solvent strength of the sample solvent and the mobile phase increases it is possible to use progressively larger and larger injection volumes This effectively allows analyte on-column sample ldquofocussingrdquo or concentration and is often employed in environmental analysis to assist in the detection of trace quantities of pesticides

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012

Exceptions to the above are whenever the mobile phase contains trace additives then it is always advisable to inject samples dissolved in the mobile phase eg ion-pair separations rely on the equilibrium between the ion pair reagent in the mobile phase and the stationary phase Injecting a sample in a solvent that doesnrsquot contain the ion pair shifts this equilibrium to the mobile phase with resulting peak distortion

Peak distortion can occur due to a mismatch between injection solvent and mobile phase strength In particular when large amounts of sample are injected in a solvent that is much ldquostrongerrdquo than the mobile phase The diagram below shows typical peak distortion problems

Figure 10 Peak shape abnormalities currently found when solvent strength is not considered when injecting

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Ionisable CompoundspH Considerations

The mobile phase pH can be used to influence the charge state of ionisable species in solution The extent of analyte ionisation can be used to affect retention and selectivity The pH of a solution will influence the charge state of an acidic or basic analyte

For example addition of an acid to an aqueous solution of a basic analyte will increase the concentration of charged analyte in solution as the hydrogen ion concentration increases Conversely raising the pH by addition of a base will increase the concentration of the neutral form of the basic analyte

Take the example of homovanillic acid shown below ndash equilibrium between the ionised and non-ionisedforms of the analyte will be established according to the solution pH

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Adding an acid to a buffered solution of homovanillic acid (ie lowering the mobile phase pH) will cause the equilibrium to shift to the left and the analyte will become less ionised (ion suppressed) as the analyterecombines to reduce the effect of the added hydrogen ions (protons) from the acidic species Adding a base to a buffered solution of homovanillic acid (ie increasing the mobile phase pH) will cause the analyte to become more ionised as the solution attempts to regain equilibrium by producing more hydrogen ions to neutralise the added base This principle was first described by Le Chetalier and the converse applies to acidic analyte species

The 2 pH rule

It can be shown that at 1 pH unit away from the analyte pKa the change in extent of ionisation is approximately 90 At 2 pH units away from the pKa the change in extent of ionisation is approximately 99 at 3 pH units 999 etc Therefore ndash a rule a thumb known as the ldquo2 pH rulerdquo is useful in predicting extent of ionisation

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Basic Analytes amp Ion Suppression

Unlike the dissociated (ionised) acids which when charged elute rapidly from the column protonated bases often have long retention times and poor peak shape This retention behaviour is due to the interaction with residual silanol species on the silica surface

Separations of basic compounds however are not usually carried out under ion suppression conditions The analyst would have to raise the pH to produce the neutral molecule High pH mobile phases can damage traditional silica columns unless specifically designed to cope with high pH

Traditionally the analysis of weak bases has been carried out at low pH ndash essentially because the surface silanol species are non-ionised (pKa approx 35 ndash 45) and peak tailing is improved somewhat

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically) highly surface active base such as Triethylamine (TEA) Piperazine NNNrsquoNrsquo-Tetramethylethylenediamine (TEMED) or Dimethyloctylamine (DMOA) These bases interact with the surface silanol species in preference to the analyte molecule and are called lsquosacrificial basesrsquo They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times

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Figure 13 Tailing factor versus concentration of TEAcarlo

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Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

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Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

carlo

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

carlo

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

carlo

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012

As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

carlo

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

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2

Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

carlo

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

carlo

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

rlope

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2

Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

carlo

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012

Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

carlo

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

carlo

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012

By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

rlope

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2

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

carlo

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012

Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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012

So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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012

To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

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012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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hum

ax-2

012

carlo

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012

carlo

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012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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012

carlo

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012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 18: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Matching Injection Volume with Sample Solvent Strength

Under ideal conditions the strength of the sample solvent used would make no difference to the chromatographic separation because the solvent would be diluted instantaneously to the same composition as the mobile phase However in practice it takes a finite period of time for the injected sample solvent to become diluted with the mobile phase

The following table provides guidelines for sample injection volumes based on the sample solvent strength with respect to that of mobile phase

100 Strong Solvent

In this demanding situation it is necessary to keep the injection volume as small as possible thereby minimising peak distortion The recommendation is for no more than 10μL

When the sample solvent is greater than 80 of the mobile phase it may be possible to inject larger volumes as the compositional difference between the sample solvent and mobile phase is correspondingly less

Stronger than Mobile Phase

When the sample solvent is no more than approximately 25 stronger than the mobile phase then injection volumes as high as 25μLshould be possible However always examine the chromatogram for possible peak distortion given the inter-relationship of this and the preceding rule

Generally analyte solubility issues are the primary reason for increasing the sample solvent strength utilised To minimise such solvent strength variations on injection dissolve the analyte at a high concentration in the strong solvent then dilute with the weaker solvent to progressively correct for the difference in eluotropic strength In a worst-case scenario dissolve in 100 strong solvent and inject as small a volume as possible

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Mobile Phase

The best injection scenario is when the sample solvent and the mobile phase are matched both in terms of eluotropicstrength and pH In this situation given the solvent homogeneity you donrsquot have to be concerned with dilution effects

However the injection volume is limited The contribution of the sample injection volume to the chromatographic peak width can be determined by the following equation

Weaker than Mobile Phase

To assess the effect of the mobile phase strength consider the ldquoRule of Threerdquo which states that a 10 change in the strong solvent will result in an approximate threefold change in analyte retention time Therefore if a chromatographic peak had a retention time of 5min in a mobile phase of 4060 wateracetonitrile then in a mobile phase of 6040 wateracetonitrile its retention time would increase to approximately 45min (3 times 3 times 5min = 45min)

Consequently if the sample were to be injected in 6040 wateracetonitrile instead then the analyte molecules would travel much more slowly through the column until the sample solvent was fully diluted with the mobile phase

Chromatographic bands travelling more slowly than the mobile phase tend to compress because as mobile phase reaches the analyte molecules at the peak ldquotailrdquo they will begin to move faster down the column catching those analyte molecules further down the column that are still effectively residing in a weaker mobile phase strength

As the difference between the solvent strength of the sample solvent and the mobile phase increases it is possible to use progressively larger and larger injection volumes This effectively allows analyte on-column sample ldquofocussingrdquo or concentration and is often employed in environmental analysis to assist in the detection of trace quantities of pesticides

carlo

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012

Exceptions to the above are whenever the mobile phase contains trace additives then it is always advisable to inject samples dissolved in the mobile phase eg ion-pair separations rely on the equilibrium between the ion pair reagent in the mobile phase and the stationary phase Injecting a sample in a solvent that doesnrsquot contain the ion pair shifts this equilibrium to the mobile phase with resulting peak distortion

Peak distortion can occur due to a mismatch between injection solvent and mobile phase strength In particular when large amounts of sample are injected in a solvent that is much ldquostrongerrdquo than the mobile phase The diagram below shows typical peak distortion problems

Figure 10 Peak shape abnormalities currently found when solvent strength is not considered when injecting

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Ionisable CompoundspH Considerations

The mobile phase pH can be used to influence the charge state of ionisable species in solution The extent of analyte ionisation can be used to affect retention and selectivity The pH of a solution will influence the charge state of an acidic or basic analyte

For example addition of an acid to an aqueous solution of a basic analyte will increase the concentration of charged analyte in solution as the hydrogen ion concentration increases Conversely raising the pH by addition of a base will increase the concentration of the neutral form of the basic analyte

Take the example of homovanillic acid shown below ndash equilibrium between the ionised and non-ionisedforms of the analyte will be established according to the solution pH

carlo

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012

Adding an acid to a buffered solution of homovanillic acid (ie lowering the mobile phase pH) will cause the equilibrium to shift to the left and the analyte will become less ionised (ion suppressed) as the analyterecombines to reduce the effect of the added hydrogen ions (protons) from the acidic species Adding a base to a buffered solution of homovanillic acid (ie increasing the mobile phase pH) will cause the analyte to become more ionised as the solution attempts to regain equilibrium by producing more hydrogen ions to neutralise the added base This principle was first described by Le Chetalier and the converse applies to acidic analyte species

The 2 pH rule

It can be shown that at 1 pH unit away from the analyte pKa the change in extent of ionisation is approximately 90 At 2 pH units away from the pKa the change in extent of ionisation is approximately 99 at 3 pH units 999 etc Therefore ndash a rule a thumb known as the ldquo2 pH rulerdquo is useful in predicting extent of ionisation

carlo

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012

Basic Analytes amp Ion Suppression

Unlike the dissociated (ionised) acids which when charged elute rapidly from the column protonated bases often have long retention times and poor peak shape This retention behaviour is due to the interaction with residual silanol species on the silica surface

Separations of basic compounds however are not usually carried out under ion suppression conditions The analyst would have to raise the pH to produce the neutral molecule High pH mobile phases can damage traditional silica columns unless specifically designed to cope with high pH

Traditionally the analysis of weak bases has been carried out at low pH ndash essentially because the surface silanol species are non-ionised (pKa approx 35 ndash 45) and peak tailing is improved somewhat

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically) highly surface active base such as Triethylamine (TEA) Piperazine NNNrsquoNrsquo-Tetramethylethylenediamine (TEMED) or Dimethyloctylamine (DMOA) These bases interact with the surface silanol species in preference to the analyte molecule and are called lsquosacrificial basesrsquo They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times

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Figure 13 Tailing factor versus concentration of TEAcarlo

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Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

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Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

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As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

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Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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2

carlo

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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carlo

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012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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carlo

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ax-2

012

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Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 19: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Mobile Phase

The best injection scenario is when the sample solvent and the mobile phase are matched both in terms of eluotropicstrength and pH In this situation given the solvent homogeneity you donrsquot have to be concerned with dilution effects

However the injection volume is limited The contribution of the sample injection volume to the chromatographic peak width can be determined by the following equation

Weaker than Mobile Phase

To assess the effect of the mobile phase strength consider the ldquoRule of Threerdquo which states that a 10 change in the strong solvent will result in an approximate threefold change in analyte retention time Therefore if a chromatographic peak had a retention time of 5min in a mobile phase of 4060 wateracetonitrile then in a mobile phase of 6040 wateracetonitrile its retention time would increase to approximately 45min (3 times 3 times 5min = 45min)

Consequently if the sample were to be injected in 6040 wateracetonitrile instead then the analyte molecules would travel much more slowly through the column until the sample solvent was fully diluted with the mobile phase

Chromatographic bands travelling more slowly than the mobile phase tend to compress because as mobile phase reaches the analyte molecules at the peak ldquotailrdquo they will begin to move faster down the column catching those analyte molecules further down the column that are still effectively residing in a weaker mobile phase strength

As the difference between the solvent strength of the sample solvent and the mobile phase increases it is possible to use progressively larger and larger injection volumes This effectively allows analyte on-column sample ldquofocussingrdquo or concentration and is often employed in environmental analysis to assist in the detection of trace quantities of pesticides

carlo

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012

Exceptions to the above are whenever the mobile phase contains trace additives then it is always advisable to inject samples dissolved in the mobile phase eg ion-pair separations rely on the equilibrium between the ion pair reagent in the mobile phase and the stationary phase Injecting a sample in a solvent that doesnrsquot contain the ion pair shifts this equilibrium to the mobile phase with resulting peak distortion

Peak distortion can occur due to a mismatch between injection solvent and mobile phase strength In particular when large amounts of sample are injected in a solvent that is much ldquostrongerrdquo than the mobile phase The diagram below shows typical peak distortion problems

Figure 10 Peak shape abnormalities currently found when solvent strength is not considered when injecting

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012

Ionisable CompoundspH Considerations

The mobile phase pH can be used to influence the charge state of ionisable species in solution The extent of analyte ionisation can be used to affect retention and selectivity The pH of a solution will influence the charge state of an acidic or basic analyte

For example addition of an acid to an aqueous solution of a basic analyte will increase the concentration of charged analyte in solution as the hydrogen ion concentration increases Conversely raising the pH by addition of a base will increase the concentration of the neutral form of the basic analyte

Take the example of homovanillic acid shown below ndash equilibrium between the ionised and non-ionisedforms of the analyte will be established according to the solution pH

carlo

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012

Adding an acid to a buffered solution of homovanillic acid (ie lowering the mobile phase pH) will cause the equilibrium to shift to the left and the analyte will become less ionised (ion suppressed) as the analyterecombines to reduce the effect of the added hydrogen ions (protons) from the acidic species Adding a base to a buffered solution of homovanillic acid (ie increasing the mobile phase pH) will cause the analyte to become more ionised as the solution attempts to regain equilibrium by producing more hydrogen ions to neutralise the added base This principle was first described by Le Chetalier and the converse applies to acidic analyte species

The 2 pH rule

It can be shown that at 1 pH unit away from the analyte pKa the change in extent of ionisation is approximately 90 At 2 pH units away from the pKa the change in extent of ionisation is approximately 99 at 3 pH units 999 etc Therefore ndash a rule a thumb known as the ldquo2 pH rulerdquo is useful in predicting extent of ionisation

carlo

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012

Basic Analytes amp Ion Suppression

Unlike the dissociated (ionised) acids which when charged elute rapidly from the column protonated bases often have long retention times and poor peak shape This retention behaviour is due to the interaction with residual silanol species on the silica surface

Separations of basic compounds however are not usually carried out under ion suppression conditions The analyst would have to raise the pH to produce the neutral molecule High pH mobile phases can damage traditional silica columns unless specifically designed to cope with high pH

Traditionally the analysis of weak bases has been carried out at low pH ndash essentially because the surface silanol species are non-ionised (pKa approx 35 ndash 45) and peak tailing is improved somewhat

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically) highly surface active base such as Triethylamine (TEA) Piperazine NNNrsquoNrsquo-Tetramethylethylenediamine (TEMED) or Dimethyloctylamine (DMOA) These bases interact with the surface silanol species in preference to the analyte molecule and are called lsquosacrificial basesrsquo They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times

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Figure 13 Tailing factor versus concentration of TEAcarlo

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Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

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Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

carlo

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

carlo

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

carlo

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012

As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

carlo

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

rlope

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max

-201

2

Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

carlo

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

carlo

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

rlope

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-201

2

Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

carlo

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012

Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

carlo

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

carlo

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012

By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

rlope

z-hu

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-201

2

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

carlo

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012

Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

pez-

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ax-2

012

In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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ax-2

012

Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

carlo

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ax-2

012

2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

pez-

hum

ax-2

012

So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

pez-

hum

ax-2

012

To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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-201

2

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 20: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Exceptions to the above are whenever the mobile phase contains trace additives then it is always advisable to inject samples dissolved in the mobile phase eg ion-pair separations rely on the equilibrium between the ion pair reagent in the mobile phase and the stationary phase Injecting a sample in a solvent that doesnrsquot contain the ion pair shifts this equilibrium to the mobile phase with resulting peak distortion

Peak distortion can occur due to a mismatch between injection solvent and mobile phase strength In particular when large amounts of sample are injected in a solvent that is much ldquostrongerrdquo than the mobile phase The diagram below shows typical peak distortion problems

Figure 10 Peak shape abnormalities currently found when solvent strength is not considered when injecting

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Ionisable CompoundspH Considerations

The mobile phase pH can be used to influence the charge state of ionisable species in solution The extent of analyte ionisation can be used to affect retention and selectivity The pH of a solution will influence the charge state of an acidic or basic analyte

For example addition of an acid to an aqueous solution of a basic analyte will increase the concentration of charged analyte in solution as the hydrogen ion concentration increases Conversely raising the pH by addition of a base will increase the concentration of the neutral form of the basic analyte

Take the example of homovanillic acid shown below ndash equilibrium between the ionised and non-ionisedforms of the analyte will be established according to the solution pH

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Adding an acid to a buffered solution of homovanillic acid (ie lowering the mobile phase pH) will cause the equilibrium to shift to the left and the analyte will become less ionised (ion suppressed) as the analyterecombines to reduce the effect of the added hydrogen ions (protons) from the acidic species Adding a base to a buffered solution of homovanillic acid (ie increasing the mobile phase pH) will cause the analyte to become more ionised as the solution attempts to regain equilibrium by producing more hydrogen ions to neutralise the added base This principle was first described by Le Chetalier and the converse applies to acidic analyte species

The 2 pH rule

It can be shown that at 1 pH unit away from the analyte pKa the change in extent of ionisation is approximately 90 At 2 pH units away from the pKa the change in extent of ionisation is approximately 99 at 3 pH units 999 etc Therefore ndash a rule a thumb known as the ldquo2 pH rulerdquo is useful in predicting extent of ionisation

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Basic Analytes amp Ion Suppression

Unlike the dissociated (ionised) acids which when charged elute rapidly from the column protonated bases often have long retention times and poor peak shape This retention behaviour is due to the interaction with residual silanol species on the silica surface

Separations of basic compounds however are not usually carried out under ion suppression conditions The analyst would have to raise the pH to produce the neutral molecule High pH mobile phases can damage traditional silica columns unless specifically designed to cope with high pH

Traditionally the analysis of weak bases has been carried out at low pH ndash essentially because the surface silanol species are non-ionised (pKa approx 35 ndash 45) and peak tailing is improved somewhat

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically) highly surface active base such as Triethylamine (TEA) Piperazine NNNrsquoNrsquo-Tetramethylethylenediamine (TEMED) or Dimethyloctylamine (DMOA) These bases interact with the surface silanol species in preference to the analyte molecule and are called lsquosacrificial basesrsquo They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times

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Figure 13 Tailing factor versus concentration of TEAcarlo

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Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

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Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

carlo

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

carlo

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012

As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

rlope

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-201

2

Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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2

carlo

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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ax-2

012

carlo

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ax-2

012

carlo

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ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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012

carlo

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012

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012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 21: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Ionisable CompoundspH Considerations

The mobile phase pH can be used to influence the charge state of ionisable species in solution The extent of analyte ionisation can be used to affect retention and selectivity The pH of a solution will influence the charge state of an acidic or basic analyte

For example addition of an acid to an aqueous solution of a basic analyte will increase the concentration of charged analyte in solution as the hydrogen ion concentration increases Conversely raising the pH by addition of a base will increase the concentration of the neutral form of the basic analyte

Take the example of homovanillic acid shown below ndash equilibrium between the ionised and non-ionisedforms of the analyte will be established according to the solution pH

carlo

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Adding an acid to a buffered solution of homovanillic acid (ie lowering the mobile phase pH) will cause the equilibrium to shift to the left and the analyte will become less ionised (ion suppressed) as the analyterecombines to reduce the effect of the added hydrogen ions (protons) from the acidic species Adding a base to a buffered solution of homovanillic acid (ie increasing the mobile phase pH) will cause the analyte to become more ionised as the solution attempts to regain equilibrium by producing more hydrogen ions to neutralise the added base This principle was first described by Le Chetalier and the converse applies to acidic analyte species

The 2 pH rule

It can be shown that at 1 pH unit away from the analyte pKa the change in extent of ionisation is approximately 90 At 2 pH units away from the pKa the change in extent of ionisation is approximately 99 at 3 pH units 999 etc Therefore ndash a rule a thumb known as the ldquo2 pH rulerdquo is useful in predicting extent of ionisation

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Basic Analytes amp Ion Suppression

Unlike the dissociated (ionised) acids which when charged elute rapidly from the column protonated bases often have long retention times and poor peak shape This retention behaviour is due to the interaction with residual silanol species on the silica surface

Separations of basic compounds however are not usually carried out under ion suppression conditions The analyst would have to raise the pH to produce the neutral molecule High pH mobile phases can damage traditional silica columns unless specifically designed to cope with high pH

Traditionally the analysis of weak bases has been carried out at low pH ndash essentially because the surface silanol species are non-ionised (pKa approx 35 ndash 45) and peak tailing is improved somewhat

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically) highly surface active base such as Triethylamine (TEA) Piperazine NNNrsquoNrsquo-Tetramethylethylenediamine (TEMED) or Dimethyloctylamine (DMOA) These bases interact with the surface silanol species in preference to the analyte molecule and are called lsquosacrificial basesrsquo They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times

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Figure 13 Tailing factor versus concentration of TEAcarlo

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Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

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Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

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As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

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Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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-201

2

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

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012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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012

carlo

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ax-2

012

carlo

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Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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carlo

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 22: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Adding an acid to a buffered solution of homovanillic acid (ie lowering the mobile phase pH) will cause the equilibrium to shift to the left and the analyte will become less ionised (ion suppressed) as the analyterecombines to reduce the effect of the added hydrogen ions (protons) from the acidic species Adding a base to a buffered solution of homovanillic acid (ie increasing the mobile phase pH) will cause the analyte to become more ionised as the solution attempts to regain equilibrium by producing more hydrogen ions to neutralise the added base This principle was first described by Le Chetalier and the converse applies to acidic analyte species

The 2 pH rule

It can be shown that at 1 pH unit away from the analyte pKa the change in extent of ionisation is approximately 90 At 2 pH units away from the pKa the change in extent of ionisation is approximately 99 at 3 pH units 999 etc Therefore ndash a rule a thumb known as the ldquo2 pH rulerdquo is useful in predicting extent of ionisation

carlo

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012

Basic Analytes amp Ion Suppression

Unlike the dissociated (ionised) acids which when charged elute rapidly from the column protonated bases often have long retention times and poor peak shape This retention behaviour is due to the interaction with residual silanol species on the silica surface

Separations of basic compounds however are not usually carried out under ion suppression conditions The analyst would have to raise the pH to produce the neutral molecule High pH mobile phases can damage traditional silica columns unless specifically designed to cope with high pH

Traditionally the analysis of weak bases has been carried out at low pH ndash essentially because the surface silanol species are non-ionised (pKa approx 35 ndash 45) and peak tailing is improved somewhat

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically) highly surface active base such as Triethylamine (TEA) Piperazine NNNrsquoNrsquo-Tetramethylethylenediamine (TEMED) or Dimethyloctylamine (DMOA) These bases interact with the surface silanol species in preference to the analyte molecule and are called lsquosacrificial basesrsquo They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times

carlo

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Figure 13 Tailing factor versus concentration of TEAcarlo

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Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

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Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

carlo

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

carlo

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

carlo

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

carlo

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012

As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

carlo

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

rlope

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2

Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

carlo

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

carlo

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

carlo

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

carlo

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

rlope

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2

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

carlo

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012

Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

pez-

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ax-2

012

In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

carlo

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ax-2

012

2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

pez-

hum

ax-2

012

So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

pez-

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ax-2

012

To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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-201

2

carlo

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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012

carlo

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012

carlo

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Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 23: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Basic Analytes amp Ion Suppression

Unlike the dissociated (ionised) acids which when charged elute rapidly from the column protonated bases often have long retention times and poor peak shape This retention behaviour is due to the interaction with residual silanol species on the silica surface

Separations of basic compounds however are not usually carried out under ion suppression conditions The analyst would have to raise the pH to produce the neutral molecule High pH mobile phases can damage traditional silica columns unless specifically designed to cope with high pH

Traditionally the analysis of weak bases has been carried out at low pH ndash essentially because the surface silanol species are non-ionised (pKa approx 35 ndash 45) and peak tailing is improved somewhat

The unwanted secondary silanol retention may be reduced (or even eliminated) by the addition of a small (sterically) highly surface active base such as Triethylamine (TEA) Piperazine NNNrsquoNrsquo-Tetramethylethylenediamine (TEMED) or Dimethyloctylamine (DMOA) These bases interact with the surface silanol species in preference to the analyte molecule and are called lsquosacrificial basesrsquo They are added to the mobile phase in sufficient concentration to ensure that the silica surface is fully deactivated at all times

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Figure 13 Tailing factor versus concentration of TEAcarlo

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Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

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Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

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As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

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Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

rlope

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2

Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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2

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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2

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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012

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012

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012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 24: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Figure 13 Tailing factor versus concentration of TEAcarlo

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Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

carlo

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Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

carlo

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

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As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

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Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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2

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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012

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Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 25: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Buffers for Reverse Phase HPLC

In order to control the retention of weak acids and bases the pH of the mobile phase must be strictly controlled This usually involves meticulous preparation and adjustment of the mobile phase to the correct pH Most workers will use a buffer to resist small changes in pH that may occur within the HPLC system (ie at the head of column when sample diluent and mobile phase mix or via evaporation of the organic solvent in a pre-mixed mobile phase ingress of CO2 into the mobile phase etc) Some common HPLC buffers are listed in the table below

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Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

carlo

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

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As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

rlope

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-201

2

Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

carlo

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

rlope

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-201

2

Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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-201

2

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

carlo

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

carlo

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

carlo

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

carlo

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

carlo

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carlo

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

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012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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-201

2

carlo

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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012

carlo

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ax-2

012

carlo

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012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 26: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Users of LC-MS require volatile buffers to avoid fouling of the atmospheric pressure interface and reduced maintenance intervals The use of trifluoroacetic acid (TFA) is to be avoided for small molecule work due to its ion suppression effects

A particular buffer is only reliable in the pH ranges given ndash usually around 1pH either side of the buffer pKa value (note some buffers have more than one ionisable functional group and therefore more than one pKa value)

The concentration of the buffer must be adequate but not excessive In general HPLC buffers range in concentration from 25 to 100 mM Buffers prepared at below 10mM can have very little impact on chromatography whilst those at high concentration (gt50mM) risk precipitation of the salt in the presence of high organic concentration mobile phases (ie gt60 MeCN) which may damage the internal components of the HPLC system The closer you are to the buffers pKa value then the more effectivey it can operate and the lower concentration required

carlo

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Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

carlo

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

carlo

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

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As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

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Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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2

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

carlo

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

carlo

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012

2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

pez-

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ax-2

012

So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

pez-

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ax-2

012

To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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012

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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012

Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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-201

2

carlo

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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012

carlo

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012

carlo

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Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 27: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Column Considerations

The HPLC column lies at the heart of every chromatographic separation and the stationary phase is used to control retention The quality of the column packing the physical attributes of the packing material and the quality of the column packing all have a direct influence over the efficiency of the analyte peaks produced

Problems with the column hardware and packed bed can result in poor peak shape

Silica as a Packing Material

Silica is often used as a support material for adsorption (normal phase) chromatography and with chemical modification of the surface for partition (reverse phase) normal phase ion-exchange chiral and size exclusion chromatography

Porous silica has a high surface area that leads to high efficiency columns (through a higher number of possible surface interactions ndash theoretical plates)

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

Whilst most manufacturers are able to provide good bonded phase surface coverage even the best manufacturing techniques will still leave a high number of silanol groups unreacted (due to steric effects) The remaining silanol groups (depending upon their conformation) are able to interact with analytescontaining ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactions

Manufacturers may choose to end-cap the column surface which involves reacting the silanol groups with a sterically small but highly reactive reagent that lsquocapsrsquo the polar surface silanol with a non-polar (and much less reactive) trimethylsilyl group

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Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

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As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

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Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

carlo

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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2

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

carlo

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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-201

2

carlo

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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ax-2

012

carlo

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ax-2

012

carlo

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012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 28: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Reverse Phase Stationary Phases

Reverse phase separations are characterized by having a stationary phase that is less polar than the mobile phase

Several popular reverse phase bonded stationary phases are shown Octadecylsilyl (or C18) is commonly used as it is a highly robust hydrophobic phase which produces good retention with hydrophobic (non-polar) analyte molecules This phase can also be used for the separation of polar compounds when used with mobile phase additives which will be discussed later

carlo

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In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

carlo

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

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As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

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2

Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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2

Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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2

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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2

carlo

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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ax-2

012

carlo

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012

carlo

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012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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012

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012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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012

Page 29: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

In general shortening the alkyl chain will shorten the retention time There are only very slight selectivity differences between for example C18 and C8 columns

The use of more polar phases such as cyano phenyl or amino phases show altered selectivity compared to the alkyl phases These phases are able to interact with polar analyte functional groups via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions

Silanols and Peak Tailing

Fully hydroxylated silica will have a Silanol surface concentration of ~8micromolm2 Following chemical modification gt 4micromolm2 of these silanols may remain even with optimum bonding conditions due to steric limitations of the modifying ligands This indicates that on a molar basis there are more residual silanolsremaining than actual modified ligand In order to remove some of these residual silanols an end-capping process may be undertaken Short chain less sterically hindered hydrophobic ligands (commonly trimethyl tri-iodo chlorosilanes or similar) are chemically reacted with the remaining unbounded silanol species leading to improved peak shape with polar and ionisable analytes ndash as previously described

carlo

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This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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012

Figure 16 Basic polar analytes analysed using Type I and Type II silica

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

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As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

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Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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2

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 30: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

This is only a partial solution however as not all of the surface silanol groups will be reacted even using sterically very small liagnds and optimized bonding conditions also the end-capping ligand is prone to hydrolysis especially at low pH Silanol groups are present in numerous conformations with some being more active than others at causing analyte peak tailing and or irreversible retention

Acidic (lone) surface silanol groups give rise to the most pronounced secondary interactions with polar and ionisable analytes Modern silica is designated as being Type I (Type A) or Type II (Type B) which primarily describes the nature of the silanol surface Type I silica is ldquohigh energyrdquo (non-homogenous) and contains a higher density of lone silanol groups whereas Type II silica is much more homogenous (bridged) and therefore gives rise to much improved peak shapes In order to create a more uniform (homogeneous) silica surface manufacturers ensure the silica surface is fully hydroxylated prior to chemical modification The incorporation of an acid wash step and avoidance of treatments at elevated temperature renders the majority of the surface in the lower energy geminal and bridged (vicinal) confirmation creating Type II silica The figure below shows how various basic and polar analytes are affected when analysed using Type I silica Hypersil as compared to Type II silica (Hypersil BDS)carlo

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Figure 16 Basic polar analytes analysed using Type I and Type II silica

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

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As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

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2

Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

carlo

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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2

Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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2

carlo

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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012

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Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 31: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Figure 16 Basic polar analytes analysed using Type I and Type II silica

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Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

carlo

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012

As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

carlo

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

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-201

2

Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

carlo

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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2

Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

rlope

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2

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

carlo

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

carlo

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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-201

2

carlo

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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012

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012

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Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 32: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Mobile phase pH will affect the degree of silanol ionisation and therefore the degree with which they interact with polar and ionisable analytes causing peak tailing Typically the pKa of surface silanol species lies in the range pH 38 ndash 45 and at eluent pH le3 all but the most acidic will be fully protonated and therefore peak tailing will be at a minimum (please refer to the figure below)

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As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

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2

Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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2

Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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2

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

carlo

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

carlo

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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-201

2

carlo

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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012

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012

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Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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012

Page 33: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

As the eluent pH increases the degree of ionisation (through deprotonation) increases and peak tailing becomes more pronounced as the silanol groups interact with charged and polar species in solution (please refer to the figure below)

Figure 18 Silanol ndash Base interactions and effect on peak shape at different pH conditions (left hand side pH lt 30 right hand side pH gt 30)

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End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

rlope

z-hu

max

-201

2

Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

rlope

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-201

2

Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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2

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

carlo

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

carlo

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carlo

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

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012

As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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-201

2

carlo

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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ax-2

012

carlo

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ax-2

012

carlo

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012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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012

Page 34: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

End Capping

The surface of non-hybrid silica is covered with silanol groups that are used in adsorption (normal phase) chromatography to interact with polar molecules or in reversed phase (as well as ion exchange chiral etc) chromatography to attach the bonded phase material

The remaining silanol groups (depending upon their conformation) are able to interact with analytes containing ionic or polar functional groups to give rise to peak tailing through unwanted secondary interactionsca

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2

Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

carlo

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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2

Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

carlo

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

carlo

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012

carlo

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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-201

2

carlo

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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ax-2

012

carlo

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ax-2

012

carlo

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012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 35: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Carbon Load

Carbon load is a measure of the amount of bonded phase bound to the surface of the packing In general terms

bull high carbon loads provide greater column capacities and resolutionbull low carbon loads render less retentive packing and faster analysis

Frits

A major cause of column deterioration and damage is the build-up of particulate and chemical contamination at the head of the packed stationary phase bed This can lead to increased back pressure and poor analyte peak shape (usually tailing or in the worst cases split peaks) HPLC Columns normally contain stainless steel inlet and outlet frits (acting as filters) which retain the column packing and allow the passage of the mobile phase The pore size of the frit must be smaller than the particle diameter of the packing eg a 05 μm frit for 18 μm packing Sample depositing on the column inlet frit can lead to peak shouldering as demonstrated below

The simplest solution is to replace the frit sometimes however you may be able to clean the frit in an ultrasonic bath

carlo

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Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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2

Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

carlo

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012

Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

carlo

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ax-2

012

carlo

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012

Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

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012

As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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-201

2

carlo

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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ax-2

012

carlo

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ax-2

012

carlo

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Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 36: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Channelling

Channels occur when pockets of air under high pressure are forced through the packed bed ndash causing pathways with little or no packing material ndash creating a ldquopath of least resistancerdquo The presence of channels will cause peak fronting as solvent (and solutes) will travel faster through them than in the rest of the column Further ndash the available surface through these channels is limited so overloading of this pathway quickly occurs and analytes undergo little (or reduced) retention in comparison with the majority of the sample band

Figure 22 The presence of channels will generate speed migration differences between different components of mobile phase thus yielding peak shape problems

carlo

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In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

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2

Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

carlo

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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2

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

carlo

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

carlo

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

carlo

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hum

ax-2

012

carlo

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012

Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

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012

As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

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012

carlo

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

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012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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-201

2

carlo

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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012

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012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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ax-2

012

carlo

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ax-2

012

carlo

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012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 37: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

In order to avoid channelling you should not

bull jar the columnbull shock the column through pressure changes or by jumping to immiscible solventsbull reverse the column flow or make sudden changes to flow rates

Remember to degas your mobile phase and prevent any particulate matter from entering the column (use an in-line filter where necessary)

Column Overload

This condition will cause different problems poor peak shapes (broadening tailing fronting and asymmetry) variable retention times selectivity issues etc and occurs when the sample concentration or volume saturates the stationary phase surface in the area of the analyte band ndash causing unusual retention effects Column capacity depends upon many factors however the table below provides the means to quickly identify situations where the possibility of column overload exists

Note that Table 5 is intended to indicate the possibility of overloading your column For more accurate information for particular applications consult with your column manufacturer

There are two forms of column overloading concentration and volume overloading Both of them lead to a decrease in chromatographic resolutionca

rlope

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-201

2

Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

carlo

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

carlo

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

carlo

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

rlope

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2

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

carlo

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012

Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

carlo

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ax-2

012

carlo

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012

Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

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012

As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

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ax-2

012

carlo

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012

Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

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012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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-201

2

carlo

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012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

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012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 38: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Voids in the Stationary Phase

Working outside the ideal pH range of your column could compromise the integrity of the stationary phase (silica dissolution) so voids are likely to develop at the head of the column due to bed compression

Silica is soluble under high pH conditions (typically pH 75 and above for traditional silica based phases) therefore silanol accessible functional groups (which are present in all silica based columns) can be attacked by strong bases while developing voids in the packing material with the consequent loss of efficiency

HPLC columns are designed to operate under high pressure conditions however columns can be damaged by sudden variations in pressure Reasons for pressure variation include

bull Slow rotation of the sample injection valvebull Fast start-up of the pumpbull Column switching operations

Voids are also formed when silica dissolution occurs and particles collapse causing bed compression when the column is pressurized Peak shouldering and broadening is one of the most common problems due to voids in the stationary phase

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Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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2

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

carlo

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012

carlo

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012

Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

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012

As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

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012

carlo

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012

Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

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012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

pez-

hum

ax-2

012

First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

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012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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012

carlo

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012

carlo

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Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 39: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Whereas in older style and cartridge type columns the inlet frit could be removed and loose stationary phase added as temporary fix newer columns do not allow this with end fittings being permanently fixed in place

Reasons for peak shouldering and splitting include

bull Column void

bull Column contamination

bull Contaminated or partially blocked frit

bull Injection solvent stronger than mobile phase

Note that if peak shouldering affects only one peak within the chromatogram then rather than stationary phase voids co-elution should be suspected

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Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

carlo

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

carlo

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carlo

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

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carlo

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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-201

2

carlo

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012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

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ax-2

012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

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012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

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012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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012

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012

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Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

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2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 40: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Temperature

Temperature plays an important role in HPLC this is because both the kinetics and thermodynamics of the chromatographic process are temperature dependent

In nearly all reversed phase separations an increase in temperature will reduce analyte retention

Additionally solvent viscosity is reduced at elevated temperature which in turn means lower backpressure

Temperature and Flow rate Cnsiderations

Figure 24 Effect of temperature on the HPLC separation Column 50 cm times 46 mm 18μm solute α-naphthol mobile phase 60 acetonitrilendash40 water

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By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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-201

2

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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2

carlo

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

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012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

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012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

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ax-2

012

carlo

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012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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012

carlo

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hum

ax-2

012

carlo

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carlo

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012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 41: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

By increasing the temperature the amount of organic solvent in the mobile phase can be reduced to maintain retention In some cases a small increase in temperature (of only a few degrees Celsius) produces a similar effect on retention as changing mobile phase composition

In the application below a mixture of parabens were separated at three different temperatures (the optimum flow rate for each temperature was selected)

Figure 25 HPLC analysis of a mixture of parabens (200 Bar) Column C18 (21mm IDtimes50 mm 17μm) mobile phase water + 30acetonitrile Sample a Methylparaben b Ethylparaben c Propylparaben d Butylparaben

Important due to lab temperature variations some form of column temperature control withmobile phase pre-heating is strongly recommendedcarlo

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012

Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

rlope

z-hu

max

-201

2

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

carlo

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

carlo

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012

2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

pez-

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012

So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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012

To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

carlo

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012

Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

carlo

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hum

ax-2

012

carlo

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012

Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

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012

As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

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012

carlo

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012

Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

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012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

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012

First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

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-201

2

carlo

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012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

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012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

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012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

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012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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carlo

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carlo

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012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

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012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

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ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

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ax-2

012

carlo

pez-

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ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 42: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Flow Rate

Keeping constant linear velocity is one of the key parameters when trying to reproduce a chromatographic separation on a different column Do not expect retention time similarities or same chromatographic efficiency when changing to a different column (dimensions packing material etc)

As expected there is a relationship between the column dimensions more specifically column ID and its optimum flow rate Table 6 gives typical flow rates for selected HPLC columns

Interestingly even if the same number of sample molecules is injected in each case smaller diameter columns tend to provide higher sensitivity than larger diameter columns The main reason being that analytes are more concentrated in the mobile phase when using small diameter columns due to the reduction column volume

Remember the use of non pure solvents in HPLC causes irreversible adsorption of impurities at the column head Filters guard columns and frits should be used to prevent this situationca

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2

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

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Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

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2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

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Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

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Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

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2

carlo

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Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

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Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

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If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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ax-2

012

carlo

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ax-2

012

carlo

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012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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012

carlo

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012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 43: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Retention Time

Introduction

The retention time of peaks within a chromatogram can aid in the identification of many problems that are associated with HPLC system components Variable retention time may result from changes in mobile phase flow rate mobile phase composition column stationary phase and column temperature Analyte retention times can fluctuate increase or decrease and can be critical in the diagnosis and elimination of HPLC system problems

Troubleshooting retention time variation requires that we first identify if the issue arises from the HPLC system or from the separation processes occurring within the HPLC column From first principles it can be generally stated that

bull If the void (hold-up) time (t0) and analyte retention time (tR) vary together suspect a flow rate change In this scenario the analyte capacity factor (k) will remain constant

bull If only the analyte retention time varies with the void (hold-up) time remaining constant then k will change also In this scenario suspect a change in the selectivity or retentivity of the separation system

carlo

pez-

hum

ax-2

012

Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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012

In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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012

Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

pez-

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012

Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

carlo

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012

2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

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012

So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

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012

To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

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Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

carlo

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Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

carlo

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012

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012

Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

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As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

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Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

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If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

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012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

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First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

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012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

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012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

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012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

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Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

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012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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012

carlo

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carlo

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012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

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Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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ax-2

012

carlo

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hum

ax-2

012

carlo

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012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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carlo

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012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 44: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Consider the following possibilities

bull Mobile phase degradationbull Selective evaporation of at least one mobile phase componentbull Incorrectly prepared mobile phasebull Improper mobile phase mixingbull Incorrect mobile phasebull Absorbtion of sample or eluent components onto the stationary phase selection and installation of the wrong column

Figure 26 Retention time variation in all peaks except in t0carlo

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In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

carlo

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012

2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

pez-

hum

ax-2

012

So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

pez-

hum

ax-2

012

To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

pez-

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

carlo

pez-

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012

Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

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012

As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

pez-

hum

ax-2

012

If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

pez-

hum

ax-2

012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

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012

First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

pez-

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012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

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hum

ax-2

012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

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ax-2

012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

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012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

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012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

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012

carlo

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012

carlo

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ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

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012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

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012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

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012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

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hum

ax-2

012

carlo

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hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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012

carlo

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012

carlo

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carlo

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012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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012

Page 45: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

In this case fresh mobile phase should be prepared if the problem persists implement solvent degassing but care needs to be taken sometimes mobile phase components evaporate when improperly degassed If only selected peaks change position then a change in the mobile phase pH or buffer concentration should be suspected

Figure 27 Retention time variation in selected peaks (t0 remains constant)carlo

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Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

carlo

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012

2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

pez-

hum

ax-2

012

So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

pez-

hum

ax-2

012

To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

carlo

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012

carlo

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012

carlo

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ax-2

012

Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

carlo

pez-

hum

ax-2

012

Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

pez-

hum

ax-2

012

As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

pez-

hum

ax-2

012

If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

pez-

hum

ax-2

012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

pez-

hum

ax-2

012

First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

pez-

hum

ax-2

012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

pez-

hum

ax-2

012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

pez-

hum

ax-2

012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

pez-

hum

ax-2

012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

pez-

hum

ax-2

012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

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012

carlo

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012

carlo

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012

carlo

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ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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Page 46: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Fluctuating Retention Times

Analyte retention time variation can be caused by changes in the mobile phase composition and is typically the result of inconsistent on-line solvent mixing or insufficient column equilibration in gradient analysis A 5minus10 reduction in analyte retention by reversed phase is possible for every 1 increase in the proportion of mobile phase organic solvent This variation is well recognized and is often practically implemented to effect gross changes in retention (and sometimes selectivity) within a separation during method development

The figure below illustrates the retention time variation for consecutive injections of a four-component system suitability standard mix using a low pressure mixing solvent delivery system The initial part of the separation method uses a 12min isocratic hold at 62 B

Figure 28 Retention time variationcarlo

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Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

carlo

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012

2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

pez-

hum

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012

So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

pez-

hum

ax-2

012

To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

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Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

carlo

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012

Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

carlo

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012

Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

carlo

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ax-2

012

carlo

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012

Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

pez-

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012

As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

pez-

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012

If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

pez-

hum

ax-2

012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

pez-

hum

ax-2

012

First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

pez-

hum

ax-2

012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

pez-

hum

ax-2

012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

pez-

hum

ax-2

012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

pez-

hum

ax-2

012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

pez-

hum

ax-2

012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 47: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Assuming that the solvent reservoir contents were correctly prepared an incorrect (or inconsistent) mobile phase composition typically results from one of several possible issues as listed below

1 A restriction in one or more of the solvent channel lines leading from the reservoir(s) to the gradient proportioning valve leading to incorrect mobile phase composition Assess this by removing the fitting that connects the inlet line with the proportioning valve (you may need to do this for two sets of tubing if you have an in-line degasser installed in the system) Once disconnected liquid should siphon freely if it does not then there is a restriction Loosen the solvent reservoir cap if sealed to relieve any partial vacuum in the reservoir

If insufficient pressurization was the problem then the solvent will now siphon freely If flow is still restricted remove the inlet solvent frit (filter) and check for siphoning again If the line siphons freely the frit is blocked If the frit is of the sintered glass variety clean by soaking in 6M nitric acid then rinse thoroughly first with H2O then MeOH then finally with H2O again before reinstalling

Do not sonicate as the glass may fracture due to the ultrasonic frequency applied If a solvent inlet line were restricted then less solvent would be introduced generating a slight vacuum in the gradient proportioning valve Consequently when the second valve opened there would be a surge of that solvent to relieve this partial vacuum with the result that the proportion of solvent introduced would vary An excess of solvent B in the mobile phase would elute the analytes earlier the effect observed in the example chromatographic trace from figure 28

carlo

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ax-2

012

2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

pez-

hum

ax-2

012

So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

pez-

hum

ax-2

012

To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

pez-

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012

Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

carlo

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ax-2

012

carlo

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hum

ax-2

012

carlo

pez-

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ax-2

012

Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

carlo

pez-

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ax-2

012

Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

pez-

hum

ax-2

012

As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

pez-

hum

ax-2

012

If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

pez-

hum

ax-2

012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

pez-

hum

ax-2

012

First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

pez-

hum

ax-2

012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

pez-

hum

ax-2

012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

pez-

hum

ax-2

012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

pez-

hum

ax-2

012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

pez-

hum

ax-2

012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 48: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

2 If solvent delivery is not restricted then a problem with the controlling software or a mechanical problem with the proportioning valve might be suspected Low pressure mixing systems operate by opening one proportioning valve for a given time closing it and then opening a second valve etc according to the lsquoduty cyclersquo of the pumping or proportioning system In the example shown in Figure 30 if the total valve cycle was 100ms and an isocratic mobile phase of 38 A62 B is required then proportioning valve A would remain open for 38ms and proportioning valve B for 62ms To check for a gradient proportioning valve failure prime all the channel lines with the same solvent then with equal volumes in their reservoirs run a 1111 (vv) quaternary gradient for a fixed time at a fixed flow rate The volume reduction in each solvent reservoir will be the same if the proportioning valves are operating correctly

3 Mobile phase inconsistencies can also occur if improperly recycled solvent is used Ensure the solvent reservoir contains a minimum of about three litres of mobile phase and that it is constantly stirred In this way sample contaminants or other small changes in the column eluent are effectively diluted out before passing through the system the next time In general it is better not to recycle mobile phase

4 In gradient analysis if the re-equilibration time is not sufficient the initial mobile phase within the column will change from run to run This will cause variations (both earlier and later elution are possible on a run to run basis) in the retention time (and possibly the selectivity of the separation) One should ensure that 10 column volumes of mobile phase at the starting composition of the gradient should pass through the column for proper re-equilibration of the whole packed bed (this may be shortened through experimentation) Two important numbers are required to achieve this the internal volume of your column (sometimes called the lsquointerstitial volumersquo) and the gradient dwell time (the time it takes for the system to change back from the final to the initial gradient composition and pump it to the inlet of your column)carlo

pez-

hum

ax-2

012

So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

pez-

hum

ax-2

012

To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

pez-

hum

ax-2

012

Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

carlo

pez-

hum

ax-2

012

Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

pez-

hum

ax-2

012

As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

pez-

hum

ax-2

012

If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

pez-

hum

ax-2

012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

pez-

hum

ax-2

012

First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

pez-

hum

ax-2

012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

pez-

hum

ax-2

012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

pez-

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ax-2

012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

pez-

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ax-2

012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

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012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

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012

carlo

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hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

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012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

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012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

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hum

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012

carlo

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012

carlo

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012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

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012

Page 49: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

So at a flow rate of 05mLmin an equilibration of ten column volumes would mean allowing 2 minutes equilibration

Now for that second important number ndash the gradient dwell volume We will show you how to calculate this is a future newsletter ndash however a well plumbed LC system will have a dwell volume below 05mL and some UPLC systems will be significantly lower However if we err on the side of caution and assume 05mL ndash this will add a further 1 minute to our equilibration time at an eluent flow rate of 05 mLmin

Therefore a total of 3 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity

5 Improve the reliability of any LC analysis with respect to both analyte retention time variation and peak shape by ensuring that the buffering capacity of any mobile phase is sufficient to compensate for any changes in pH

Generally a buffer will operate with greatest buffering efficiency when within 1 pH unit of its pka Importantly phosphate buffers do not give such a desired buffering capacity in the pH range 3minus6 This pH range could be filled by using either an acetate buffer (pka 48) or citrate as this buffer exhibits three pkarsquos in the pH range typical of reversed phase separations However citrate suffers from the fact that it is highly corrosive to stainless steel surfaces

carlo

pez-

hum

ax-2

012

To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

pez-

hum

ax-2

012

Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

carlo

pez-

hum

ax-2

012

Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

pez-

hum

ax-2

012

As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

pez-

hum

ax-2

012

If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

pez-

hum

ax-2

012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

pez-

hum

ax-2

012

First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

pez-

hum

ax-2

012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

pez-

hum

ax-2

012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

pez-

hum

ax-2

012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

pez-

hum

ax-2

012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

pez-

hum

ax-2

012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 50: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

To maintain adequate buffering strength for analyte samples start with a buffer concentration of between 25minus50mM Do not use less than 20mM unless mass spectrometry is being used as the detection mechanism Consider changing the buffer system used to one of a volatile nature ie ammonium acetate or ammonium formate Volatile buffer systems will help prevent contamination and potential ldquofoulingrdquo of the mass spectrometer source improving sensitivity and detection efficiency

The figure below illustrates the detrimental effect varying pH has on both analyte retention time and peak shape The two compounds being chromatographed are benzoic acid and sorbic acid respectively At a buffered pH of 35 these weak acid compounds are fully protonated (ion suppressed) and consequently they exhibit long retention times on a reversed phase column (Trace A) At a buffered pH of 70 the acids are fully de-protonated (ionized) They now possess a much greater degree of polarity than at a pH of 35 and consequently their retention time decreases dramatically (Trace B) However if an unbuffered pH of 70 is used then the ionisation state of these acid analytes are not controlled effectively and as the dynamic equilibrium is constantly changing between their respective ion-suppressed and ionised forms then both their peak shape and retention times vary dramatically (Trace C)

Figure 29 Effect of pH on peak shape and retention timecarlo

pez-

hum

ax-2

012

Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

carlo

pez-

hum

ax-2

012

Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

pez-

hum

ax-2

012

As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

pez-

hum

ax-2

012

If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

pez-

hum

ax-2

012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

pez-

hum

ax-2

012

First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

pez-

hum

ax-2

012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

pez-

hum

ax-2

012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

pez-

hum

ax-2

012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

pez-

hum

ax-2

012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

pez-

hum

ax-2

012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 51: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Temperature Effects

Retention time variation may also be caused by fluctuations in temperature A 1minus2 change in retention time is possible for every 10 C change in column temperature The simplest way to eliminate this potential problem is to ensure that both the column and mobile phase are thermostatically controlled Check for potential temperature problems by using a calibrated thermometer and determine if any observed temperature fluctuations correlate with changes in analyte retention time

Column aging may also contribute to retention time variation The combined action of high temperatures and aggressive mobile phases (for example most HPLC silica based columns should be used with mobile phase pH between 2 and 8) will accelerate the natural column degradation process that is responsible for many problems such as reduced selectivity reduced efficiency peak shape problems inconsistent retention time etc Using a guard column may extend the effective lifetime of a column However the use of a guard column is recommended for only one specific reason to act as a chemical filter in removing any strongly retained material(s) that could potentially contaminate the analytical column

Running check standards more frequently may allow a data capture system to effectively ldquokeep uprdquo with the observed retention time drift Nominate a sample chromatographic peak as a reference peak The use of internal standards may also help especially if relative rather than absolute retention times are used The table below illustrates the observed effect of changing separation conditions on analyte tR

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

carlo

pez-

hum

ax-2

012

Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

pez-

hum

ax-2

012

As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

pez-

hum

ax-2

012

If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

pez-

hum

ax-2

012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

pez-

hum

ax-2

012

First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

pez-

hum

ax-2

012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

pez-

hum

ax-2

012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

pez-

hum

ax-2

012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

pez-

hum

ax-2

012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

pez-

hum

ax-2

012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 52: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

carlo

pez-

hum

ax-2

012

Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

pez-

hum

ax-2

012

As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

pez-

hum

ax-2

012

If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

pez-

hum

ax-2

012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

pez-

hum

ax-2

012

First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

pez-

hum

ax-2

012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

pez-

hum

ax-2

012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

pez-

hum

ax-2

012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

pez-

hum

ax-2

012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

pez-

hum

ax-2

012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 53: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

carlo

pez-

hum

ax-2

012

Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

carlo

pez-

hum

ax-2

012

Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

pez-

hum

ax-2

012

As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

pez-

hum

ax-2

012

If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

pez-

hum

ax-2

012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

pez-

hum

ax-2

012

First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

pez-

hum

ax-2

012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

pez-

hum

ax-2

012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

pez-

hum

ax-2

012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

pez-

hum

ax-2

012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

pez-

hum

ax-2

012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 54: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Decreasing Retention Times

If both the void time (t0) and retention time (tR) are decreasing then the analytersquos capacity factor (krsquo) will remain constant In this scenario suspect a progressive increase in the flow rate However ndash letrsquos consider the situation in which analyte retention time tR is decreasing but the hold-up time t0 is not

Typically this situation will occur when the analyte retention mechanism is affected This most often will be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndash usually due to an incorrect mobile phase pH (too high for acidic species or too low for bases) Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check the pH of any buffered mobile phase being used Unless specifically stated by the column manufacturer the operating pH should be kept within the pH range 2minus8 Below approximately pH of 2 the siloxane bond attaching the stationary phase ligand to the silica support will be broken and loss of bonded phase will result (phase bleed) Above a pH of approximately 8 dissolution of the silica support may occur In both instances analyte retention times will commonly decrease please do note that enhanced retention of basic and polar analytes can be observed when anlaysed on column that has experienced high phase bleed due to extra hydrogen bonding and cation exchange via the exposed silanols One would also observe a reduction in analyte peak efficiency under these cricustances Consider switching to a column type specifically designed for use at extremes of pH

carlo

pez-

hum

ax-2

012

Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

pez-

hum

ax-2

012

As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

pez-

hum

ax-2

012

If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

pez-

hum

ax-2

012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

pez-

hum

ax-2

012

First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

pez-

hum

ax-2

012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

pez-

hum

ax-2

012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

pez-

hum

ax-2

012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

pez-

hum

ax-2

012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

pez-

hum

ax-2

012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 55: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Ensure the column has not been overloaded ndash the peak apex retention time can often be seen to move to earlier retention as the degree of column overload worsens Decrease the absolute amount of analyte being applied by diluting the sample or reducing the injection volume

If performing gradient elution ensure that the solvent components are being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

The table below helps you to identify the origin and propose remedial actions for decreasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

pez-

hum

ax-2

012

As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

pez-

hum

ax-2

012

If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

pez-

hum

ax-2

012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

pez-

hum

ax-2

012

First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

pez-

hum

ax-2

012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

pez-

hum

ax-2

012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

pez-

hum

ax-2

012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

pez-

hum

ax-2

012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

pez-

hum

ax-2

012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 56: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

carlo

pez-

hum

ax-2

012

Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

pez-

hum

ax-2

012

As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

pez-

hum

ax-2

012

If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

pez-

hum

ax-2

012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

pez-

hum

ax-2

012

First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

pez-

hum

ax-2

012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

pez-

hum

ax-2

012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

pez-

hum

ax-2

012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

pez-

hum

ax-2

012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

pez-

hum

ax-2

012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 57: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Increasing Retention Times

If both the void time (t0) and retention time (tR) are increasing then suspect a progressive decrease in the flow rate Check the method operating parameters and instrument flow rate calibration Letrsquos consider the situation in which analyte retention time tR is increasing but the hold-up t0 isnrsquot

A retention time increase may be due to a change in eluotropic strength of the mobile phase (organic content too high or buffer strength (concentration) too low) or the degree of ionization of the analyte molecule ndashusually due to an incorrect mobile phase pH (too high for acidic species or too low for bases)

When pre-mixed mobile phases are allowed to stand the more volatile solvent component(s) can evaporate thereby changing the overall retention characteristics typically leading to increasing retention times This problem is exacerbated with longer analytical campaigns as the gaseous headspace above the mobile phase increases as the level within the reservoir is depleted Ensure that the eluent reservoir is capped and ensure as little headspace as possible is maintained above the eluent liquid

Check that the mobile phase has been correctly prepared (check organic aqueous ratio pH and buffer strength) and that ingress of CO2 into eluent reservoir has not reduced the eluent pH on standing

Check all pump-head components for leaks and if necessary perform a volumetric check of instrument flow rate using a calibrated electronic flow meter or by measuring the time take to deliver 10 mL of eluent into a measuring cylinder For gradient elution check that the gradient is being mixed and proportioned correctly Loosen the solvent reservoir cap(s) to relieve any potential partial vacuum formation ensuring full pressurization and solvent line filling

carlo

pez-

hum

ax-2

012

As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

pez-

hum

ax-2

012

If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

pez-

hum

ax-2

012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

pez-

hum

ax-2

012

First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

pez-

hum

ax-2

012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

pez-

hum

ax-2

012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

pez-

hum

ax-2

012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

pez-

hum

ax-2

012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

pez-

hum

ax-2

012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 58: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

As the column gets older secondary interactions are promoted by an increased number of exposed silanolgroups so retention time of polar and or basic analytes may increase ndash this should be apparent in the first instance by an increase in the peak tailing of more polar analytes Eliminate any column secondary interactions by using a mobile phase modifier or buffering the mobile phase appropriately Triethylamine can be added as a lsquocompeting basersquo to eliminate polar analyte interactions with residual silanol groups as well as increasing the effective carbon loading of the column or switching to an endcapped type II silica column

The table below helps you to identify the origin and propose remedial actions for increasing retention time related problems

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

pez-

hum

ax-2

012

If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

pez-

hum

ax-2

012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

pez-

hum

ax-2

012

First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

pez-

hum

ax-2

012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

pez-

hum

ax-2

012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

pez-

hum

ax-2

012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

pez-

hum

ax-2

012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

pez-

hum

ax-2

012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 59: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

carlo

pez-

hum

ax-2

012

Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

pez-

hum

ax-2

012

If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

pez-

hum

ax-2

012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

pez-

hum

ax-2

012

First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

pez-

hum

ax-2

012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

pez-

hum

ax-2

012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

pez-

hum

ax-2

012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

pez-

hum

ax-2

012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

pez-

hum

ax-2

012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 60: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Peak Shapes issues

The shape of the chromatographic peaks can aid in the identification of many problems that are associated with the system components

Peak shape problems may not adversely affect the resultant data determine if the analytical results are truly compromised before taking actions

For more information on peak shape related problems please visit the links below[15 16 17]

Peak Broadening

Correcting broadened chromatographic peaks requires information on whether the peak broadening is the result of a change in the HPLC system column deterioration or a ldquolate elutingrdquo peak from an earlier injection

If all of the separated peaks are broadened with early peaks exhibiting more pronounced broadening then the problem may have been simply caused by the introduction of a large extra-column volume in the system

bull Addition of a disproportionate length of tubing or wider ID tubingbull A poorly seated capillary or column connective fittingbull Worn PEEK finger-tight nuts poorly made (non-zero dead volume) connections

carlo

pez-

hum

ax-2

012

If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

pez-

hum

ax-2

012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

pez-

hum

ax-2

012

First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

pez-

hum

ax-2

012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

pez-

hum

ax-2

012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

pez-

hum

ax-2

012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

pez-

hum

ax-2

012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

pez-

hum

ax-2

012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 61: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

If gradual peak broadening has been observed over a longer period of time then it is indicative of a general deterioration in the column itself

Column efficiency or plate number (N) is used as a mathematical measure of the deterioration of a column over time and injection number Measuring the plate number for a nominated sample compound or evaluating the chromatographic peaks of a set of system suitability standards recommended by the column manufacturer and comparing them to logbook records will indicate the extent of the deterioration Providing the mobile phase and system settings have not been changed analyte retention time should not vary significantly when efficiency drops

Column efficiency can be calculated by applying the following equation

bull If N is seen to increase with increasing analyte retention time then the column is the biggest contributor to the system dwell volume and the HPLC is performing satisfactorily

bull If N is seen to decrease or is random with increasing analyte retention time then the HPLC is the biggest contributor to the system dwell volume and any contributor to extra column volume should be reduced (consider tubing length and id number of connections and flow cell internal volume in the first instance)

carlo

pez-

hum

ax-2

012

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

pez-

hum

ax-2

012

First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

pez-

hum

ax-2

012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

pez-

hum

ax-2

012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

pez-

hum

ax-2

012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

pez-

hum

ax-2

012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

pez-

hum

ax-2

012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 62: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Evaluate whether other system components may also have contributed to the broadening peaks -including deteriorating guard columns for example high molecular weight compounds especially proteins may have broad peaks on columns with pore sizes of lt 80A ensure gt 300A pore size is used with such analyte species

A late eluting peak from a previous sample injection should be suspected whenever a single broad band appears in a chromatogram with otherwise narrow bands (efficient peaks) Importantly this peak may not appear in all analyses and therefore may not be noticed initially especially in complex multi-component separations

Given the ldquolate eluterrdquo in question is suffering simply from an increased capacity factor (krsquo) and therefore much increased diffusion then one simple approach to identifying its origin is to allow the chromatogram to run for several times the normal run time The potential problem then arises of what happens if the ldquolate-eluterrdquo is not observed in every sample run

A more efficient approach to identifying a late eluting peak is to calculate its true retention time first This approach has the advantage that it allows you to identify with which particular sample injection the peak is actually associated

carlo

pez-

hum

ax-2

012

First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

pez-

hum

ax-2

012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

pez-

hum

ax-2

012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

pez-

hum

ax-2

012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

pez-

hum

ax-2

012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

pez-

hum

ax-2

012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 63: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

First determine the plate number of a known peak in the chromatographic trace As an example we will take a peak possessing a retention time of 12 min with a PWHH (Wfrac12) of 015 min Using the equation previously described this gives a plate number of

By measuring the peak width at half height maximum of the late eluting peak it is possible to predict its retention time

In this example suppose the peak width of the problem peak is 05min Using this peak width and the plate number for the column previously determined from the known chromatographic peak of 35456 the calculated retention time is 40 min This means that the late eluting peak is associated with an injection made approximately 40 min previously Re-injecting the suspect sample and altering the runtime accordingly can confirm this retention time value

Often it is not possible to eliminate these late-eluting peaks Therefore instead of modifying the separation conditions or waiting until the peak elutes before making the next injection it is usually more convenient to modify the run time so that the late eluting peak falls in an unimportant region of a later chromatogramcarlo

pez-

hum

ax-2

012

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

pez-

hum

ax-2

012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

pez-

hum

ax-2

012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

pez-

hum

ax-2

012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

pez-

hum

ax-2

012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 64: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Table 11 helps you to identify the origin and propose remedial actions when peak broadening occursca

rlope

z-hu

max

-201

2

carlo

pez-

hum

ax-2

012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

pez-

hum

ax-2

012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

pez-

hum

ax-2

012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

pez-

hum

ax-2

012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 65: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

carlo

pez-

hum

ax-2

012

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

pez-

hum

ax-2

012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

pez-

hum

ax-2

012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

pez-

hum

ax-2

012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 66: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Peak Shouldering and Splitting

If all peaks in the chromatographic trace are doublets then the column has probably developed a void at the head of the packed bed or has been subjected to ldquochannellingrdquo Substituting the column will quickly confirm this problem

If a void is suspected reverse the column and wash in 100 strong solvent to remove any contamination from the column inlet filter and direct to waste bypassing the detector Check the manufacturerrsquos instructions as to whether the column should remain in the reversed flow orientation

As a partial blockage of the column inlet frit is generally caused by deposition of particulates from the mobile phase sample solvent pump seals or rotor seal ensure adequate filtering and include an inline filter between the injector and column head where required

If only one peak in the chromatogram is a doublet then a co-eluting or interfering peak should be suspected Confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles or an alternative selectivity (different stationary phase chemistry)

Peak shoulders usually indicate co-eluting compounds Adjust the selectivity shown to the co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating the outcome If required flush your column (consult your column manufacturer)

carlo

pez-

hum

ax-2

012

Figure 32 Recommended flushing procedure for an HPLC columncarlo

pez-

hum

ax-2

012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

pez-

hum

ax-2

012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 67: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Figure 32 Recommended flushing procedure for an HPLC columncarlo

pez-

hum

ax-2

012

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

pez-

hum

ax-2

012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 68: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Routinely flush the column with a high elution strength solvent when performing isocratic separations to wash any strongly retained non-polar compounds off the column This is illustrated in the figure below where the peak shape of a variety of basic drug compounds being separated isocratically in a 25mM Na2HPO4 (pH30)MeOH (6040) mobile phase are significantly improved following a column wash with 100 acetonitrile

carlo

pez-

hum

ax-2

012

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 69: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column The consequence will be the formation of doublet peaks or peak shouldering Try to match the sample diluents strength to the initial eluent solvent strength where solubility allows You can perform serial dilutions from a more concentrated sample altering the eluent strength with each dilution to prevent precipitation in many instances Alternatively one should restrict the injection volume to below 10 μL

Figure 34 Peak distortion due to injection solvent and mobile phase mismatch The sample is a mixture of parabens Column C18 30 x 50 mm 18 μm Temperature 45 degC Mobile phase 10 mLmin (30 - 65 CH3CN)carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 70: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 71: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

carlo

pez-

hum

ax-2

012

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 72: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Peak Tailing

Tailing peaks are the most common chromatographic peak shape distortion A peak is classified as tailing if its asymmetry is greater than 12 although peaks with As values as high as 15 are acceptable for many assays

The primary cause of peak tailing is due to the occurrence of more than one mechanism of analyte retention In reversed-phase separations the main mode of analyte retention is through non-specific hydrophobic interactions with the stationary phase However polar interactions with any ionized residual silanol groups residing on the silica support surface are also common Compounds possessing amine and other basic functional groups interact strongly with such ionised silanol groups producing tailing peaks This is illustrated in the figure shown below at a mobile phase pH gt30

Figure 36 Strong interactions (at high pH values) between analyte molecules and silanol groups from the stationary phase will lead to peak tailing

carlo

pez-

hum

ax-2

012

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 73: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Such secondary interactions can be minimised by performing the chromatographic separation at a lower pH thereby ensuring the full protonation of such ionisable residual silanol groups

Figure 37 Reduction of secondary interactions through pH adjustment (low pH in this case)carlo

pez-

hum

ax-2

012

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 74: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Alternatively an end-capped column can be utilized as previously discussed

If all chromatographic peaks tail then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

Evaluate the column for the possibility that the cause is either the development of a column void or a partially blocked inlet frit Substituting the column will quickly confirm the problem

If only one or some of the chromatographic peaks tail then consider the possibility that the column is exhibiting secondary partition or retention effects

Figure 38 The reduction of pH (right hand side chromatogram) can improve peak shape when dealing with basic analytes

carlo

pez-

hum

ax-2

012

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 75: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Adding triethylamine (TEA) to the mobile phase at approximate concentration levels of 5minus10 mM will also reduce or possibly eliminate tailing of basic compounds The TEA competes with basic polar analytes for binding to the residual silanol groups performing a pseudo end-capping function However this approach is less favored as the columnrsquos stationary phase will be irreversibly altered by the addition of such a modifier affecting subsequent selectivity shown by the separation system

Ion-pair chromatography may solve the tailing problem for acidic or basic analytes An ion-exchange mode of chromatography is another option for ionic analyte species

A single tailing peak can also result from a small peak eluting just after the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak tailing should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating using the procedure described under Peak Broadening

The table below helps you to identify the origin and propose remedial actions when peak tailing occurscarlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 76: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 77: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

carlo

pez-

hum

ax-2

012

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 78: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

Peak Fronting

If all chromatographic peaks front then consider the possibility that the column has been mass overloaded If necessary use a higher capacity stationary phase ie increased carbon or pore size use a column with an increased column diameter or decrease the absolute sample amount or volume injected

If too great a sample volume has been injected and the sample solvent and mobile phase are not correctly matched then two distribution equilibriums will occur resulting in differential sample partitioning on-column[18]

Reference to the empirical rules in respect of matching injection volume with sample solvent strength will aid in eliminatingthis phenomena

A single fronting peak can also result from a small peak eluting just before the chromatographic peak of interest The possible occurrence of an interfering compound is the primary reason why peak fronting should not be ignored As was described under peak splitting and shouldering confirm the presence of an interferent by changing the detection wavelength or improve the resolution of the separation by using a column offering greater efficiency ie a longer column or a column packed with smaller sized particles

Adjust the selectivity shown to any co-eluting compounds by the separation system by altering the mobile phase composition or type separation temperature or the column stationary phase Use a sample clean-up procedure ie SPE to remove any interfering contaminants Evaluate if any co-eluting peak may have come from a previous chromatographic run by making a blank injection and evaluating

The table below helps you to identify the origin and propose remedial actions when peak fronting occurs

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 79: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 80: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

carlo

pez-

hum

ax-2

012

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 81: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

carlo

pez-

hum

ax-2

012

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012

Page 82: HPLC Troubleshooting Separations Retention Time ...quimica.udea.edu.co/~carlopez/hplc/hplc-troubleshooting-separations... · This essential guide examines the common causes of retention

2 ldquoPractical HPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2010

3 ldquoHPLC Troubleshooting amp Maintenancerdquo Crawford Scientific 2008

4 G Guiochon in C Horvaacuteth (Editor) High Performance Liquid Chromatography Advances and Perspectives Vol 2 Academic Press New York 1980

6 Dao T-T Nguyen Davy Guillarme Sabine Heinisch Marie-Pierre Barrioulet Jean-Louis Rocca Serge Rudaz Jean-Luc Veuthey ldquoHigh throughput liquid chromatography with sub-2μm particles at high pressure and hightemperaturerdquo Journal of Chromatography A 1167 (2007) 76ndash84

7 A-M Siouffi ldquoAbout the C Term in the Van Deemterrsquos Equation of Plate Height in Monolithsrdquo Journal of Chromatography A 1126 (2006) 86ndash94

8 Alain Bertho and Alain Foucault ldquoComments on Van Deemter Plot in High Speed CountercurrentChromatographyrdquo Journal of Liquid Chromatography amp Related Technologies 2001 24(13) Pp 1979 ndash 1985

12 Justin Chow John W Dolan ldquoRetention Changesrdquo January 2012

13 John W Dolan ldquoTroubleshooting Basics Part 3 Retention Problemsrdquo December 2011

14 John W Dolan ldquoVariability mdash How to Control It Why Arent Retention Times Constantrdquo December 2007

15 John Dolan ldquoPeak Shape Problemsrdquo July 2008

16 John Dolan ldquoSplit Peaks mdash A Case Studyrdquo January 2005

17 Ryan D Morrison and John W Dolan ldquoPeak Fronting Column Life and Column Conditioningrdquo April 2005

18 John Dolan ldquoThe Power of Mobile Phase Strengthrdquo July 2006carlo

pez-

hum

ax-2

012