chapter 11 column liquid chromatography 540-061... · types of liquid chromatography ... examples:...
TRANSCRIPT
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Chapter 11
Column Liquid Chromatography
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Types of Liquid Chromatography
• Partition chromatography
• Adsorption or liquid-solid chromatography
• Ion Exchange chromatography
• Size exclusion or gel chromatography
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Adsorption Chromatography
The stationary phase is solid. Separation is
due to adsorption/desorption steps
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•Adsorbent can be
packed in a column
spread on a plate,
or impregnated in a
A porous paper
•Both solutes and
solvents will be
attracted to the
stationary phase
•If the solutes have
different degrees of
attraction
separation would be
achieved
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Partition chromatography
Separation is based on solute partitioning
between two liquid phases
• More highly retained species have greater affinity
(solubility) for the stationary phase compared to the
mobile phase (solvent)
• Separation of solutes is based on the difference in
the relative solubilities
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Modes of separation
• Normal phase partition chromatography
Polar stationary phase and nonpolar solvent
• Reverse phase partition chromatography
Nonpolar stationary phase and polar solvent
• Reverse phase is now more common
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Changing the mode of separation will lead to a change
In the elution order of the solutes that could be almost
Reversed.
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• Separation is based on the affinity of ions in solution for
oppositely charged ions on the stationary phase
• Stationary phase has charged surface opposite
that of the eluents
Ion exchange chromatography
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Separation by ion exchange chromatography
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Size exclusion chromatography
• Separation is based on molecular size. Stationary
phase is a material of controlled pore size. It is also
called Gel permeation chromatography
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Size exclusion chromatography
• Columns are made to match the separation of
specific size ranges
• Larger species will elute first. They cannot pass
through many pores so their path is shorter
• Size exclusion liquid chromatog. Is useful for
determining size, size range and molecular weights
of polymers and proteins.
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Separation by size exclusion chromatography
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LC Solvents (Mobile phase)
• LC solvents depend upon the type of
chromatographic mode used:
* Normal Phase
* Reversed phase
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Solvent selection
• If the sample is water insoluble or nonpolar- normal phase mode is used
• If the sample is water soluble or not soluble but polar- use the reverse phase mode
• It is seldom to find a single solvent does the job. Thus mixtures of two or more solvents are used
• Two factors are considered:
– Solvent strength, (o) A measure of relative solvent polarity (ability to
displace a solute). It is the adsorption energy per unit area of
solvent. o for silica is about 0.8 of those on alumina
– Polarity index, (P’) • Solvents that interact strongly with solutes are strong or polar solvents
• Polarity of solvents has been expressed by many terms, one of which is the polarity index. Thus, the P’ value measures the relative polarity of various solvents
Used for reversed phase methods
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o P’
Solvent strength and polarity index
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Solvents used as mobile phase in liquid chromatography
Snyder classified solvents based on acidity and basicity,
dipole and chemical properties
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Gradient elution
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Advantages of gradient elution
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Gradient elution
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Gradient elution
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Mobile Phases for adsorption chromatography
• Common mobile phases on alumina
hexane; chloroform; 2-propanol
Example: separation of amines
• Common mobile phases on Silica
hexane; chloroform; 2-propanol
Examples: separation of ethers, esters,
prophyrins, vitamins
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Mobile Phases for partition chromatography
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Ion exchange phases
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High Performance Liquid
Chromatography
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Column Liquid Chromatography
• LC techniques are: Classical LC and
HPLC or HSLC (S = speed)
• Both techniques have same basic
principle for separation but differ in
apparatus and practice used
• HPLC gives high speed, high
resolution, high sensitivity and
convenient for quantitative Analysis.
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HPLC
originally refered to:
High Pressure Liquid Chromatography
currently refers to:
High Precision Liquid Chromatography
– the high pressure allows using small
particle size to allow proper separation at
reasonable flow rates
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Features of HPLC compared to Classical LC
Particle size of the packing substance
• Classical LC utilises large porous particles that make it difficult to speed up the flow rate by pumping due to a decrease in resolution that results from the mass transfer limitation in the deep pores. These high capacity particles are good for preparative chromatography
• Since the mass transfer coefficient is a function of the square of dp (van deemter eq.) thus the HPLC was based on using pellicular and porous microparticles. Pellicular particles when packed into narrow columns will lead to an increase in column efficiency of 10 to 100 folds
• Pellicular particles have dense solid cores thus they are easily packed
• Vs is significantly reduced and the sample capacity is reduced to 0.05 to 0.1 of the totally porous packing
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Effect of particle packing size on column efficiency
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Column length
• Efficiency is very high due to packing thus
shorter columns are used (~ 20 cm)
• For difficult separations longer (50-100 cm)
are used with smallest available particles and
high pressure solvent feed
Effect of sample size on column efficiency
• Efficiency increases as the sample size
decreases
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Effect of sample mass on column efficiency
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Columns
• LC columns could be made of stainless steel, glass and glass lined stainless steel that is used for extremely inert surfaces.
• In classical LC, elution takes place under gravity or low pressure by using small pumps
• Columns can be thermostated by placing in an oven or using a water jacket
• HPLC columns are mainly made of stainless steel packed with the microparticles
• When same amount is injected in the HPLC column, narrower and longer peaks are obtained that leads to greater detector sensitivity
• In HPLC solvent consumption is reduced
• HPLC columns facilitate coupling to MS that requires flow rates <50 L/min to avoid over pressuring the ion source in the MS
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Components
Solvent Reservoir and Degassing System
Pumps
Precolumns
Sample Injection System
Columns
Temperature Control
Detectors
Readouts
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Schematic diagram of a typical high performance liquid
chromatograph
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Schematic of Liquid Chromatograph
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Solvent Reservoir and Degassing System – isocratic elution - single solvent
separation technique
– gradient elution - 2 or more solvents,
varied during separation
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Improvement in separation
efficiency by gradient
elution. Column: 1m x
2.1mm id, precision-bore
stainless. Sample: 5L of
chlorinated benzenes in
isopropanol. Detector: UV
photometer (254 nm).
Conditions: temperature
60oC, pressure, 1200 psi.
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Pumping systems
Requirements for pumping system
• Generation of pressure up to 6000 psi
• Pulse free output
• Flow rates of 0.1 to 10 ml/min
• Flow control and flow reproducibility of 0.5% relative or better
• Corrosion resistance components
Types of pumps:
• Direct pressure pumps (pneumatic pumps)
• Syringes (displacement pumps)
• Reciprocating pumps
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They are limited to pressures less
Than 2000 psi
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They are of limited solvent capacity (about 250 ml)
and inconvenience when the solvent is changed.
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Widely used pumping; motor driven piston that pumps the solvent into
the chamber. The two ball check valves (f) Open and close alternatively
to control the solvent into and out of the chamber.
•It has the disadvantage of producing pulsed flow which must be damped
because its presence is manifested as baseline noise
• They have small internal
volume (35-400 L)
• High output pressure (10000 psi)
• Readily adoptable to gradient
elution
• Constant flow rates that are
independent of column back
pressure and solvent viscosity
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Reciprocating pump
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there
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Sample Loop
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Detectors
Properties of good detectors
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Types of Detectors
• Absorbance (UV with Filters, UV with
Monochromators)
• IR Absorbance
• Fluorescence
• Refractive-Index
• Evaporative Light Scattering Detector
(ELSD)
• Electrochemical
• Mass-Spectrometric
• Photo-Diode Array
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Ultraviolet detector
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Refractive-Index Detector
Schematic of a differential refractive-index detector
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Mass spectrometric detectors
• LC and MS appear to be incompatible.
HPLC MS
Liquid phase operation Gas phase operation
20-25 oC operation 100-350 oC operation
Almost no sample limitation same volatility desired
Relatively inexpensive Expensive
Uses inorganic buffers can’t tolerate inorganic
buffers
Conventional flow rate Accepts 10 ml/min
produce 550 ml/min gas at STP
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Features of LS-MS
• Its sensitivity approaches sub nanograms
• The significant problem here is 1 ml of hexane,
methanol, or water generates respectively, 180, 350
and 1250 ml/min gas
• Since MS operates under vacuum the sample vapor
must be removed without removing a substantial
amount of solute
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LC-MS interface techniques
• Direct liquid inlet: small portion of LC effluent is directed into
the ion source
• Moving belt method: LC effluent is deposited onto a moving
belt, the solvent is evaporated, and the sample is volatilized
from the belt into the ion source
• Thermospray ionization: ions are created when aqueous
buffered mobile phase is passed through a heated stainless
steel capillary creating a supersonic jet of vapor with
subsequent evaporation of the mobile phase from charged
liquid droplets
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Applications of Liquid Chromatography
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Applications • Preparative HPLC -the process of isolation and purification of compounds. • analytical HPLC-obtain information about the sample compound.
– identification, – quantification, – resolution of a compound.
• Chemical Separations - using HPLC
– certain compounds have different migration rates given a particular column and mobile phase.
• Purification - the process of separating or extracting the target compound from other (possibly structurally related) compounds or contaminants.
– Each compound should have a characteristic peak under certain chromatographic conditions.
– choose the conditions, such as the proper mobile phase, to allow adequate separation to collect or extract the desired compound as it elutes from the stationary phase.
• Identification of compounds by HPLC is a crucial part of any HPLC assay.
– accomplished by researching the literature and by trial and error.
– Identification of compounds can be assured by combining two or more detection methods.
• Quantification - the process of determining the unknown concentration of a compound in a known solution. – inject a series of known concentrations of the standard compound
solution – chromatograph of these known concentrations – peaks that correlate to the concentration of the compound injected
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Adsorption chromatography
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Adsorption chromatography
• It is LSC and oldest chromatographic method introduced by Tswett that became the HPLC technique
• Silica and alumina are mostly used as the stationary phases in thin layer or column
• How do adsorbents separate compounds?
• Consider the surface of the most widely adsorbent, silica gel….
• Silica gel is a stable porous solid terminated at the surface with silanol (Si-OH) or siloxane (Si-O-Si) bonds
• The slightly acidic silanol groups are of importance in separation however, siloxane bonds are of little or no influence
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Most acidic
Silanol groups
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Silanol groups and interactions with solutes
• Silanol groups have varying degrees of acidity
• The most acidic ones are located at adjacent silicon atoms with intermolecular H-bonding. These lead to undesirable effects like chemisorption and peak tailing
• To avoid problems, polar modifier such as water is added in order to deactivate the strongest adsorption sites
• Interactions between adsorbent surface and solute vary from nonspecific (dispersion or vander Waal’s forces) to specific ones (electrostatic interactions such as permanent dipoles or electron donor acceptor interactions such as hydrogen bonding)
• Retention on silica gel or alumina is governed mainly by interactions with the polar functional groups of the solute
• Compounds of different chemical types (hydrocarbons and alcohols) are easily separated by LSC
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• Homologous or other mixtures differing
in the extent of aliphatic substitution
(no change in polarity) cannot be
differentiated
• LSC is unique in its ability to separate
polyfunctional compounds especially
positional isomers
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• With a few exceptions, the order of
retention times on silica and alumina
is:
olefins < aromatic hydrocarbons <
halides, sulfides < ethers < nitro
compounds < esters ~ aldehydes ~
ketones < alcohols amines < sulfones <
sulfoxides < amides < carboxylic acids
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Intramolecular hydrogen
bonding; i.e, less intermolecular
interaction with the surface
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Solvent Selection for Adsorption Chromatography
• In liquid-solid chromatography, the
only variable available to optimize the
retention factor and the selectivity
factor is the composition of the mobile
phase (in contrast to partition
chromatography, where the column
packing has a pronounced effect on the
selectivity factor)
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Influence of the Mobile Phase in LSC:
Gradient Elution
• Interactions in LSC involve a competition between the solute
molecules (X) and the molecules (S) of the mobile phase
adsorption sites. This equilibrium is illustrated by
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• Thus, stronger adsorption of the mobile
phase decreases adsorption of the solute.
• Solvents can be classified according to their
strength of adsorption (solvent or eluent
strength, o).
• Such a quantitative classification is referred
to as an eluotropic series.
• An eluotropic series can be used to find an
optimum solvent strength for a particular
separation.
• Using a solvent of constant composition is
called isocratic elution.
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Solvent Strength
• The polarity index, P', used in partition chromatography can also serve as a rough guide to the strengths of solvents for adsorption chromatography.
• Solvent (Eluent) strength °, which is the solvent adsorption energy per unit surface area is a much better index.
• This parameter depends upon the adsorbent. ° values for silica are about 0.8 of those on alumina.
• Note that solvent-to-solvent differences in ° roughly parallel those for P'.
• For a given isocratic elution, the initial solvent is selected by matching the relative polarity to that of sample components.
• Solvent is chosen to match the most polar functional group. Alcohols for –OH group and amines for amino acids.
• If in an isocratic elution the k'-values for the solutes are too small (sample elutes rapidly) then a weaker (low °, less polar) solvent is selected
• On the other hand, if the sample does not elute in a reasonable time because of high k’ values then a stronger (high °, more polar) solvent would be selected.
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Choice of Solvent Systems (binary mixture)
• Binary solvent mixtures may be used to find an optimum value of the solvent-strength parameter °
• Two compatible solvents are chosen, one of which is too strong (° too large) and the other is too weak. A suitable value for k' is then obtained by varying the volume ratio of the two.
• a mixture of isooctane (° = 0.01) and methylene chloride (° = 0.42) can be matched with an isocratic solvent strength similar to that of carbon tetrachloride (° = 0.18).
• Unfortunately ° does not vary linearly (because of solvent-solvent and preferential solvent-surface interactions) with volume ratios. Thus, calculating an optimal mixture is more difficult.
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General Elution Problem
• This problem appears with isocratic solvent systems and multicomponent samples with widely differing k'-values.
• If a strong isocratic mobile phase is selected that will adequately elute strongly retained compounds, then the weakly retained ones will be eluted too quickly and will be poorly separated
• Conversely, if a weak mobile phase is chosen, so that weakly retained sample components will be retained and separated, then very strongly retained solutes may not be eluted at all-or only very slowly
• The most common solution is using a technique called solvent programming or gradient elution. Here, elution is begun with a weak solvent and the solvent strength is increased with time. The changes are made either stepwise or continuously
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Applications of Adsorption Chromatography
Adsorption chromatography is best suited for nonpolar compounds having molecular weights less than perhaps 5000.
Although some overlap exists between adsorption and partition chromatography, the methods tend to be complementary.
Generally, liquid-solid chromatography is best suited to samples that are soluble in nonpolar solvents and correspondingly have limited solubility in aqueous solvents such as those used in the reversed-phase partition procedure.
A particular strength of adsorption chromatography, which is not shared by other methods, is its ability to differentiate among the components of isomeric mixtures.
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Partition Chromatography
(Liquid-liquid and Bonded phase Chromatography)
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Most of the applications have been to nonionic , polar compounds of low to moderate molecular weight (usually <3000).
Recently, however, methods have been developed (derivatization and ion pairing) for separations to ionic compounds.
Partition chromatography can be subdivided into liquid-liquid and bonded-phase chromatography. The difference in these techniques lies in the method by which the stationary phase is held on the support.
With liquid-liquid, a liquid stationary phase is retained on the surface by physical adsorption.
With bonded-phase, the stationary phase is bonded chemically to the support surfaces.
•
Features of Partition Chromatography
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• Liquid-liquid chromatography is limited to compounds with comparatively low values of K (or k'), because the stationary phase must be a good solvent for the sample but a poor solvent for the mobile phase.
• In practice, increasing solvent strength in order to elute compounds with high K- (or k'-) values will increase the solubility of the stationary phase and remove the stationary phase from its support.
• When the solvent strength is high enough to dissolve an appreciable amount of stationary phase, presaturation is made difficult. – in conventional LLC solvent programming is ruled out.
• Even with its limitations, LLC is a very useful technique because it can resolve minute differences in the solubility of the solute.
• Many solvent pairs are available, and the choice of the proper ones allows great selectivity to be achieved.
• Both Paper chromatography and TLC are examples of LLC.
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Liquid-liquid chromatography
• The stationary and mobile phases are selected so as to have little or no mutual solubility.
• Therefore, they generally are quite different in their solvent properties. For example one might choose water as the stationary phase and pentane as the mobile phase for normal LLC.
– However, water does have a finite (though very slight) solubility in pentane.
• Using pentane will slowly remove the water and change the nature of the separation mechanism.
• For this reason, the mobile phase must be presaturated with the stationary phase before it enters the column (or plate).
• Presaturation can be done by stirring the two phases together until equilibration takes place; but, in LC, it is more conveniently done by placing a precolumn before the injector and the chromatographic column.
• The precolumn should contain a high-surface-area packing, such as silica gel, coated with a high percentage (say 30 to 40% by weight) of the stationary phase used in the analytical column.
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Drawbacks of LLC:
• finding immiscible solvent pairs,
• presaturing the mobile phase to avoid removal of coated stationary phase,
• the impossibility of using gradient elution to solve the general elution problem
Solution
• Use of chemically bonded stationary phases. Bondedphase chromatography (BPC) now dominates in use all modes of HPLC.
• Microparticulate silica gel is the base material used for the synthesis of almost all chemically bonded phases.
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Bonded-Phase Chromatography
• The supports are prepared from rigid silica, or silica-based,
compositions.
• The surface of fully hydrolyzed silica (hydrolyzed by heating
with 0.1 M HCl for a day or two) is made up of chemically
reactive silanol groups.
The most useful bonded-phase coatings are siloxanes
formed by reaction of the hydrolyzed surface with an
organochlorosilane
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R : alkyl group or a substituted alkyl group.
• The unreacted SiOH groups, unfortunately, impart an undesirable polarity to the surface, which may lead to tailing of chromatographic peaks, particularly for basic solutes.
• To lessen this effect, siloxane packings are frequently capped by further reaction with chlorotrimethylsilane that, because of its smaller size, can bond many of the unreacted silanol groups.
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• The -Si-O-Si-C bond is stable under most conditions used in LC but is attacked by hydrolysis under basic conditions (pH > 7).
• Bonded phase stationary phase can be used with gradient elution. This is a major advantage of BPC.
• Two main techniques can be classified, based on the relative polarities of the stationary and mobile phases: (a) Normal phase BPC, and (b) reversed-phase BPC.
• Normal-phase BPC is used when the stationary phase (e.g., aminopropyl) is more polar (as evidenced by the predominant functional group) than the mobile phase (e.g., hexane).
• Reversed-phase BPC is used when the stationary phase is nonpolar (e.g., octadecylsilane) and the mobile phase is polar (e.g., water-methanol). Solute-elution order is often the reverse of that observed with normal-phase BPC. – The technique is ideally suited to substances insoluble or only
sparingly soluble in water but soluble in alcohols or other water-miscible organic solvents.
– Because many organic compounds show this solubility behavior, reversed-phase BPC is the most widely used mode of HPLC, accounting for about 60%of the published applications.
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Reversed-Phase and Normal-Phase Bonding
Phase Chromatography
Based upon the relative polarities of the mobile and stationary phases, two types of partition chromatography are distinguishable.
normal-phase BP chromatography Highly polar stationary phase such as water or
triethyleneglycol supported on silica or alumina particles; a relatively nonpolar solvent such as hexane serves as the mobile phase.
The least polar component is eluted first because in a relative sense, it is the most soluble in the mobile phase
Increasing the polarity of the mobile phase has the effect of decreasing the elution time.
• Normal-phase BPC can replace LSC on silica gel in many applications
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Reversed-phase chromatography
• The stationary phase is nonpolar, often a
hydrocarbon, and the mobile phase is relatively
polar (such as water, methanol, or acetonitrile).
• The most polar component appears first, and
increasing the mobile phase polarity increases
the elution time.
• Perhaps three quarters of all highperformance
liquid chromatography is currently being
carried out in columns with reversed-phase
packings.
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Reasons for the wide usage of RP-BPC
• Nonionic, ionic, and ionizable compounds can often be separated, sometimes at the same time, using a single column and mobile phase.
• Bonded-phase columns are relatively stable provided certain precautions, especially pH control, are taken.
• The predominant mobile phase, water, is inexpensive and plentiful.
• The most frequently used organic modifier, methanol, can be obtained at a reasonable price and of sufficient purity in most places in the world.
• The elution order is often predictable because retention time usually increases as the hydrophobic character of the solute increases.
• Columns equilibrate rapidly, thereby permitting faster method development and sample turnaround after gradient elution.
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Relationship between polarity and elution times for
normal phase and reversed phase chromatography
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Effect of chain length of the alkyl group of the bonded
phase upon performance
•
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• Longer chains produce packings that are more retentive. In addition, longer chain lengths permit the use of larger samples.
• In most applications of reversed-phase chromatography, elution is carried out with a highly polar mobile phase such as an aqueous solution containing various concentrations of such solvents as methanol, acetonitrile, or tetrahydrofuran.
• In this mode, care must be taken to avoid pH values greater than about 7.5 because hydrolysis of the siloxane takes place, which leads to degradation or destruction of the packing.
•
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Applications of Partition Chromatography
• Reversed-phase bonded packings, when
used in conjunction with highly polar
solvents (often aqueous), approach the ideal,
universal system for liquid chromatography.
• Because of their wide range of applicability,
their convenience, and the ease with which k'
and a can be altered by manipulation of
aqueous mobile phases, these packings are
frequently applied before all others.
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• Typical applications of bonded-phase chromatography. (a) Soft-drink additives. Column: 4.6 x 250 mm packed with polar (nitrile) bonded-phase packings. Isocratic solvent: 6% HOAC/94% HZO. Flow rate: 1.0 cm3/min. (b) Organophosphate insecticides. Column: 4.5 x 250 mm packed with 5-wm, C8, bonded-phase particles. Gradient: 67% CH30H/33% H20 to 80 CH3/20% H20. Flow rate 2 mL/min. Both used 254-nm UV detectors.
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Derivative Formation
• In some instances, the components of a
sample are converted to a derivative before, or sometimes after, chromatographic separation is undertaken for the following reasons:
• Reduce the polarity of the species so that partition rather than adsorption or ion-exchange columns can be used
• Increase the detector response and thus sensitivity, for all of the sample components
• Enhance the detector response to certain components of the sample.
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Use of derivatives to reduce polarity and enhance sensitivity
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Ion-Pair Chromatography
• Ion-pair (or paired-ion) chromatography is a type of reversed-phase partition chromatography that is used for the separation and determination of ionic species.
• The mobile phase in ion-pair chromatography consists of an aqueous buffer containing an organic solvent such as methanol or acetonitrile and an ionic compound containing a counter ion of opposite charge to the analyte.
• A counter ion is an ion that combines with the analyte ion to form an ion pair, which is a neutral species that is retained by a reversed-phase packing.
• Most of the counter ions contain alkyl groups to enhance retention of the resulting ion pair on the nonpolar stationary phase.
• Elution of the ion pairs is then accomplished with an aqueous solution of methanol or other water soluble organic solvent.
• Applications of ion-pair chromatography frequently overlap those of ion-exchange chromatography.
• An example of where the ion-pair method provides better separations is for analyzing mixtures of chlorate and nitrate ion. For this pair of solutes, selectivity with an ion-exchange packing is poor.
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ION-EXCHANGE CHROMATOGRAPHY • Ion-exchange chromatography (IC), which is often
shortened to ion chromatography refers to modern and efficient methods of separating and determining ions based upon ion-exchange resins.
• Ion chromatography was first developed in the mid-1970s when it was shown that anion or cation mixtures can be readily resolved on HPLC columns packed with anion-exchange or cation-exchange resins.
• At that time, detection was generally performed with conductivity measurements. Currently, other detectors are also available for ion chromatography.
• Ion chromatography was an outgrowth of ion-exchange chromatography,
•
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Ion-Exchange Equilibria
Ion-exchange processes are based upon exchange equilibria between ions in solution and ions of like sign on the surface of an essentially insoluble, high-molecular weight solid.
Natural ion-exchangers, such as clays and zeolites, have been recognized and used for several decades.
Synthetic ion-exchange resins were first produced in the mid-1930s for water softening, water deionization, and solution purification.
The most common active sites for cation-exchange resins are the sulfonic acid group -SO3
- H+, a strong acid, and the carboxylic acid group -COO- H+, a weak acid.
Anionic exchangers contain tertiary amine groups
-N(CH3)3OH- or primary amine groups -NH3+OH-;
• the former is a strong base and the latter a weak one.
When a sulfonic acid ion-exchanger is brought in contact with an aqueous solvent containing a cation Mx+ :
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Similarly a strong base exchanger interacts with the
anion Ax- as shown by the reaction
Cation exchanger
Anion exchanger
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Influences on Distribution Coefficients and Selectivity
• Ion-exchange chromatography involves more
variables than other forms of chromatography.
• Distribution coefficients and selectivities are functions of:
pH,
solute charge and radius,
resin porosity,
ionic strength and type of buffer,
type of solvent,
temperature, and so forth.
• The number of experimental variables makes ion-exchange chromatography a very versatile technique, since each may be used to effect a better separation, but a difficult one because of the time needed to optimize a separation.
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By selecting a common reference ion such as H+, distribution ratios for different ions on a given type of resin can be experimentally compared.
Such experiments reveal that polyvalent ions are much more strongly held than singly charged species. Within a given charge group, however, differences appear that are related to the size of the hydrated ion as well as to other properties.
Thus, for a typical sulfonated cationexchange resin, values for Kex decrease in the order
• For anions, Kex for a strong base resin decreases in the order
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Ion Chromatography
• Ion chromatography (IC) is an ion-exchange technique that uses, most popularly, a low-capacity column combined with a conductivity detector
• Its most frequent practical application is the determination of trace anions in aqueous solution.
• The low-capacity column allows the use of a buffer with a low ionic strength.
• There are two forms of IC practiced today:
(a) suppressed or dual-column IC,
(b) nonsuppressed or single column IC.
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Ion Chromatography with Eluent
Suppressor Columns
The widespread application of ion chromatography for the determination of inorganic species was inhibited by the lack of a good general detector.
Conductivity detectors are an obvious choice for this task.
They can be highly sensitive, they are universal for charged species, and, as a general rule, they respond in a predictable way to concentration changes.
The only limitation arises from the high electrolyte concentration required to elute most analyte ions in a reasonable time.
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As a consequence, the conductivity from the mobile-phase components tends to swamp that from analyte ions, thus greatly reducing the detector sensitivity.
The problem of high eluent conductance was solved by the introduction of a so-called eluent suppressor column immediately following the analytical ion-exchange column.
The suppressor column is packed with second
ion-exchange resin that effectively converts the ions of the solvent to a molecular species of limited
ionization without affecting the analyte ions.
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For example, when cations are being separated and determined, hydrochloric acid is often chosen as the eluting reagent, and the suppressor column is an anion-exchange resin in the hydroxide form. The product of the reaction in the suppressor is water. That is,
H+(aq) + Cl-(aq) + Resin+OH-(s) ---> Resin+Cl-(s) + H2O
The analyte cations are, of course, not retained by this second column.
For anion separations, the suppressor packing is the acid form of a cation-exchange resin. Sodium bicarbonate or carbonate may serve as the eluting agent. The reaction in the suppressor is then
Na+(aq) + HCO 3- (aq) + Resin-H+(s) -> Resin-Na+(s) + H2CO3(aq)
Here, the largely undissociated carbonic acid does not contribute significantly to the conductivity.
An inconvenience associated with the original suppressor columns was the need to regenerate them periodically (typically, every 8 to 10 hr) in order to convert their packings back to the original acid or base form.
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Single-Column Ion Chrornatography
Equipment has also become available commercially for ion chromatography in which no suppressor column is used.
This approach depends upon the small differences in conductivity between the eluted sample ions and the prevailing eluent ions.
To amplify these differences, low-capacity exchangers are used, which make possible elution with species having low equivalent conductances.
Single-column chromatography tends to be somewhat less sensitive and to have a more limited range than ion chromatography with a suppressor column.
An indirect photometric method that permits the separation and detection of nonabsorbing anions and cations without a suppressor column has recently been described.
Here also, no suppressor column is used, but instead, anions or cations that absorb Uv or Vis radiation are used to displace the analyte ions from the column.
When the analyte ions are displaced from the exchanger, their place is taken by an equal number of eluent ions (provided, of course, that the charge on the analyte and eluent ions is the same).
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Size-exclusion Chromatography
Gel-permeation or gel-filtration chromatography
applicable to high molecular weight species
Packing is small silica or polymer particles containing a network of uniform pores into which solute and solvent molecules can diffuse.
The average residence time of analyte molecules in the pores depends upon the effective size of these molecules
Molecules that are larger than the average pore size will not be retained
Molecules with sizes smaller than those of the pores will be retained
Intermediate size molecules will penetrate according to their sizes. Thus fractionation occurs.
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• The process is almost always carried out in a column, but it also has been performed on a thin layer.
• Column packing materials with pores of different (controlled) sizes are generally used.
• The materials can be soft gels, semirigid gels, or rigid materials.
• The soft and semirigid gels can change their pore sizes, depending on the solvent used as a mobile phase.
• The soft gels, of the polydextran or agarose type, can swell to many times their dry volume, whereas the semirigid gels of the polyvinylacetate or polystyrene type swell to 1.1 to 1.8 times their dry volume. Rigid materials, such as porous glass or porous silica beads, have fixed pore sizes and do not swell at all.
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Theory of Size-Exclusion Chromatography
• The total volume Vt of a column packed with a porous polymer or silica gel is given by
Vt = Vg + Vi + Vo
Vg is the volume occupied by the solid matrix of the gel,
Vi is the volume of solvent held in its pores,
Vo is the free volume outside the gel particles.
Assuming no mixing or diffusion, Vo also represents the theoretical volume of solvent required to transport through the column those components too large to enter the pores of the gel.
In fact, however, some mixing and diffusion will occur, and as a consequence the nonretained components will appear in a Gaussian-shaped band with a concentration maximum at Vo.
For components small enough to enter freely into the pores of the gel, band maxima will appear at the end of the column at an eluent volume corresponding to (Vi + Vo).
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Molecules of intermediate size are able to transfer into some fraction K of the solvent held in the pores; the elution volume Ve for these retained molecules is
Ve = Vo + KCVi (1)
• Equation 1 applies to all of the solutes on the column.
For molecules too large to enter the gel pores, KC = 0 and Ve = Vo; for molecules that can enter the pores unhindered, KC = 1 and Ve = (Vo + Vi).
In deriving Eq. 1, the assumption was made that no interaction, such as adsorption, occurs between the solute molecules and the gel surfaces. With adsorption, the amount of interstitially held solute will increase; with small molecules, KC will then be greater than unity.
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Eq. 1 rearranges to
• KC = (Ve - Vo)/Vi = CslCm (2)
where KC is the distribution constant
for the solute. Values of KC range from
zero for totally excluded large
molecules to unity for small molecules.
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The useful molecular weight range for a sizeexclusion packing is conveniently illustrated by means of a calibration curve such as that shown in the upper part of the Figure.
Molecular weight, which is directly related to the size of solute molecules, is plotted against retention volume VR. Note that the ordinate scale is logarithmic.
The exclusion limit defines the molecular weight of a species beyond which no retention occurs. All species having greater molecular weight than the exclusion limit are so large that they are not retained and elute together to give peak A in the chromatogram shown.
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The permeation limit is the molecular weight below which the solute molecules can penetrate into the pores completely.
All molecules below this molecular weight are so small that they elute as the single band labeled D.
As molecular weights decrease from the exclusion limit, solute molecules spend more and more time, on the average, in the particle pores and thus move progressively more slowly.
It is in the selective permeation region that fractionation occurs, yielding individual solute peaks such as B and C in the chromatogram.
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Vo Vi
No retention
beyond this MW
Unretained
large
molecules
Limit below which solute
molecules can penetrate
completely into the pores
Permeation limit
Molecules below this
MW are so small that
they elute as the single
band D
A B
C D
Such calibration curves are supplied by manufacturers
of packing materials
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