Chapter 32

Dong-Sun Lee/ CAT-Lab / SWU 2012-Fall version

High Performance Liquid Chromatography

CSI: Crime Scene Investigation - Dark Motives (2004)

Catherine Willow is a single parent with a young daughter, Catherine’s a street-smart Las Vegas native, a veteran criminalist who juggles the job she loves with her responsibilities at home. The horrors of the crime scenes Catherine investigates bring her fears for her child into sharper focus, making her even more determined to shield her little girl from the seamier, shadowy side of Vegas that is revealed after the sun goes down.Catherine Willows is played by Marg Helgenberger.

HPLC was built by C. Harvath in 1965 at Yale University

High-Performance Liquid Chromatography (HPLC)

- HPLC is a type of chromatography that employs a liquid mobile phase and a very finely divided stationary phase. To obtain satisfactory flow rates, the liquid must be pressurized to several hundred or more pounds per square inch.

- HPLC is a process that was developed in the late 1960's. - Major technical advances that developed at that time included... 1. Solid packing particles were reduced in size from 150-200 micrometers to 5-10 micrometers.

2. Smaller particles increased the resolution of separation, allowing smaller, shorter columns.

Liquid Chromatography was first discovered in 1903 by M.S.Tswett, who used a chalk column to separate the pigments of green leaves. Only in 1960's the more and more emphasis was placed on the development of liquid chromatography.

Effect of particle size of packing and flow rate on plate height in liquid chromatography.

G SC G LC

G AS S FC

NP RP IE C

G PC G FC

SEC

Colum n

TLC P aper

P lan ar

L IQ U ID

CHROMATOG RAPHY

Classification of chromatography

COMPARISON OF CHROMATOGRAPHIES

TYPE STATIONARY PHASE MOBILE PHASE MECHANISM

Solvent Extraction Organic Aqueous Partition

K = [A]o/[A]a

Ion Exchange Resin Aqueous Partition

e.g., water softener: K = [Ca+2]resin/[Ca+2]aq

Paper/TLC Paper, Silica Gel

Alumina

Aqueous or Organic Adsorption

Column Silica Gel, Alumina Aqueous or

Organic

Adsorption

GLC Liquid: Substrate Gas Partition

GSC Alumina, Silica Gel Gas Adsorption

EC Molecular Sieves Gas Size

TYPE STATIONARY PHASE MOBILE PHASE MECHANISM

HPLC Liquid:Substrate Liquid Partition

HPLC Silica, Alumina Liquid Adsorption

HPLC Gel Liquid Size

HPLC as compared with the classical technique is characterized by:

• small diameter (2-5 mm), reusable stainless steel columns;

• column packings with very small (3, 5 and 10 mm) particles and the continual development of new substances to be used as stationary phases;

• relatively high inlet pressures and controlled flow of the mobile phase;

• precise sample introduction without the need for large samples;

• special continuous flow detectors capable of handling small flow rates and detecting very small amounts;

• automated standardized instruments;

• rapid analysis; and

• high resolution.

Types of HPLC

There are many ways to classify liquid column chromatography. If this classification is based on the nature of the stationary phase and the separation process, three modes can be specified.

In adsorption chromatography the stationary phase is an adsorbent (like silica gel or any other silica based packings) and the separation is based on repeated adsorption-desorption steps.

In ion-exchange chromatography the stationary bed has an ionically charged surface of opposite charge to the sample ions. This technique is used almost exclusively with ionic or ionizable samples. The stronger the charge on the sample, the stronger it will be attracted to the ionic surface and thus, the longer it will take to elute. The mobile phase is an aqueous buffer, where both pH and ionic strength are used to control elution time.

In size exclusion chromatography the column is filled with material having precisely controlled pore sizes, and the sample is simply screened or filtered according to its solvated molecular size. Larger molecules are rapidly washed through the column; smaller molecules penetrate inside the porous of the packing particles and elute later. Mainly for historical reasons, this technique is also called gel filtration or gel permeation chromatography although, today, the stationary phase is not restricted to a "gel".

Concerning the first type, two modes are defined depending on the relative polarity of the two phases: normal and reversed-phase chromatography.

• In normal phase chromatography, the stationary bed is strongly polar in nature (e.g., silica gel), and the mobile phase is nonpolar (such as n-hexane or tetrahydrofuran). Polar samples are thus retained on the polar surface of the column packing longer than less polar materials.

• Reversed-phase chromatography is the inverse of this. The stationary bed is nonpolar (hydrophobic) in nature, while the mobile phase is a polar liquid, such as mixtures of water and methanol or acetonitrile. Here the more nonpolar the material is, the longer it will be retained.

Above mentioned types cover almost 90% of all chromatographic applications. Eluent polarity plays the highest role in all types of HPLC.

There are two elution types: isocratic and gradient. In the first type constant eluent composition is pumped through the column during the whole analysis. In the second type, eluent composition (and strength) is steadily changed during the run.

Application of liquid chromatography

Instrumentation

Components of High Performance Liquid Chromatography

Liquid phase samples (mixtures) are injected onto an LC column usually using a syringe and specially devised injection valve. The sample is swept onto the chromatographic column by the flowing mobile phase and chromatographic separation occurs as the mixture travels down the column. Normal HPLC detectors detect the elution of a compound from the end of the column based on some physical characteristic such as ultraviolet light absorption, ability to fluoresce, or the difference in index of refraction between the analyte and the mobile phase itself. The majority of HPLC systems work this way.

An example schematic of an HPLC system is shown below:

Mobile phase

ReservoirPump Injector Pre-Column Column Detector

Data

Typical apparatus for HPLC

Mobile phase reservoir, filtering

The most common type of solvent reservoir is a glass bottle. Most of the manufacturers supply these bottles with the special caps, Teflon tubing and filters to connect to the pump inlet and to the purge gas (helium) used to remove dissolved air. Sparging is a process in which dissolved gases are swept out of a solvent by bubbles of an inert, insoluble gas. Helium purging and storage of the solvent under helium was found not to be sufficient for degassing of aqueous solvents. It is useful to apply a vacuum for 5-10 min. and then keep the solvent under a helium atmosphere.

Mobile phase of the HPLC components: One or more glass, plastic or stainless steel solvent reservoirs to hold mobile phase. a. Mobile phase is usually degassed via a sparger; a vacuum system, heating/stirring device may also be used.

b. Mobile phase solutions are usually pre-filtered before use to remove dust and particulates.

c. Elution may be ... 1) Isocratic - a constant composition mobile phase.

2) Gradient elution - two (or more) solvents whose mix ratio is changed in a programmed way. Solvents of different polarity and usually mixed to generate a gradient of changing polarity in the column.

As the solute capacity, or k', for the compound decreases, the compound begins to migrate through the stationary phase. Each of the other compounds in the sample subsequently migrate as their k' values decrease. Compared with isocratic elution, resolution and separation are improved, and bandwidths are nearly equal: 

Isocratic and Gradient Elution

• Isocratic elution

– a single solvent (or constant mixture)

• Gradient elution – continuous change of solvent composition to increase eluent strength.

Mobile phase selection

Solvent strength (o ) is a quantitative representation of solvent strength.

For non-electrolytic solvents, solvent strength is synonymous with “polarity” in normal phase LC. Strong or polar solvents are characterized by their ability to dissolve polar samples and to elute solutes with low capacity factors(k’) in normal phase LC. In reversed phase LC there is an inverse relationship between solvent strength and chromatographic elution power.

For example, water is a strong solvent but has little elution power in RP-LC and must, therefore, be mixed with a less polar organic midifier to elute a solute with a low k’ value.

The eluotropic series gives the chromatographer a method of influencing the k’ values of sample components.

Decreasing the solvent strength, k’ values may be increased. To increase chromatographic resolution per unit time during the course of a chromatographic run, the solvent strength can be increased in a stepwise or continuous manner causing k’ values to change. This is known as solvent programming.

Solvent UV cut-off

(nm)

Refractive Index

Boiling Point

(C)

Viscosity

(cP, 25 C)

Solvent Polarity Parameter(P’)

Solvent Strength Parameter()

Selectivity

Group

Isooctane 197 1.389 99 0.47 0.1 0.01 -

N-Hexane 190 1.372 69 0.30 0.1 0.01 -

Methyl t-butyl ether

210 1.369 56 0.27 2.5 0.35 Ⅰ

Benzene 278 1.501 81 0.65 2.7 0.32 Ⅶ

Methylene chloride

233 1.421 40 0.41 3.1 0.42 Ⅴ

n-Propanol 240 1.385 97 1.9 4.0 0.82 Ⅱ

Tetrahydrofuran 212 1.405 66 0.46 4.0 0.82 Ⅱ

Ethyl acetate 256 1.370 77 0.43 4.4 0.58 Ⅵa

Chloroform 245 1.443 61 0.53 4.1 0.40 Ⅷ

Dioxane 215 1.420 101 1.2 4.8 0.56 Ⅵa

Acetone 330 1.356 56 0.3 5.1 0.56 Ⅵa

Ethanol 210 1.359 78 1.08 4.3 0.88 Ⅱ

Acetic acid 1.370 118 1.1 6.0 Large Ⅳ

Acetonitrile 190 1.341 82 0.34 5.8 0.65 Ⅵa

Methanol 205 1.326 65 0.54 5.1 0.95 Ⅱ

Water 1.333 100 0.89 10.2 very large Ⅷ

Properties of some common solvents used in HPLC.

Solvent characteristics of mixed solvent.

Solvent Selectivity Solvent strength weighting factor( S i)

Group Reverse Phase* Normal Phase

Methanol II 2.6 5.1

Acetonitrile VI 3.2 5.8

Tetrahydrofuran III 4.5 4.0

Water --- 0 10.2

Chloroform VIII 4.1

Methylene chloride V 3.1

Methyl t-butylether I ca.2.5

Ethyl ether I 2.8

Hexane --- 0

* Approximate value for some other common solvents are acetone(3.4), dioxane(3.5), ethanol(3.6), and isopropanol(4.2).

ST = i Siqi

ST = total solvent strength of the mixture

Si = solvent strength weighting factor

qi= volume fraction of solvent in the mixture

Example) methanol/water ( 60 : 40 v/v %)

ST = i Siqi = SCH3OHqCH3OH + SH2OqH2O

ST = (2.6)(0.6) + (0)(0.4) = 1.56

iso-eluotropic solvents

Binary system

acetonitrile/water

1.56 = (3.2)(qCH3CN) (0)(qH2O)

(qCH3CN) = 0.49 = 49 %

Ternary system

methanol / tetrahydrofuran / water (35:10:55)

Stationary Phase

The stationary phase in HPLC refers to the solid support contained within the column over which the mobile phase continuously flows. The sample solution is injected into the mobile phase of the assay through the injector port. As the sample solution flows with the mobile phase through the stationary phase, the components of that solution will migrate according to the non-covalent interactions of the compounds with the stationary phase. The chemical interactions of the stationary phase and the sample with the mobile phase, determines the degree of migration and separation of the components contained in the sample. For example, those samples which have stronger interactions with the stationary phase than with the mobile phase will elute from the column less quickly, and thus have a longer retention time, while the reverse is also true. Columns containing various types of stationary phases are commercially available. Some of the more common stationary phases include: Liquid-Liquid, Liquid-Solid (Adsorption), Size Exclusion, Normal Phase, Reverse Phase, Ion Exchange, and Affinity.

Liquid-Solid operates on the basis of polarity. Compounds that possess functional groups cabable of strong hydrogen bonding will adhere more tightly to the stationary phase than less polar compoounds. Thus, less polar compounds will elute from the column faster than compounds that are highly polar.

Liquid-Liquid operates on the same basis as liquid-solid. However, this technique is better suited for samples of medium polarity that are soluble in weakly polar to polar organic solvents. The separation of non-electrolytes is achieved by matching the polarities of the sample and the stationary phase and using a mobile phase which possesses a markedly different polarity.

Size-Exclusion operates on the basis of the molecular size of compounds being analyzed. The stationary phase consists of porous beads. The larger compounds will be excluded from the interior of the bead and thus will elute first. The smaller compounds will be allowed to enter the beads and will elute according to their ability to exit from the same sized pores they were internalized through. The column can be either silica or non-silica based. However, there are some size-exclusion that are weakly anionic and slightly hydrophobic which give rise to non-ideal size-exclusion behavior.

Normal Phase operates on the basis of hydrophilicity and lipophilicity by using a polar stationary phase and a less polar mobile phase. Thus hydrophobic compounds elute more quickly than do hydrophilic compounds.

Reverse Phase operates on the basis of hydrophilicity and lipophilicity. The stationary phase consists of silica based packings with n-alkyl chains covalently bound. For example, C-8 signifies an octyl chain and C-18 an octadecyl ligand in the matrix. The more hydrophobic the matrix on each ligand, the greater is the tendancy of the column to retain hydrophobic moieties. Thus hydrophilic compounds elute more quickly than do hydrophobic compounds.

Normal-phase chromatography

Reversed-phase chromatography

Polar stationary phase nonpolar stationary phase

More polar solvent has higher eulent strength

Less polar solvent has higher eulent strength

Ion-Exchange operates on the basis of selective exchange of ions in the sample with counterions in the stationary phase. IE is performed with columns containing charge-bearing functional groups attached to a polymer matrix. The functional ions are permanently bonded to the column and each has a counterion attached. The sample is retained by replacing the counterions of the stationary phase with its own ions. The sample is eluted from the column by changing the properties of the mobile phase do that the mobile phase will now displace the sample ions from the stationary phase, (ie. changing the pH).

Affinity operates by using immobilized biochemicals that have a specific affinity to the compound of interest. Separation occurs as the mobile phase and sample pass over the stationary phase. The sample compound or compounds of interest are retained as the rest of the impurities and mobile phase pass through. The compounds are then eluted by changing the mobile phase conditions.

High performance ion-exchange chromatography

1. The column is packed with ... a. A polystyrene ion-exchange resin of beaded particles <10 micrometers available

b. A silica-based ion-exchange resin (particles).

The R groups would be coupled through the silane chemistry described previously.

2. Samples are run through the columns packed with these particles and ion exchange would occur. For example,...

a. In the top diagram above, the resin would be exhaustively washed with acid to "load" the cation exchange sites (sulfonic acid groups in the example above) with protons (and elute any bound cations). b. In the lower diagram, a sodium-containing sample is then loaded on the column. The sodium competes and displaces the protons and binds to the resin. The protons are then eluted. c. The amount of sodium bound can be determined by titrated the eluted acid. d. The law of mass action applies in all cases: high concentrations of one species shift equilibrium, favoring binding of the higher concentration species and elution of the other.

3. Ion-exchange is also commonly performed in open columns (without the pumps and automated controls of the HPLC).

a. Polystyrene-type resins may be used, such as... 1) DOWEX 50 (a strong, cation exchanger with a sulfonic acid functional group). 2) IRC-150 (a weak, cation exchanger with a carboxylic acid functional group). 3) DOWEX 1 (a strong, anion exchanger with a quaternary amine functional group). 4) IR-45 (a weak anion exchanger with a methylammonium functional group).b. Polysaccharide-type resins may be used, such as... 1) DEAE-cellulose and DEAE-Sephadex (weak anion exchangers with the diethylaminoethyl functional group). Sephadex is a trade name of a beaded, cross-linked dextran bead. 2) CM-cellulose and CM-Sephadex (weak cation exchangers with the carboxymethyl functional group). 3) Phospho-cellulose and phospho-Sephadex (strong cation exchangers with a phosphoryl group).c. Polysaccharide-type resins are commonly used in protein applications with open columns, but can also be used in HPLC work. However, polysaccharides cannot stand up to high pressures. (The HPLC must be run at pressures less than ~150 pounds per square inch).

Size-Exclusion Chromatography

A. This is the newest HPLC technique. B. It is also called gel permeation, gel filtration, exclusion, molecular sieving and steric exclusion chromatography. C. In this technique, columns are packed with porous particles with different pore diameters. The particles usually are made of ... 1. Polystyrene that is cross-linked with divinylbenzene (semirigid) to withstand the high pressures. a. Organic solvents are commonly used (e.g., acetone, tetrahydrofuran, etc.) since water will not wet polystyrene. b. Slow flow rates are common.

2. Glass or silica. a. Silica allows high flow rates and high pressures. b. The pH must be kept below ~7.5 or the silica will dissolve.

3. Soft gels (cross-linked dextrans, polysaccharides or similar polymers). a. This would include... 1) Sephadex (dextran) 2) Sepharose (agarose) 3) BioGel P (polyacrylamide) b. Pressures must be kept low (<150 p.s.i.) to prevent crushing the gels. c. These are most useful for water-soluble species of molecular weights between 100 - 25,000,000 grams/mole (e.g., proteins, enzymes, etc.)

Counter ion and ion pair chromatography

Q+ + X- = Q Xaq = QXorg

( mobile phase ) ( stationary phase)

k' = ([QX]org / [X]aq) (VS / VM)

= (VS / VM) E [Q+]aq

Counter ion ( ca 0.1 w/w % )

Acidic(cathionic) sample : tetraethyl ammonium salt,

tetrabutyl ammonium phosphate

Basic (anionic) sample : octyl sulfonate, perchloric acid

Chromatograms illustrating separations of mixtures of ionic and nonionic compounds by ion pair chromatography

Hydrophobic interaction chromatography

Two stationary phases or hydrophobic interaction chromatography.

SOLVENT DELIVERY SYSTEM TO PROVIDE PULSE-FREE FLOWS

The requirements for an analytical HPLC pump include

(i) pulse-free flows,

(ii) flow rates ranging from 0.1-10 mL/min,

(iii) accurate flow control with good reproducibility,

(iv) generation of high pressure (up to 6000 psi), and

(v) corrosion- and solvent- resistant components.

Pumping system.

a. The pumping system must... 1) Generate pressures up to ~6,000 pounds per square inch. 2) Give a pulse-free output (via a pulse damper).

3) Give flow rates of ~0.1 - 10 mL/minute.

4) Give flow reproducibility of 0.5% or better.

5) Resist corrosion by a variety of solvents.

b. Note that explosion due to high pressure is not a concern. Liquids do not compress like gases.

c. Fire from a solvent leak is more likely.

d. Priming syringes are usually used to purge the pumps and pump lines of trapped gas bubbles.

HPLC Pumps

There are several types of pumps available for use with HPLC analysis, they are: Reciprocating Piston Pumps, Syringe Type Pumps, and Constant Pressure Pumps.

Reciprocating Piston Pumps consist of a small motor driven piston which moves rapidly back and forth in a hydraulic chamber that may vary from 35-400 µL in volume. On the back stroke, the separation column valve is closed, and the piston pulls in solvent from the mobile phase reservoir. On the forward stroke, the pump pushes solvent out to the column from the reservoir. A wide range of flow rates can be attained by altering the piston stroke volume during each cycle, or by altering the stroke frequency. Dual and triple head pumps consist of identical piston-chamber units which operate at 180 or 120 degrees out of phase. This type of pump system is significantly smoother because one pump is filling while the other is in the delivery cycle.

Syringe Type Pumps are most suitable for small bore columns because this pump delivers only a finite volume of mobile phase before it has to be refilled. These pumps have a volume between 250 to 500 mL. The pump operates by a motorized lead screw that delivers mobile phase to the column at a constant rate. The rate of solvent delivery is controlled by changing the voltage on the motor.

In Constant Pressure Pumps the mobile phase is driven through the column with the use of pressure from a gas cylinder. A low-pressure gas source is needed to generate high liquid pressures. The valving arrangement allows the rapid refill of the solvent chamber whose capacity is about 70 mL. This provides continuous mobile phase flow rates.

High-pressure piston pump for HPLC. Solvent at the left passes through an electronic valve synchronized with the large piston and designed to minimize the formation of solvent vapor bubbles during the intake stroke. The spring-loaded outlet valve maintains a constant outlet pressure, and the damper further reduces pressure surge. Pressure surge from the first piston are decreased in the damper that “breathes” against a constant outside pressure, Pressure surge are typically <1% of the operating pressure. As the large piston draws in liquid, the small piston propels liquid to the column. During the return stroke of the small piston, the large piston delivers solvent into the expanding chamber of the small piston. Part of the solvent fills the chamber, while the remainder flows to the column. Delivery rate is controlled by the stroke volumes.[courtesy Hewlett-Packard Co., Palo Alto, CA.]

Solvent delivery system

JASCO LC-1580 Pump and UV-1570/1575 UV/Visible Detector

http://www.jasco.co.jp/English/main/main.html

http://www.jasco.co.uk/lcpumps.htm

Two methods for generating binary solvent gradients.

SAMPLE INJECTOR

A manual sample injector that is typically used comprises of a 6-port 2-position valve that includes a 10 or 500 µl fixed sample loop. In one configuration, the flow from the pump is sent directly into the column; when the position is switched the flow from the pump is diverted via the sample loop into the column, thereby performing a sample injection. Valves with electrically or pneumatically actuated position switches are also commercially available.

Automated sample injectors (autosamplers) that can store and sequentially inject multiple samples are popular in quality control applications and for high-throughput screening.

Injection valve for HPLC. Replaceable sample loop comes in various fixed-volume sizes.

Rheodyne manual sample injector and sample loops (ss or PEEK)

Scale Sample Volume

Micro 2 µL to 500 µL

Analytical 1.0 µL to 5.0 mL

Preparative 100 µL to 10 mL

http://www.rheodyne.com/sampinj.html

Several different needle point styles are offered on Hamilton syringe and needle products depending upon the application.

Point Style 3: Blunt needle point for use with HPLC injection valves and for sample pipetting.

Point Style 4: 10°-12° beveled needle point recommended for life science applications

http://www.hamiltoncomp.com/product/syringe/point.html

HPLC Sample Injection Loop 

The injection loop is a critical component of an HPLC and is potentially one of the largest sources of excess HPLC system volume.  The sample is injected into the loop while the loop is switched out of the HPLC flow path.  After the loop is filled with sample it is switched back into the flow path and the sample is swept onto the head of the HPLC column for later elution or the sample is injected directly into a mass spectrometer as part of a flow injection analysis.

PEEK™  Loop

Note:PEEK™ (polyetheretherketone) is a registered trade mark of Victrex plc.

Columns

There are various columns that are secondary to the separating column or stationary phase. They are: Guard, Derivatizing, Capillary, Fast, and Preparatory Columns.

Guard Columns are placed anterior to the separating column. This serves as a protective factor that prolongs the life and usefulness of the separation column. They are dependable columns designed to filter or remove: 1) particles that clog the separation column; 2) compounds and ions that could ultimately cause "baseline drift", decreased resolution, decreased sensitivity, and create false peaks; 3) compounds that may cause precipitation upon contact with the stationary or mobile phase; and 4) compounds that might co-elute and cause extraneous peaks and interfere with detection and/or quantification. These columns must be changed on a regular basis in order to optimize their protective function. Size of the packing varies with the type of protection needed.

Derivatizing Columns- Pre- or post-primary column derivatization can be an important aspect of the sample analysis. Reducing or altering the parent compound to a chemically related daughter molecule or fragment elicits potentially tangible data which may complement other results or prior analysis. In few cases, the derivatization step can serve to cause data to become questionable, which is one reason why HPLC was advantageous over gas chromatography. Because GC requires volatile, thermally stabile, or nonpolar analytes, derivatization was usually required for those samples which did not contain these properties. Acetylation, silylation, or concentrated acid hydrolysis are a few derivatization techniques.

Capillary Columns- Advances in HPLC led to smaller analytical columns. Also known as microcolumns, capillary columns have a diameter much less than a millimeter and there are three types: open-tubular, partially packed, and tightly packed. They allow the user to work with nanoliter sample volumes, decreased flow rate, and decreased solvent volume usage which may lead to cost effectiveness. However, most conditions and instrumentation must be miniaturized, flow rate can be difficult to reproduce, gradient elution is not as efficient, and care must be taken when loading minute sample volumes. 

Microbore and small-bore columns are also used for analytical and small volumes assays. A typical diameter for a small-bore column is 1-2 mm. Like capillary columns, instruments must usually be modified to accommodate these smaller capacity columns (i.e., decreased flow rate). However, besides the advantage of smaller sample and mobile phase volume, there is a noted increase in mass sensitivity without significant loss in resolution. ---Capillary Electrophoresis

Fast Columns- One of the primary reasons for using these columns is to obtain improved sample throughput (amount of compound per unit time). For many columns, increasing the flow or migration rate through the stationary phase will adversely affect the resolution and separation. Therefore, fast columns are designed to decrease time of the chromatographic analysis without forsaking significant deviations in results. These columns have the same internal diameter but much shorter length than most other columns, and they are packed with smaller particles that are typically 3 µm in diameter. Advantages include increased sensitivity, decreased analysis time, decreased mobile phase usage, and increased reproducibility. 

Preparatory Columns- These columns are utilized when the objective is to prepare bulk (milligrams) of sample for laboratory preparatory applications. A preparatory column usually has a large column diameter which is designed to facilitate large volume injections into the HPLC system. Accessories important to mention are the back-pressure regulator and the fraction collector. The back-pressure regulator is placed immediately posterior to the HPLC detector. It is designed to apply constant pressure to the detector outlet which prevents the formation of air bubbles within the system. This, in turn, improves chromatographic baseline stability. It is usually devised to operate regardless of flow rate, mobile phase, or viscosity.The fraction collector is an automated device that collects uniform increments of the HPLC output. Vials are placed in the carousel and the user programs the time interval in which the machine is to collect each fraction. Each vial contains mobile phase and sample fractions at the corresponding time of elution. Packings for columns are diverse since there are many modes of HPLC. They are available in different sizes, diameters, pore sizes, or they can have special materials attached (such as an antigen or antibody for immunoaffinity chromatography). Packings available range from those needed for specific applications (affinity, immunoaffinity, chiral, biological, etc.) to those for all-purpose applications. The packings are attached to the internal column hull by resins or supports, which include oxides, polymers, carbon, hydroxyapatite beads, agarose, or silica, the most common type(Brown, 1990).

Columns

a. Columns are usually stainless steel tubes. b. Lengths are typically 10 - 30 centimeters.

c. Inside diameters of 4 - 40 mm are typical; newer high-performance columns are 1 - 4.6 mm.

d. Particulate packing size is typically 5 - 10 micrometers.

HPLC column with replaceable guard column to collect irreversibly adsorbed impurities. Titanium frits distribute the liquid evenly over the diameter of the column.

Stationary Phases (Adsorbents)

HPLC separations are based on the surface interactions, and depends on the types of the adsorption sites (surface chemistry). Modern HPLC adsorbents are the small rigid porous particles with high surface area.

Main adsorbent parameters are:

• Particle size: 3 to 10 m

• Particle size distribution: as narrow as possible, usually within 10% of the mean;

• Pore size: 70 to 300 m

• Surface area: 50 to 250 m2/g

• Bonding phase density (number of adsorption sites per surface unit): 1 to 5 per 1 nm2

The last parameter in the list represents an adsorbent surface chemistry. Depending on the type of the ligand attached to the surface, the adsorbent could be normal phase (-OH, -NH2), or reversed-phase (C8, C18, Phenyl), and even

anion (NH4+), or cation (-COO-) exchangers.

Plate height as a function of flow rate for stationary-phase particle sizes of 10, 5, and 3m.

Scanning electron micrographs of silica chromatography particles.

(a) Aggregate of spherical particles (50% porosity, 150 m2/g surface area)

(b) Spongelike structure (70% porosity, 300m2/g surface area)

Schematic structure of sillica gel.

The Stationary Phase

Most common support –highly pure, spherical, microporous particles of permeable silica, several hundred m2/g

Bonded stationary phase

The octadecyl (C18) stationary phase is by far the most common in HPLC. It is frequently designated ODS, for octadecylsilane

Common polar phases Common nonpolar phases

R = (CH2)3NH2 amino

R = (CH2)17CH3 octadecyl

R = (CH2)3C≡N cyano R = (CH2)7CH3 octyl

R = (CH2)2OCH2(OH)CH2OH diol R = (CH2)3C6H5 phenyl

Silica surface dimethyl silane R= C8, C18, ……

Bulky isobutyl groups protect siloxane bonds from hydrolysis at low pH. Siloxane (Si-O-SiR) bond hydrolyze below pH2 PH 2-8

Molecular model of octadecyl siloxane

DETECTORS

Today, optical detectors are used most frequently in liquid chromatographic systems. These detectors pass a beam of light through the flowing column effluent as it passes through a low volume ( ~ 10 ml) flowcell. The variations in light intensity caused by UV absorption, fluorescence emission, or change in refractive index (depending on the type of detector used) from the sample components passing through the cell, are monitored as changes in the output voltage. These voltage changes are recorded on a strip chart recorder and frequently are fed into an integrator or computer to provide retention time and peak area data.

The most commonly used detector in LC is the ultraviolet absorption detector. A variable wavelength detector of this type, capable of monitoring from 190 to 460-600 nm, will be found suitable for the detection of the majority samples.

Other detectors in common use include: refractive index (RI), fluorescence (FL), electrochemical (EC) and mass-spectrometric (MS). The RI detector is universal but also the less sensitive one. FL and EC detectors are quite sensitive (up to 10-15 mole) but also quite selective. The MS detector is the most powerful one but it still the most complicated and most expensive.

UV/Vis Absorbance Detectors:

Most modern UV/Vis detectors consist of a scanning spectrophotometer with grating optics. The independent or combined use of a Deuterium source (UV range, 190-360 nm) with a Tungsten source (visible range, 360-800 nm) provides a simple means of detecting absorbing species as they emerge from the column.

Several operational modes can be chosen –

1. the entire chromatogram may be obtained at a selected wavelength; or,

2. when the eluted peaks are suitably separated, different wavelengths may be selected for observing each peak

3. simultaneous dual wavelength detection or the ratio of absorbances at two different wavelengths may be used for more accurate determinations

Ultra-Violet (UV) detectors measure the ability of a sample to absorb light. This can be accomplished at one or several wavelengths:  

A) Fixed Wavelength measures at one wavelength, usually 254 nm

B) Variable Wavelength measures at one wavelength at a time, but can detect over a wide range of wavelenths

C) Diode Array measures a spectrum of wavelengths simulateneously

UV detectors have a sensitivity to approximately 10-8 or 10 -9 gm/ml.

Light path in a micro flow cell of a spectrophotometric detector. Cell that have a 0.5cm pathlength and contain only 8 L of liquid are common.

- most common

-simple systems : intense 254nm emission of a mercury lamp.

- high-quality detectors full-scale absorbance range 0.0005-3 absorbance units with a noise level near 1% of full scale.

- good for gradient elution with nonabsorbing solvents

PDA Detectors:

The most powerful UV/Vis absorbance detectors in use today are photodiode-array (PDA) based instruments that permit very rapid collection of data over a selected spectral range. Thus, spectral data for each chromatographic peak can be collected and stored. This stored data may then be compared with the spectrum of a pure standard from a library - a spectral analysis study of peak purity. The PDA detector is very useful for the identification of components that are difficult to resolve (overlapping peaks) since the characteristic spectrum for each of the unresolved components is likely to be different.

Photodiode array ultraviolet detector for HPLC.(a) Dual-beam optical system uses grating polychromator, one diode array for the sample spectrum, and another diode array for the reference spectrum. Photodiode arrays are described in Section 20-3. (b) Reversed-phase chromatography (using c-18-silica) of sample containing 0.2ng of anthracene, with detection at 250nm. Full-scale absorbance is 0.001. (c) Spectrum of anthracene recorded as it emerged from the column.

Fluorescence Detectors:

These instruments are most useful for the detection of components that exhibit a chemiluminescent property such as fluorescence or phosphorescence. They are more sensitive than UV absorbance detectors by at least one order of magnitude. Fluorescence is typically observed by detection of the grating-isolated emission radiation at a 90-degree angle to the excitation beam. Fluorescent compounds are typically encountered in the petroleum industry (PAH), pharmaceuticals and natural products (aflatoxins).

Often the number of fluorescing species can be enhanced by post-column derivatization (PCD) reaction of the eluted compounds (or pre-column derivatization reaction of the sample itself) with special reagents; e.g., analysis of amino acids, carbamates, glyphosates.

Refractive Index (RI) Detector:

RI detectors have the significant advantage of responding to nearly all solutes. The difference in the refractive index of the reference mobile phase versus the column effluent results in the detection of separated components as peaks on the chromatogram. Because of its extreme sensitivity to the mobile phase, this detector may not be used without adequate pulse-damping within the LC pump, nor is it suitable for gradient applications because of the changing mobile phase composition. The detection limits are usually lower than those observed with absorbance detectors.

Refractive Index Detector

- responds to almost every solute

- Detection limit : 1000times poorer than that the uv detector

- useless in gradient elution

- sensitive to changes in pressure and temperature(0.01)℃

- not useful for trace analysis

- small linear range

Deflection-type refractive index detector

Electrochemical Detector:

Electrochemical detectors measure compounds that undergo oxidation or reduction reactions. Usually accomplished by measuring gain or loss of electrons from migrating samples as they pass between electrodes at a given difference in electrical potential.

Has sensitivity of 10-12 to 10-13 g/ml

Detection based on amperometry is the most common electro-analytical method used in HPLC. Although these detectors have not yet been exploited to the same extent as optical detectors, they offer the advantage of wide applicability in addition to sensitivity. Potentially detectable organic functional groups by this method include hydrocarbons, olefins, amides, amines, diazo groups, nitro compounds, phenols, quinolines, halogens, ethers, esters, ketones, and aldehydes. The typical amperometric detector is made up of a simple thin-layer type of flow-through cell (1-5 microliter volume), and the working (indicator) electrode is usually one of glassy carbon, gold, or platinum.

Amperometric thin layer cell for HPLC

Conductivity Detector:

This instrument provides universal, reproducible, high-sensitivity detection of all charged species. This detector may be used with an HPLC system for the simple and reliable quantification of anions, cations, metals, organic acids, and surfactants down to the ppb level. The addition of a chemical suppressor between the column and conductivity detector serves to reduce the eluant conductivity, allowing the use of gradient elution and the determination of ppb levels with minimum baseline drift.

For a typical determination of low levels of anions, the eluant is converted to its weakly ionized low-conductivity acid (e.g., Na2CO3 to carbonic acid),

reducing the background noise. At the same time, the analyte anions are converted to their corresponding high-conductivity acids (e.g., NaCl to HCl), increasing the relative analyte signal.

EVAPORATIVE LIGHT SCATTERING

Evaporative Light Scattering Detectors involves nebulization of the column effluent to an aerosol, followed by solvent vaporization to produce a small solute droplets, and then these droplets detected in the light scattering cell. System consists of three parts the nebulizer, the drift tube, and the light scattering cell.

Analytical column outlet is connected directly to the nebulizer . The column effluent is mixed with a stream of nebulizing gas to form an aerosol. The aerosol consists of a uniform dispersion of droplets. The lower the mobile phase flowrate, the less gas and heat are needed to nebulize and evaporate it. Reduction of flowrate by using 2.1mm I.D. column should be considered when sensitivity is important. The gas flowrate will also regulate the size of the droplets in the aerosol. Larger droplets will scatter more light and increase the sensitivity of the analysis. The lower the gas flow used, the larger the droplets will be. It is also important to remember that the larger the droplet, the more difficult it will be to vaporize in the drift tube. Unvaporized mobile phase will increase baseline noise. There will be an optimum gas flowrate for each method which will produce the highest signal-to-noise ratio.

Volatile components of the aerosol are evaporated in the drift tube .

LIGHT SCATTERING CELL

The nebulized column effluent enters the light scattering cell. In the cell, the sample particles scatter the laser light, but the evaporated mobile phase does not. The scattered light is detected by a silicone photodiode located at a 90o

angle from the laser. The photodiode produces a signal which is sent to the analog outputs for collection. A light trap is located 180o from the laser to collect any light not scattered by particles in the aerosol stream.

Operation of an evaporative light-scattering detector

Evaporative Light-Scattering Detector

- responds to less volatile than the mobile phase

- Mass of analyte

- with an uv detector, a small mass of strongly absorbing analyte gives a strongly signal than a large mass of weakly absorbing analyte

- nonlinear

- compatible with gradient elution

- no peaks associated with the solvent front

LC/MS system

Tandem MS system

Selecting the Separation Mode

Solute dissolve in water or organic solvents ?

Guide to HPLC mode selection

After optimizing the solvent, you might still need to improve resolution. To increase resolution, you can

- Decrease flow rate

- Increase column length

- Decrease particle size

Temperature as a variable

- Affect the relative retention of different compounds

- Influential for ionic compounds and molecules with multiple, polar substituents

Gradient Separations

Dwell volume – the volume between the point at which solvents are mixed and the beginning of the column

Dwell time – the time required for the gradient to reach the column

dwell volume (mL) tD = flow rate (mL.min)

Measurement of dwell time

Gradient Elution Is a Fine Way to Begin Method Development

- The first run on a new mixture should be a gradient

- If ∆ t/tG > 0.25, use gradient elution.

- If ∆ t/tG < 0.25, use isocratic elution.

- The isocratic solvent should have the composition applied to the column halfway through the period ∆ t

Steps in developing a Gradient Separation

1. Run a wide gradient (e.g., 5 to 100% B) over 40-60min. From this run, decide whether gradient or isocratic elution in best.

2. If gradient elution is chosen, eliminate portions of the gradient prior to the first peak and following the last peak. Use the same gradient time as in step 1.

3. If the separation in step 2 is acceptable try reducing the gfadient time to reduce the run time

More difficult approaches

- changing the solvent

- using a longer column

- using a small particle size

- changing the separation phase

Typical applications of bonded-phase chromatography. (a) Soft-drink additives.

(b) Organophosphate insecticides.

Ion chromatogram of a mixture of cations. Ion chromatogram of a mixture of anions.

Gel-filtration chromatogram for glucose(G), fructose(F), and sucrose(S) in canned juices.

Gel-permeation separation of components in an epoxy resin.

Separation of fullerenes

Seapartion of tocopherols by HPLC with electrochemical detection.

Column: TSKgel ODS-80 (4.6mm, 15cm); Mobile phase: 25mM NaClO4 in acetonitrile;

Flow rate: 0.800 ml/min, Temperature: 35oC; Voltage : 800mV; Pressure: 34 kgf/cm2.