Separation Techniques

The different types of liquid chromatography can be classified into four main classes based on the solute-stationary phase interaction. These are (1) adsorption, (2) partition,

(3) ion exchange, and (4) size exclusion. For the first three, the liquid mobile phase has a major role in governing solute retention. The fundamental principles and important packings for each of these LC modes will be described as well as providing typical application chromatograms. In addition, chiral separations will be discussed in a separate section.

A. Adsorption LC

Adsorption LC or liquid-solid chromatography (LSC) is principally carried out in the normal phase mode. It involves no partitioning of the sample solute in the stationary phase. Instead, the polar groups of each organic solute interact through primarily hydrogen bonding forces with the polar sites of the stationary phase. Therefore, careful adjustment of the polarity of the mobile phase for stable activity of the polar sites is needed for reproducible separation.

The most common packing materials for LSC are porous silica (SiO2)x or alumina (Al2O3)x. Both of these materials have numerous surface hydroxyls that act as the adsorption sites. Silica particles have a high surface area averaging 400 m2/g and are quite acidic in nature with a surface pH of about 5. This pH is usually not so low as to cause acid-catalyzed solute degradation reactions during the chromatography. As expected, retention of organic bases such as various anilines or nitrogen heterocycles is particularly good on silica. Alumina on the other hand, is

FIGURE 15 Analytical (a) and preparative (b) isolation of vitamin B-12 intermediates. (a) Column, 180 x 0.2-cm i.d., Corasil II, 37-50 ^m; mobile phase, hexane/isopropanol/methanol. (b) Column, 240 x 2.3 cm i.d. 37-80 ^m silica: mobile phase, hexane/isopropanol/methanol (5:2:1), flow rate 34 ml/min; injected sample, 5 g. [From Snyder, L. R., and Kirkland, J. J. (1979). "Introduction to Modern Liquid Chromatography, 2nd ed." Wiley, New York, p. 655. Reprinted with permission.]

FIGURE 15 Analytical (a) and preparative (b) isolation of vitamin B-12 intermediates. (a) Column, 180 x 0.2-cm i.d., Corasil II, 37-50 ^m; mobile phase, hexane/isopropanol/methanol. (b) Column, 240 x 2.3 cm i.d. 37-80 ^m silica: mobile phase, hexane/isopropanol/methanol (5:2:1), flow rate 34 ml/min; injected sample, 5 g. [From Snyder, L. R., and Kirkland, J. J. (1979). "Introduction to Modern Liquid Chromatography, 2nd ed." Wiley, New York, p. 655. Reprinted with permission.]

quite basic with a pH of about 12 and generally is lower in surface area with larger pores. Base catalyzed degradation reactions on alumina can be a problem. However, good retention of acidic organic compounds such as phenols and carboxylic acids is possible on alumina. Florisil, a magnesia-silica coprecipitate, which is strongly acidic in nature, has also been used for LSC. However, silica accounts for about 80% of all applications. Free (non-hydrogen bonded) hydroxyl groups are more reactive to solute polar groups and provide most of the retention. The siloxane, Si-O-Si, bonds are very weak in their adsorption properties. The presence of a polar solvent in the mobile phase such as water will promote hydrogen bonding, decreasing the number of active sites available for solute retention. The LSC retention mechanism can be summarized as a competition between the solute molecules (Z) and the solvent molecules (S) for the adsorption sites.

where Zm represents solute molecules in the mobile phase, Sads solvent molecules adsorbed on the packing,

Zads solute molecules adsorbed on the packing, and Sm solvent molecules in the mobile phase.

A quantitative log relationship between retention factor k' and mobile phase strength NB follows.

log k' = k'B - (Ax / nb) log Nb , where kB = retention factor in a pure nonpolar solvent, Ax = adsorption cross section of analyte X, nB = adsorption cross section of solvent molecule, and NB = number of polar solvent molecules. If the slope representing the number of analyte molecules/number of solvent molecules displaced is large, B is either a very polar solvent and/or the analyte is weakly retained. The converse is true if the slope is small.

This adsorption-desorption equilibrium is in operation continuously as the solute molecules pass down the column. The more polar the mobile phase, the more adsorption sites will be blocked by the solvent, causing the solute molecules to remain in the mobile phase and decreasing retention.

TABLE III Properties of Solvents for HPLC

Solvent

£ob

UV cutof (nm)

Viscosityat25°C [centipoise (cP)]

Pentane

0.0

195

0.22

Isooctane

0.01

197

0.47

Cyclohexane

0.04

200

0.90

Carbon tetrachloride

0.18

265

0.90

p-Xylene

0.26

290

0.60

Toluene

0.29

285

0.55

Benzene

0.32

280

0.60

Ethyl ether

0.38

218

0.24

Chloroform

0.40

245

0.53

Methylene chloride

0.42

233

0.41

Tetrahydrofuran

0.45

212

0.46

Acetone

0.56

330

0.30

Ethyl acetate

0.58

256

0.43

Aniline

0.62

310

3.8

Acetonitrile

0.65

190

0.34

Dimethylsulfoxide

0.75

268

2.0

Isopropanol

0.82

205

1.9

Ethanol

0.88

210

1.1

Methanol

0.95

205

0.54

Water

large

191

0.90

a Most of this data was taken with permission from a similar table in Snyder, L. R., and Kirkland, J. J. (1979). "Introduction to Modern Liquid Chromatography," Wiley,New York, p.248.

b Eluotropic series for alumina(similarrankfor silica).

a Most of this data was taken with permission from a similar table in Snyder, L. R., and Kirkland, J. J. (1979). "Introduction to Modern Liquid Chromatography," Wiley,New York, p.248.

b Eluotropic series for alumina(similarrankfor silica).

The usual order or elution of organic solutes is dependent on the type of polar functional groups, number of groups, and orientation. A listing of functional groups from low fc' (capacity factor which is proportional to retention) to high fc' follows: Alkane < olefins < aromatic & organic halides < sulfides < ethers < nitro compounds

< esters & aldehydes & ketones < alcohols & amines

< sulfones < sulfoxides < amides < carboxylic acids. As expected, this order roughly reflects the eluotropic series in Table I. A greater number of polar groups will promote retention unless their close proximity permits intramolecular hydrogen bonding.

One of the strengths of LSC is its ability to separate isomers, particularly aromatics functionalized with polar groups, in the retention order ortho < meta < para. The ortho compound is retained the least due to intramolecular hydrogen bonding. The meta functional groups can independently interact with the stationary adsorption sites but not often at the same time. The para isomer is retained longest because the two opposite functional groups can "sit down" on the adsorption surface and both simultaneously interact with the active sites. A chromatogram of nitroaniline isomers is shown in Fig. 16. As required for all types of liquid chromatography, the sample must be soluble in the mobile phase. Therefore, LSC is generally used for organic solvent extracts of solid or aqueous samples as well as characterization of product solutions from organic synthesis.

A classification of solvents to their ability to adsorb on the stationary phase is called an eluotropic series ( ). Retention of solute is reduced with mobile phase solvents of higher solvent strength parameter, e0. Solvent polarity parameters (0) are similar to e0 values. They can be used to estimate the overall polarity ofa binary solvent as PAb = 0A PA + 0B PB. For example, 0 values for hexane, diethyl ether, tetrahydrofuran, ethyl acetate, ace-tonitrile, and water are respectively 0.1, 2.8, 4.0,4.4, 5.8, and 10.2. The solvent polarity P2' required for a desired fc' can be predicted from fc1 and that solvent polarity P( using the equation £2/£'i = 10(P 1-P 2)/2. A two-fold change in P' results in a ten-fold change in fc'. One of the problems of adsorption LC is that solvent impurities (particularly water) in organic solvents can markedly affect solute retention and cause nonreproducible chromatograms. To alleviate this problem and also help reduce peak tailing, the mobile phase can be intentionally saturated with water. Alternatively, addition of a polar organic solvent at less than 1% will also work. Generally, alkanes with either chlorinated, ether, or ester solvents as modifiers are used as mobile phases for LSC. Hexane modified with 50% methylene chloride and 0.1% isopropanol or acetonitrile is considered a good mobile phase to start with.

B. Partition LC

Partition LC or liquid-liquid chromatography (LLC) involves solvation of the solute molecules in the stationary phase held by the packing or solid support. The versatility of partition LC is due to the wide variety of possible stationary phases. Partition LC, like adsorption LC, can be used in the normal phase mode but is more commonly employed for reversed-phase LC which uses a nonpolar stationary phase and a polar mobile phase. The retention between solute and stationary phases can be due to hydrogen bonding, dipole-dipole, and/or Van der Waal forces. Hydrogen bonding forces have been previously described for LSC. Dipole-dipole interactions are electrostatic in nature due to the charge asymmetry of the solute and stationary phases. Van der Waal forces, which dominate in reversed-phase HPLC, are interactions between hydrophobic or nonpolar groups of the solute and the liquid phase. Essentially, the water or miscible organic solvent molecules exist in a high-energy state when adsorbed to the nonpolar (C-18) derivatized silica surface. A larger aromatic solute molecule will preferentially displace many adsorbed solvent molecules in an entropy driven process, resulting in a lower energy state. A general rule of thumb is the

FIGURE 16 LSC separation of nitroaniline isomers on 10-xm alumina, 15 cm x 2.4 mm column, 40% CH2Cl2 in hexane mobile phase, flow rate 1.7 ml/min, 1 xg of each isomer. [From Majors, R. E. (1973). Anal. Chem. 45, 757. Reprinted with permission by the American Chemical Society.]

greater the number of hydrophobic groups (CH3 or CH2) or the lower the number of hydrophilic groups, the greater the expected retention. It has been shown that the log of kB / kA for molecules A and B which differ by the CH2 group in structure is proportional to the surface tension of the mobile phase. However, the partitioning mechanism of retention is also important as indicated by linear plots of log k' versus number of carbons for a homologous series of solutes. As shown by Dill and Dorsey, the density of the stationary phase cannot be too high to permit entry of the solute between the C-18 chains.

A quantitative relationship between log k' and fraction of the nonpolar solvent B (usually water) in the mobile phase (0B) is given by log k' = log kw - S0b , where k'w = retention factor for solute in pure water, and S = solvent strength parameter (a measure of nonpolarity).

The earliest LLC packings were simply a solid support such as silica coated with the liquid of choice such as oxypropionitrile. The advantages of these packings were ease of preparation, wide choice of liquid phases, and high sample capacity. However, the lifetime and reproducibil-ity of these columns were often poor due to the gradual stripping of the stationary phase by the mobile phase. Saturation of the mobile phase with the stationary phase only partially alleviated the problem. Therefore, bonded-phase packings that have the liquid phase covalently attached to the solid support were developed and now are almost exclusively used. Reversed-phase packings are synthesized by reaction of the desired organochlorosilane with the hy-droxyl groups of porous silica to form a siloxane bond as

0 ch3

O CH3

dry toluene

0 ch3

O ch3

where R often = CH3(CH2)3, ch3(ch2)t, CH3(CH2)n, or phenyl. An organic base such as pyridine is often added to neutralize the HCl produced and drive the reaction to the right. Recently, sonication during the bonding reaction has improved coverage. Ligand loading for a C-18 column is about 2-4 |mol/m2 silica. Specialty normal phase bonded silica packings such as amino or cyano functionalized materials can be made in an analogous fashion using 4-aminobutyltriethoxysilane and 3-cyanopropyltriethoxysilane. In these reactions, ethanol is produced and the silane reagent can potentially bond to three silica sites. However, if incomplete bonding results, the remaining Si—OCH2CH3 moieties will hydrolyze to deleterious Si—OH groups. Unfortunately, reactions of the surface hydroxyls only proceed to an extent of about 50% and the residual Si—OH groups can hydrogen bond with polar groups of solute molecules, causing peak tailing. To partially alleviate this problem, trimethylchlorosilane (TMCS), because of its smaller size, is reacted to "end-cap" many of the remaining hydroxyls. Kirkland and coworkers have found it is important to fully hydroxylate the silica packing before silanization to minimize the number of isolated acidic silanols, which, in particular, cause peak tailing for basic solutes. The C-18 packing is probably the most widely used reversed-phase packing. Gilpin as well as others have shown the orientation of these bonded hydrocarbon chains changes as a function of temperature and solvent. The shorter chain hydrocarbon packings as well as phenyl silica are used when lower retention is desired. Since siloxane bands are cleaved by strong acid, mobile phase pH constraints between 2 and 8 remain a limitation of silica-bonded phase packings.

To improve the lifetime of bonded phase silica packings, reactions with di- or trichloroorganosilanes have been

carried out in the presence of water with the usual resultant formation of a polymeric layer:

These reactions are often difficult to control because both cross-linking and linear polymerization are possible. The polymer layer may be too thick to permit good chromato-graphic mass transfer or too thin to give adequate sample capacity. In addition, residual silanols will be formed if not all the Si—Cl groups react; an end capping reaction with TMCS is recommended.

Alternatively, bifunctional chlorosilanes with an ether bridging group or simply sterically protected monochlorosilanes such as chlorodiisopropyloctyl silane have both provided protection of the siloxane bond between the silane and silica surface from acid hydrolysis. Using a low pH mobile phase required for the reversed-phase separation of peptides and proteins, essentially no change in column performance was observed between the first and forty-first injection. Polymers have also been cross-linked on the silica surface to form stable packings.

Mobile phases for reversed-phase chromatography are often methanol-water or acetonitrile-water binary mixtures because the organic solvent has good miscibility with water and has a low UV wavelength cut-off. The organic solvent should also have a low viscosity (see Table III) to reduce column backpressure and to minimize the Cm term of the Van Deemter equation by maximizing Dm. This is particularly important because the viscosity of a binary organic solvent-water mixture is generally higher than either the pure solvent or water. Essentially solvent strength as ordered in Table III should be reversed; the greater the polarity of the mobile phase, the stronger the hydrophobic interaction of the nonpolar solute groups with the reversed phase packing. Increasing the water content will enhance retention, while increasing the organic content will reduce retention. Often, a starting mobile phase of 50-50 methanol-water is tried if appropriate mobile phase composition information for a particular sample is lacking. The solvent polarity P2 required for a desired k2 can be predicted from kf1, and solvent polarity P| by using the equation k2/ kf1 = 10(P 2-P 1)/2. Again a two-fold change in P' results in a ten-fold change in k'. An example of reversed-phase HPLC for the separation of beverage additives is shown in Fig. 17.

The water-organic mobile phase should be modified for ionizable solutes. Adding a buffer to control the solvent

FIGURE 17 Reversed-phase separation of beverage additives on 10-^m C-18 silica (Partisil-10 ODS-2). Column 4.6 mm x 25 cm, mobile phase 50:50 methanol-water, flow rate 0.6 ml/min., pressure 529 psi, UV detection at 254 nm. Peaks: (a) Saccharin, (b) Theobromine, (c) Theophylline, and (d) Caffeine. [Reprinted by permission from Whatman, Inc.]

FIGURE 17 Reversed-phase separation of beverage additives on 10-^m C-18 silica (Partisil-10 ODS-2). Column 4.6 mm x 25 cm, mobile phase 50:50 methanol-water, flow rate 0.6 ml/min., pressure 529 psi, UV detection at 254 nm. Peaks: (a) Saccharin, (b) Theobromine, (c) Theophylline, and (d) Caffeine. [Reprinted by permission from Whatman, Inc.]

pH will suppress ionization of either weak organic acids or bases and minimize peak tailing. Strong organic acids and bases often exhibit poor hydrophobic retention and cannot be neutralized in the pH range from 2 to 7.5. For solute anions, a quaternary ammonium salt such as tetrabutylam-monium hydrogen sulfate is added to the mobile phase to form an ion pair that can hydrophobically partition with the reversed-phase packing. Ion-pair formation for solute cations is accomplished using a sulfonated alkane such as hexane sulfonic acid. However, the mechanism for ion-pair chromatography is not this simple and immobilization of the ion-pair reagent on the hydrophobic reversed-phase packing with the ionic group oriented out is likely. This in-situ ion exchange phase can retain the solute ion through electrostatic means. In any case, ion-pair chromatography is very effective as seen in Fig. 18.

Micellar liquid chromatography is the use of a surfactant such as sodium dodecyl sulfate (SDS) in the mobile phase at a concentration above the critical micelle concentration (CMC) of about 10-2 M. At the CMC, aggregation of60-100 surfactant monomers occurs with the hydropho-bic part of the molecule oriented toward the center of the assembly and the hydrophilic tail exposed to the solution. Other surfactants used have been cationic or nonionic in nature, such as cetyltrimethylammonium ion and Brij-35, respectively. For reversed-phase HPLC, the surfactant can

FIGURE 18 Separation of catecholamines and interfering compounds. Ultrasphere C-18 column, 25 cm x 4.6 mm, mobile phase: 10% methanol, 90% 0.1 M potassium phosphate, pH 3.0, 0.2 mM sodium octylsulfonate, at ± 0.72 V vs Ag/AgCl reference electrode, sample size 20 ^l. Peaks: (1) Ascorbic acid, (2) Dihydroxyphenylglycol, (3) Norepinephrine (4) Epinephrine, (5) Hydroxymethoxyphenylglycol, (6) Dihydroxybenzylamine, (7) Normetanephrine, (8) Dopamine, and (9) Dihydroxybenzylamine. [Reprinted from permission from Beckman/Altex Scientific.]

FIGURE 18 Separation of catecholamines and interfering compounds. Ultrasphere C-18 column, 25 cm x 4.6 mm, mobile phase: 10% methanol, 90% 0.1 M potassium phosphate, pH 3.0, 0.2 mM sodium octylsulfonate, at ± 0.72 V vs Ag/AgCl reference electrode, sample size 20 ^l. Peaks: (1) Ascorbic acid, (2) Dihydroxyphenylglycol, (3) Norepinephrine (4) Epinephrine, (5) Hydroxymethoxyphenylglycol, (6) Dihydroxybenzylamine, (7) Normetanephrine, (8) Dopamine, and (9) Dihydroxybenzylamine. [Reprinted from permission from Beckman/Altex Scientific.]

replace the typical methanol or acetonitrile modifier. One advantage of micellar liquid chromatography is in gradient elution where the reequilibration time can be dramatically shortened compared to gradient reversed-phase LC. In addition, direct injection of serum samples for drug analysis can be tolerated by the HPLC column if a micellar mobile phase is used.

In general, reversed-phase columns often have only a short lifetime when used for the analysis of drug samples in serum, due to the buildup of proteinaceous material on the hydrophobic particle surface. To overcome this problem, Pinkerton and co-workers developed the internal surface reversed-phase class of packings. This material is synthesized by first bonding a hydrophobic polypeptide containing phenylalanine to the silica surface and inside the pores. Using the enzyme carboxypeptidase A, the phenylalanine groups on the surface are cleaved off, but stearic hindrance prevents any reaction inside the pores. The small drug molecules such as phenobarbital can be still retained chromatographically inside the pores while the protein has little affinity to the surface hydrophilic phase. Recently, modifications of this concept have been directed to attachment of a hydrophilic polymer on the outside surface of the reversed-phase particles to prevent adsorption of proteins. These packings are sometimes called restricted access media (RAM).

Hydrophobic interaction chromatography (HIC) is a type of reversed-phase LC using relatively hydrophilic column packings and a high-salt content in the mobile phase. A dedicated HPLC instrument with titanium pump heads, a special injector, and plastic column hardware with PEEK connecting tubing is recommended to prevent corrosion and delecterious sample interactions caused by stainless steel. Because HIC packings have 10-100 times less density of hydrophobic groups, a high-salt concentration is necessary to enhance the hydrophobic retention. Because proteins retain their native conformation in such mobile phases, HIC is particularly useful for the separation of enzymes without denaturation. Typical experimental conditions are the use of a salt gradient from 2 to 0.1 M (NH4)2 SO4, using a propyl or phenyl column for the purification of trypsin with high activity.

Because of the great interest in aqueous samples, a wide variety of reversed-phase HPLC applications have been published. The pharmaceutical, biochemical, food and beverage, and the environmental laboratories represent only a partial listing where reversed-phase HPLC is common.

C. Chiral Separations

Because only one optical isomer of a drug may be pharmacologically active, an important application of HPLC

is in the field of chiral separations. Two approaches can be employed to separate enantiomers. One method is to derivatize the enantiomers with an optically pure chi-ral reagent, forming two chiral centers in the products. These diasteromers have different physical properties and can be separated by conventional normal-phase HPLC. The derivatizing reagent should have bulky groups attached directly to the chiral center and generate derivatives with the two chiral centers close to each other to provide a more facile resolution of the diasteromers. For example, the reagent a-methyl-p-nitro-benzylamine will permit the resolution of racemic carboxylic acids, while a-naphthylethylisocyanate can modify racemic alcohols before separation.

The second approach is to use either a chiral mobile or stationary phase to directly distinguish the optical isomers. The use of a chiral mobile phase is based on the premise that the sample compounds will form strong associations with the chiral reagent. Based on ligand exchange chromatography, D- and L-amino acids could be separated using an optically active copper (II) proline complex in the mobile phase. If a L-proline ligand is used, the L-amino acid elutes after the D-enantiomer and vice versa using the D-proline ligand. Ion-pair formation using an optically active base such as quinine has permitted the separation of acid enantiomers. In this case, the formation of an optically active dynamic ion exchange resin may also assist in the separation.

For chiral recognition, three simultaneous interactions, one of which is stereochemically based such as hydrogen bonding, dipole-dipole, and/or dipole-induced dipole of the stationary phase with the analytes, should occur. The preparation and characterization of chiral stationary phases for the separation of enantiomers by HPLC has been studied thoroughly by Pirkle and co-workers. For example, chiral N-(3,5-dinitrobenzoyl)-phenylglycine is reacted with aminopropyl silica to form a chiral packing material (Fig. 19). The electron withdrawing dinitroben-zoyl group is a good n electron acceptor favoring the separation of enantiomers with aromatic groups such as N-acetylated a-arylalkylamines. In contrast, the application of a (s)-N-1-N-naphthyl-leucine chiral phase is particularly good to set up an electron-donating type interaction. Separation of dinitrobenzoyl derivatives of amines or thiols is possible. A second type of a chiral stationary phase depends on a size exclusion mechanism. For example, a B-cyclodextrin having a molecular weight of 1000 and 35 chiral centers has secondary hydroxyl groups on the edge of the "donut" structure to preferentially hydrogen bond with an enantiomer of the right configuration Fig. 20. Dansylated D-amino acids such as phenylalanine and leucine have capacity factor values of about four compared to three for the corresponding L-amino acid. The

FIGURE 19 Interaction between chiral stationary phase and amide derivative of (R)-ibuprofen. [From Braithwaite, A., and Smith, F. J. (1996). "Chromatographic Methods, 5th Ed." Chapman & Hall, London.]

presence of an aromatic group as part of the solute structure to ensure inclusion complexation with the glycosidic oxygens is important. The other cyclodextrins shown in Fig. 20 either smaller or larger in size can also provide steric chiral recognition but are not as commonly used as the p form. Proteins such as bovine serum albumin (BSA) when bonded to silica have also been shown to provide chiral recognition of low molecular compounds, such as aromatic amino acids, coumarins and benzoin derivatives.

D. Ion-Exchange LC

Ion-exchange chromatography is still considered the dominant HPLC method for the separation of either inorganic or organic ions, particularly the former. The separation mechanism can be best explained as an equilibrium process between the charged functional groups of the stationary phase and the oppositely charged counter ions in the mobile phase as well as the solute ions. The appropriate cation and anion exchange reactions can be written as follows.

Cation exchange:

Anion exchange:

where X represents the sample ion, Y the mobile phase ion (counter ion), and Res+ or Res- the ionic site on the stationary support resin. A quantitative relationship between log k' and log of the concentration of the ionic eluent [ Ex ] is given by

FIGURE 20 Three-dimensional presentation of the geometries of cyclodextrin. [From Braithwaite, A., and Smith, F. J. (1996). "Chromatographic Methods, 5th Ed." Chapman & Hall, London.]

log k = -(y /x )log[ Ex ] + (log B )/x, where y = charge of the solute, x = charge of the eluent, and B = the product of the capacity of the packing and the equilibrium constant for the ion-exchange process.

Although silica has been used, the most common ionexchange supports are PS-DVB resins because of their stability at pH extremes. The non-cross-linked benzene rings are available for functionalization. Sulfonation of PS-DVB resin yields the strong cation exchanger, Res-SO-X+, while chloromethylation and subsequent amination forms the strong anion exchanger, Res-CH2-N+ (CH3)3X-. The capacity of these resins, the number of exchangeable groups per gram of resin, can range from 0.1 to 2 meq/g, depending on reaction conditions. Surface agglomerization is a convenient method to prepare low capacity ion exchange packings for ion chromatography. For example, sulfonated PS-DVB microspheres (5-40 ¡m) are contacted with colloidal anion exchange particles (100-1000 A) to electrostatically form a surface agglomerated anion exchange resin. The ion-exchange capacity of the resin can be controlled by changing either the size of the microspheres or of the colloidal particles, as well as the degree of functionalization of the latter particles. The corresponding weak anion exchanger Res-NH+ (CH3)2X-and cation exchanger Res-COO- X+ have also been developed for use in the separation of labile molecules such as proteins.

The mobile-phase factors of pH and ionic strength primarily control the retention of ion exchange resins. There fore, buffered solutions are almost always the major component of a mobile phase for ion-exchange LC. For weak acidic or basic solutes, the mobile phase pH controls their ionized state and ability to interact with the resin. The capacity of weak ion-exchange resins is in addition influenced by pH. All other factors considered equal, the greater the capacity of the resin, the greater the ion retention. Finally, the pH as well as the buffer salt can contribute significantly to the overall ionic strength of the mobile phase. Ionic strength is calculated by taking one-half of the sum of the ion concentration times their charges squared. As the ionic strength increases, the amount of counter ion in the mobile phase increases, driving the equilibrium back to the left. This competition of the counter ions for the stationary ionic sites results in a reduced retention of the solute ions. The lower the resin capacity, the smaller the ionic strength that is required to elute the solute ions from the column. The ionic strength is often intentionally increased gradually to improve the separation of weakly and strongly retained ions in a mixture (see Section IV, Fig. 26).

The nature of the ionic solutes often affects their ion exchange retention. As expected, polyvalent ions are held more tightly than monovalent ions. Within a given charge group, retention generally increases with the size of the ion but decreases with the size of the hydrated radius. Sol-vated ionic radii limit coulometric interactions between ions and energy must be put into the system to strip the water away. The retention order for the alkali metals is Cs+ > Rb+ > K+ > NH+ > Na+ > H+ > Li+. Because of its greater hydration, Li+ is retained less than H+.

In past years, the direct detection of inorganic anions, cations, and small aliphatic organic acids and bases after column separation has been difficult. Development of ion chromatography in the mid 1970s solved this problem. Now two approaches, both using low-capacity ion-exchange columns and a conductivity detector, are commercially available. First developed by Small, the dual-column ion chromatography system traps the ions of the mobile phase by connecting a high-capacity suppressor column downstream from the analytical ion-exchange column. For example, using a sodium hydroxide mobile phase, the separated anions elute into a protonated cation suppressor column. There the mobile phase is neutralized to water as shown in the equation Res-SO-H+ + Na+ + OH- ^ Res-SO-Na+ + H2O, and the separated anions are changed to the corresponding acids, Res-SO-^ + M+ + A- ^ Res-SO-M+ + H+ + A-. Sensitive conductivity detection of the separated ions is now possible at the sub-ppm level. The analogous system for cation analysis, in which HCl is the eluent and the suppressor column is an anion exchanger in the hydroxide form, is equally effective. Hollow fibers, and more recently membranes, have been used in place of the suppressor column. A continuous bathing stream of either acid or base eliminates the problem of periodic regeneration of the suppressor column. Now, even this process has been simplified and just water surrounding the membrane is simply allowed to undergo electrolysis to generate the necessary H+ or OH-ions. The second method, single-column or nonsuppressed ion chromatography, uses a low capacity (about 0.1 meq/g or less) ion-exchange separation column permitting low ionic strength mobile phases. The conductivity of the mobile phases is electronically zeroed out, permitting detection of only the sample ions. Although the detection limits are not quite as low as the suppressed ion chromatography method, the single-column method can be easily adapted to existing HPLC hardware and is easier to maintain. Applications of ion chromatography for waste water, boiler water, drinking water, and plating bath samples, as well as others are documented in the literature. Two examples of nonsuppressed ion chromatography are shown in Fig. 21.

The indirect detection method for IEC depends on the use of an ionic mobile phase that not only controls the retention of the sample ions but also responds to the detector of choice. For example, consider indirect photometric detection. After the ion-exchange separation and during the elution process, light-absorbing ions in the mobile phase replaced by photometrically inactive injected sample ions will cause a decreased absorbance at the detector and negative peaks to be recorded. For IPC, salicylate or naphthalenedisulfonate has been used for anion separations and Ce(III) or an aromatic amine have been used for

FIGURE 21 Separation of meta ions by ion chromatography. (A) Alkali metals using Ion-200 cation exchange column; eluent: 2.0 mM picolinic acid, pH 2.0; flow rate 2.6 ml/min; sample volume 5 /u,l; 2-6 ppm each ion; conductivity detection. (B) Inorganic anions using Ion-100 anion exchange column; 1.5 mM phthalate, pH 5.0; flow rate, 1.5 ml/min; sample volume 10 ^l; 30-80 ppm each ion; conductivity detection. [Reprinted with permission from Interactions Chemicals, Inc.]

FIGURE 21 Separation of meta ions by ion chromatography. (A) Alkali metals using Ion-200 cation exchange column; eluent: 2.0 mM picolinic acid, pH 2.0; flow rate 2.6 ml/min; sample volume 5 /u,l; 2-6 ppm each ion; conductivity detection. (B) Inorganic anions using Ion-100 anion exchange column; 1.5 mM phthalate, pH 5.0; flow rate, 1.5 ml/min; sample volume 10 ^l; 30-80 ppm each ion; conductivity detection. [Reprinted with permission from Interactions Chemicals, Inc.]

IEC of cations. If a fluorescent or an electrochemically active ionic mobile phase such as Ce(III) is used for IEC, indirect fluorometric or electrochemical detection would be possible in an analogous fashion. Indirect detection limits less than 0.1 ppm are fairly comparable to direct conductivity methods.

Ion-exclusion chromatography uses an ion-exchange column with an appropriate mobile phase to permit the penetration of nonionic substances into the liquid, both inside and between the resin beads. Retention is based on polar interactions of the solute with the resin functional groups and/or nonpolar forces between the solute and the resin backbone. For example, weak organic acids are separated using a cation-exchange resin and an acidic mobile phase to maintain solute neutrality (Fig. 22). Highly ionized simple inorganic anions pass through un-retained. Solutes with a more hydrophobic character such as longer chain hydrocarbon or aromatic monofunctional acids are retained well. In addition, organic acids elute in order of increasing pka values. Neutral hydrophilic compounds such as sugars can also be separated by ion exclusion chromatography using a Ca2+- or Pb2+-loaded

FIGURE 22 Separation of short-chained carboxylic acids by ion-exclusion chromatography. Column: ORH-801 sulfonated cation exchange; eluent: 0.01 N sulfuric acid; flow rate: 0.8 ml/min; temperature: 35°C; detection: UV at 210 nm. [Reprinted with permission from Interactions Chemicals, Inc.]

cation-exchange column and water as the mobile phase. Retention of the sugars is assisted by weak complex formation with the metal cation. Sometimes an inorganic salt is added to the mobile phase to improve retention by promoting a "salting in" phenomenon. Aliphatic alcohols and amines have also been separated by ion-exclusion chromatography.

E. Size-Exclusion LC

Size-exclusion chromatography (SEC) is used for the separation of large-molecular-weight compounds such as polymers or proteins. SEC is generally divided into two classes, gel-filtration chromatography (GFC), which uses aqueous solvents, and gel-permeation chromatography (GPC), which uses organic solvents. The separation mechanism is based on the relative size of the pores of the packing and the molecules to be separated (Fig. 23). If the molecule is large compared to the pore size, it will be excluded from the particles and pass down the column un-retained (point A). Molecules similar in size to the pores can partially penetrate the packing particles and are retained to differing extents allowing separation (region B). Molecules much smaller in size than the pores can easily penetrate all the pores of the packing particles and will be retained to the same degree (point C). Therefore, the peaks of an SEC chromatogram are ordered from highest to lowest molecular size.A quantitative relationship for Fig. 19is

retention volume

FIGURE 23 Molecular weight calibration curve for SEC.

retention volume

FIGURE 23 Molecular weight calibration curve for SEC.

Vr = Vm + KVs, where Vm is the volume between the particles, Vs is the volume within the pores of the packing, and K is the partition coefficient described by the ratio of pore volume accessible by the solute divided by the total pore volume. Since separation is based on molecular dimensions (size and shape), monodisperse samples having the same molecular weight (MW) may not be of the same size. Biopolymers such as proteins can adopt different conformations, and small molecules can be associated together depending on the solvent conditions. Therefore, careful column calibration with standards of similar structure is important to obtain reliable MW information of monodisperse samples. For a polydisperse sample as shown by a broad SEC peak, there is no well-defined MW value but instead a distribution of MW values around an average. A number average Mn or weight average MW, Mw, can be calculated knowing the number and MWs of various fractions of the broad peak. The viscosity detector developed by Yau and the laser light scattering detector have both been shown to be invaluable for obtaining reliable MW information for SEC.

Proper choice of the mobile phase and packing is important to attain a strictly steric retention mechanism. As with other types of LC, both polymers and silica packings have been used for SEC. By controlling the cross-linking during the synthesis of PS-DVB resins, polymers with different pore sizes can be prepared. Because of its hydrophobicity, PS-DVB in normally used for GPC. Sulfonated PS-DVB as well as polyacrylamide are hydrophilic enough to be used for GPC of polar solutes such as sugars. Spherical silica with pore sizes ranging from 60 to 4000 A are available for separation of molecules from about 500 to 105 in molecular weight. Although untreated silica can be used for many sample applications, particularly organic polymers, it is usually modified for the separation of biological molecules. For example, glycophase silica, Si-^Si(CH2)3OCH2-CH-(OH)CH2(OH), is preferred for the separation of proteins. The selection of an SEC packing is dependent on the range of components with different molecular weights desired to be separated. Calibration curves of polystyrene standards versus retention volume for packings of different pore sizes are well documented. For 60-A silica, a linear MW range of 102 to 104 is possible. For a 750-A silica, a linear MW range of 104 to 106 is found. To expand the range of molecular weights that can be separated, it is necessary to connect in series columns of two different pore-sized packings. For the previous example, a linear fractionation range from 102 to 106 MW would be possible.

Unlike all other modes of LC separation, the mobile phase is not chosen to control peak separation but to ensure sample solubility and minimize solute-stationary phase adsorption effects. Therefore, the sample solvent determines whether the mobile phase is predominantly organic or aqueous. In general, to minimize adsorption effects, a mobile phase that is more strongly adsorbed to the packing than the solute is advised. For example, for the separation of polyurethanes on silica, dimethyl-formamide is preferred over tetrahydrofuran. The ionic strength should generally be greater than 0.05 M when using aqueous mobile phases with silica. One important application for SEC is for the initial exploratory separation of an unknown sample to indicate how complicated it might be. For example, SEC could easily separate a biological sample into high-molecular-weight proteins and low-molecular-weight peptides and amino acids. These peaks could be collected for further HPLC study using ion exchange or reversed phase. A typical example of an SEC protein separation is shown in Fig. 24. Another major use of SEC is for the separation of polymeric oligomers such as polystyrene. In general, the peaks of an SEC chro-matogram are quite broad and resolution is only modest. However, the important role that SEC can play in an overall HPLC separation scheme will be elaborated in the next section.

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