Chapter 6 Fundamentals of ion-exchange chromatography

chapter 6

Fundamentals of ion-exchange chromatography 0. MIKEB CONTENTS Principles and terminology ....................................................... Characterization of ion exchangers ................................................. Reactions, affinity and selectivity in ion exchange ..................................... Ionexchange equilibria and kinetics ................................................ Column operation and ion-exchange chromatography ................................... Ion exclusion, ion retardation, the ionsieve process and partition chromatography on ion exchangers ................................................................ Ligandexchange chromatography .................................................. Ion exchange in non-aqueous solutions .............................................. References ...................................................................

69 73 75 77 80

83

85 85 86

PRINCIPLES AND TERMINOLOGY Ion exchangers can be defined as polyvalent materials that are insoluble in water, contain bound ionogenic groups and are capable of dissociating and exchanging ions in solutiorl. Sometimes the shortened term ionex is used instead of ion exchanger. In spite of the fact that there are natural ion exchangers, most ion exchangers have been prepared synthetically. Natural and synthetic ion exchangers may consist of inorganic or organic materials and are usually solid substances, but liquid ion exchangers are used in special circumstances. Ion exchangers and their properties have been described in thousands of papers and tens of monographs, and many of these articles also deal with chromatographic aspects. The most important publications in this field since 1960 are those by Dorfner (1963a, b), Helfferich( 1962a), Hering, Inczedy, Marinsky, Osborn, Paterspn, Reuter, Saldadze et al. and Samuelson. Griesbach (now Reuter) is producing a comprehensive German work in numerous volumes. There are specialized chapters on ion exchange in monographs on chromatographic methods by Flaschka and Barnard, Genge, Kunin, Mikes’, Morris and Morris, and Walton (1 967). Systematic reviews have been published every other year, the last three being by Walton (1968, 1970, 1972). In a typical ion exchanger (see the schematic representation in Fig. 6.1), there are two main components: a porous matrix (or network) and electrically charged, covalently bound functional ionogenic groups. Ion exchangers can be divided into four main groups, depending on the composition of the matrix: (1) inorganic exchangers, based on aluminium silicates and other suitable minerals; (2) synthetic resins of many types; (3) ion-exchange cellulose; (4) ion-exchange polydextran. References p . 8 6

69

70

FUNDAMENTALS OF ION-EXCHANGE CHROMATOGRAPHY C

A

Fig. 6.1. Schematic representation of resinous ion-exchange particles. A = Anion exchanger; C = cation exchanger. The lines illustrate the polymer chains and cross-linking of the matrix, and the charges in circles the functional ionogenic groups. Counter-ions have been omitted.

There are five principal classes of functional groups present in ion exchangers, and hence exchangers can be classified on this basis as follows: (1) cation exchangers; ( 2 ) anion exchangers; (3) amphoteric and dipolar ion exchangers; (4) chelating ion exchangers; (5) selective (or specific) ion exchangers. Cation exchangers are high-molecular-weight polyvalent insoluble anions (polyvalent acids), the ionogenic groups of which are saturated with individual soluble cations. These are able to dissociate when the exchanger comes into contact with an aqueous solution, and thus may be exchanged, e.g. : COONa COONa

-

COO-Na’ COO-Na+

CaCI2-

Ca2+

+

2 NaCl

The shortened term catex is also used instead of cation exchanger. An anion exchanger (anex) is a high-molecular-weight polyvalent insoluble cation (polyvalent base), which is able to exchange electrostatically bound soluble anions, e.g. :

Amphoteric ion exchangers are polyvalent insoluble “zwitter-ions” (polyvalent “inner salts”), which dissociate in aqueous solution without the release of ions into the solution. However, they are then able to bind dissociated salts from the solution, cations to anionic groups and anions to cationic groups, e.g. :

PRINCIPLES A N D TERMINOLOGY COO' Na' &R3CI-

*

71 $$O0-

+

NaCl

NR3

This binding represents a dynamic equilibrium. After washing with a large amount of water, the original internally neutralized form of the exchanger is regenerated and the salt is released. It is difficult to prepare these exchangers with exact equivalence of ionogenic groups. Dipolar ion exchangers can be prepared by binding amino acids (e.g., arginine via amino group) to the matrix. These dipolar ion exchangers are used advantageously for the separation of biopolymers. Chelating ion exchangers are resins prepared by incorporating complex-forming groups, e.g., iminodiacetic acid. These ion exchangers retain only specific ionic groups (e.g., heavy metals or alkaline earths) and are therefore more selective than cation exchangers. An example is given below, in which M2+represents, for example, Cuz+,NiZ+,ZnZ+,Co2+or

uop:

Selective (or specific) ion exchangers are synthesized experimentally for a specific purpose. They contain functional groups that are able to retain only one type of ion or a very limited number of types. For example, Skogseid synthesized a resin with trinitrophenyliminodinitrophenyl groups. These groups specifically bind potassium ions:

This type of ion binding cannot be considered as a true ion exchange, but is more closely related to potassium precipitation by hexanitrodiphenylamine (dipicrylamine). The principle of selective ion exchange resembles that of affinity chromatography. The homogeneity of functional groups is very important in ion-exchange chromatography. Exchangers can be classified as monofunctional if they contain only one type of ionogenic or chelating group; these are sometimes called homoionic exchangers, and are most suitable for chromatography. Cheaper polyfunctional exchangers are used only for certain technical purposes. The presence of different kinds of ionogenic (or chelating) groups leads to a loss of resolution in the chromatography. The terminology of ions should now be explained. Let us consider a homoionic exchanger, in which there is one type of charged functional group covalently bound to the matrix (cf Fig. 6.2). In every case, dissociable counter-ions with an opposite charge are bound to it by electrostatic forces and thus form part of the ion exchanger. Counterions are mentioned in the literature and in commercial pamphlets when the ionic form of an ion exchanger is illustrated. Co-ions play an important role in the mechanism of ionexchange chromatography and are ions with the same type of charge as the functional group, but they are soluble and capable of forming salts, acids, bases or water with counter-ions. The co-ions compete with the functional group in attracting counter-ions, References p.86

72

FUNDAMENTALS OF ION-EXCHANGE CHROMATOGRAPHY CI -

CI-

clCI -

CI-

Na+

CI-

-2’

car

CI -

Na’

CINa b- CI-

CI

CI-

CINa’

Na+

CI-

CI -

-

Na+

Fig. 6.2. Scheme of cation exchange. Left: bead of cation exchanger in the Na+ form (the negative circles represent carboxylic functional groups, Na’ the counter-ions in the ion-exchanger, Ca2+the counter-ions in solution and Cl- the co-ions); right: bead after exchange. The process can be expressed by the equation 2(R-COO-Na+)

+ CaCl, * (R-COO-),CaZ+ + 2 NaCl

where R = resin, Caz+are the counter-ions in the ionexchanger, Na+ the counter-ions in solution and

c1- the co-ions.

thereby facilitating the exchange of the desired counter-ion. Examples to illustrate this terminology by Helfferich (1962a) are as follows: o p e of exchanger

Cation Cation Anion Anion

Functional POUP

-coo-

- $0;

-y(CH3)3 -N(CH,

Counter-ion in ion exchanger (ionic form of exchanger)

Co-ion in solution

Counter-ion in solution [to be exchanged)

N a+ H+ C1OH-

CIc1N a+ N a+

Caz+

K+

OHCH, COO-

The first of these four examples is represented schematically in Fig. 6.2. The complete change of counter-ions in an ion exchanger is called cycling, and sometimes the term “cycle” is used instead of “ionic form”. Cycling at the end of an ionexchange operation to produce the ion exchanger in its original form is called regeneration.

73

CHARACTERIZATION OF ION EXCHANGERS

CHARACTERIZATION OF ION EXCHANGERS Information describing the properties of individual ion exchangers is necessary before they can be used for chromatography. It is usually presented in the form of tables (cf: Chapter 13). First the type of exchanger must be known. Both cation and anion exchangers are classified according to the nature of the active groups, as shown below: Ion exchanger

5Pe

Usual functional group

Cation exchanger

Strongly acidic Medium acidic Weakly acidic Strongly basic Medium basic

Sulphonic Phosphonic Carboxylic Quaternary ammonium Mixture of tertiary and quaternary ammonium groups Amines, polyamines

Anion exchanger

Weakly basic

The ionic form of a commercial ion exchanger is usually indicated by the manufacturer. Cation exchangers are produced in the acidic form, designated H'(or H*),or in the salt from @a+, L,i+ and others). Anion exchangers are produced as the free base, designated O H (or B*), or as salts (usually Cl-). The type of matrix (lattice) of an ion exchanger must be considered carefully. Polystyrene or polyacrylic types find a wide application, while phenolic types are, in general, not suitable for chromatography, because phenolic R-OH groups dissociate in alkaline media and thus form an additional cation exchanger group, R-0- , with different properties from those of the main functional group. For the chromatography of many biopolymers, polystyrene or other aromatic matrices are not suitable because of their denaturating effect. Polyacrylic types are better, but a cellulose or a polydextran matrix is usually the best. The degree of cross-linking of the matrix is very important in chromatography, and defines the average porosity of exchangers. The symbol X, accepted as a measure of the degree of cross-linking, represents the percentage of divinylbenzene in the styrene polymerizationmixtureused to prepare this type ofresin(cf: Chapter9, Ion-exchange materials). The process ofcross-linkingis easily controlled and therefore it is possible to produce a resin withaporositysuitable foragivenpurpose,and in commercial resins it varies from X1 to 16, X2 to X9 being most often used. The more cross-links present, the less an exchanger swells. The functional groups of individual exchangers have the same electric charge and therefore they have the tendency to extend the network to a maximum, and this process is accompanied by the hydration of functional groups and bound ions. The strength of the repulsing force is influenced by the type of functional group, ionic strength, pH and the nature of any bound ions, but it is strictly limited by the cross-linking. Swelling is reversible and can be considered as a state of balance between the tension of the elastic network and the osmotic pressure of the inside solution, arising from the presence of counter-ions. Volume changes in swollen resins, due to changes in the composition of the surrounding solution, some times disturb the chromatographic operation, and these difficulties occur more often with resins with a low degree of cross-linking. However, the lower cross-linked resins change ions more rapidly, but they are less selective. As a result References p.86

74

FUNDAMENTALS OF ION-EXCHANGE CHROMATOGRAPHY

of variations in the cross-linking reactions, Pepper et al. recommended the use of water regain values (W.R.) to characterize ion exchangers instead of the X values. The water regain is defined as the maximum weight in grams of water taken up to 1 g of completely dry ion exchanger. The relationshp between the X and W.R. values for strongly acidic cation exchangers is shown below: X

w.R.

X

W.R.

2 4 6 8

3.45 1.92 1.36 1.04

10 15 20 25

0.83 0.59 0.48 0.38

The cross-linking and porosity are equivalent terms used for ion-exchange resins, but this terminology is not valid in the case of macroreticular resins (cf, Chapter 9). The cross-linking defines here the composition of the matrix only; very large pores of macroreticular resins are best expressed by the maximal molecular weight of substances penetrating the beads. The capacity of an ion exchanger is a measure of the total amount of ions the resin is able to bind and is usually expressed as milliequivalents (mequiv.) per gram of dry resin (in the Hi or C1- form) or as milliequivalents per millilitre of fully swollen wet resin (in the H'or C1- form) packed in the bed. T h s value, which is a measure of all charged groups present, is not usually achieved in practice, but is lower for various reasons, e.g., operating under non-equilibrium conditions. Factors that influence the available capacity are concentration and ionic strength of the eluent, pH, temperature, the accessibility of functional groups and the nature of the counter-ions. The dependence of the ion-exchange capacity on pH is illustrated by the titration curves in Fig. 6.3. It can be seen that for strongly acidic and strongly basic exchangers, the capacity is virtually independent of pH and they can therefore be used over a wide pH range. However, the capacity of weakly acidic cation exchangers and of weakly basic anion exchangers is strongly dependent on pH, so the use of weakly acidic exchangers is therefore limited in media of low pH and weakly basic anion exchangers are not very efficient in alkaline media. A

C 1

0

5

10 HC I (rnequiv /g)

0

10

5 NaOH(mequiv/g)

Fig. 6.3. Titration curves of ion exchangers in a dilute solution of neutral salts. A = anion exchangers; C = cation exchangers; a = strongly basic anion exchanger or strongly acidic cation exchanger;b = medium basic or acidic exchanger; c = weakly basic anion exchanger (mine-type) or weakly acidic (carboxylic) cation exchanger.

75

REACTIONS, AFFINITY A N D SELECTIVITY

Particle size and particle form are important characteristics of ion exchangers. The particle size determines how quickly equilibrium is established and hence influences the sharpness of a chromatographic separation: the smaller the particle size, the sharper is the separation. If the particles are too small, the flow resistance of the chromatographic column is increased and higher pressures are required for elution and care must therefore be taken when choosing a suitable grain size. The particle form is also an important factor. Ion exchangers are delivered either in the form of grains prepared by grinding the resin gel (these have irregular shapes) or as uniform beads (spheres), prepared by polymerization in an emulsion. Generally, the latter are better for chromatography, because they do not pack the column so tightly and consequently there is a lower flow resistance. The bead form possesses better mechanical properties and hence the losses caused by friction are lower. The particle size is usually expressed in terms of the size range of dry copolymer beads before any ionic groups are attached, and is measured by standard mesh screens. Sometimes the particle size is expressed as the wet mesh range after maximal swelling". The wet mesh size depends, of course, on the many factors mentioned in the preceding paragraphs. Millimetres (mm) and microns (pm) are also used to measure grain size. For the conversion table for U S . standard mesh screens, see Table 1 1.1 (p.286). For fine chromatographic separations, it is important that the particle size should be as uniform as possible. The narrower the variation in grain size, the sharper is the separation obtained.

REACTIONS, AFFINITY AND SELECTIVITY IN ION EXCHANGE Exchange reactions of simple ions are best described in terms of an ionic redistribution between the ion-exchange gel and the aqueous phase. These reactions are always stoichiometric because the electroneutrality of the resin must be maintained. As no covalent bonds are formed or broken during this process, there is little heat evolution or absorption accompanying ion exchange. The only exceptions are neutralization reactions involving a cation exchanger in the H+ form or an anion exchanger in the O H form, in which the formation of low-dissociated water is the source of heat. Ion exchange is, in general, a reversible process and therefore an equilibrium is obtained, e.g. : 2(R-SO;Na+)

+ CaZ++ (R-SO;)?

CaZ++ 2 Na'

where R = resin. This equilibrium depends not only on the relative affinities of ions for the exchanger, but also on the relative ionic concentrations. Therefore, ions with a low affinity for the exchanger can regenerate it and replace ions of a greater affinity, if the former are present at a higher concentration. In practice, this is made use of in the regeneration of water softeners: CaZ+and Mg2+are present in natural hard water at relatively low concentrations. Because they have a higher affinity for the cation exchanger (in the Na' form), they exchange with Na' . A large amount of water can be treated in this way, to produce water that contains only monovalent ions which do not precipitate *American manufacturers use U.S. standard mesh screens as their standard, and British manufacturers B.S.S. standard mesh screens.

'References p.86

76

FUNDAMENTALS OF ION-EXCHANGE CHROMATOGRAPHY

soap. When the ion exchanger is exhausted, a small amount of concentrated sodium chloride solution quickly regenerates the ion exchanger to the original Na' form. The affinity of ions for an ion exchanger is sometimes called the "ion-exchange potential" and in dilute aqueous solutions it increases with the size of the ionic charge. Polyvalent ions are more strongly bound than monovalent ions, the affinity being proportional to the charge. For ions of the same charge, the exchange potentials are inversely proportional to the radius of the hydrated ions. Because the radii of many cations are inversely proportional to their atomic weights, the affinities of these cations can be arranged in order of atomic weights. The exchange potentials of cations are similar to or identical with the so-called lyotropic series. The affinity of anions is governed by similar rules. In addition, the exchange potentials increase with the ability of anions to polarize. Examples (cf. Kunin and Myers, and Nachod) of typical affinity sequences are given below. Composite affinity sequence for cations: (CH3)4w < Li' < Na' < NW4 < K' < Rb' < Cs' < T1' < Ag';Mg2+< CaZ+< Sr" < BaZ+;Fez+ < Co2+< NiZ+< Cuz+< Zn"; A13' < Sc3+< Lu3+< Yb" < Tm3+ < Er3+< Ho3+< Y3' < Dy" < Tb" < Gd3' < Eu3+< Sm3+< Pm3+ < Nd3' < Pr3+ < Ce3+< La3+.The position of €Ion ?strongly acidic cation exchangers is nearly the same as that of L f , and on weakly acidic (carboxylic) cation exchangers it is about the same as that of Ba". Composite affinity sequence for anions: fluoride < acetate < formate < chloride < bromide < chromate < molybdate < phosphate < arsenate
77

EQUILIBRIA AND KINETICS

Therefore, the dissociation constant of the ammonium group, pK2, determines the affinity. The behaviour of amphoteric ions is illustrated below (the decisive dissociation constant is indicated with an asterisk): Ion exchanger

Medium

Ionic form

Formula

Dissociation constant

Cation exchanger Aqueous solution Anion exchanger

Acidic Neutral Basic

Cation Zwitter-ion Anion

H3N+-CH(R)-COOH H, N'-CH(R)-COOH,N-CH(R)-COO-

PK,;PKT PK,;PKI PK:; PKI

The sequence of emergence of some amino acids in practice was found to differ from the theoretical sequence based on pK values in an ion-exchange experiment, owing to adsorption due to Van der Waals forces. The hydrophobic side-chains of amino acids are adsorbed on the aromatic network of the resin, and this process can be decreased by increasing the temperature. Sometimes these interactions can help in chromatographic separations. These rules are valid for the chromatography of low-molecular zwitter-ions (e.g., amino acids and smaller peptides). The behaviour of high-molecular-weight amphoteric substances and of their fragments (e.g.,proteins and large polypeptides) follows other rules (Porath and Fryklund). They usually exist only in two alternative states in contact with ion exchangers: either completely sorbed or not sorbed at all. The state depends on the conditions in the solution (pH and ionic strength). The choice of a suitable composition of the buffer used for the sorption and for the selective desorption of proteins is explained in Chapter 10 (p.264). Certain valuable results on the resolution of proteins and large peptide fragments have been reported by Novotnjl and by Novotny et al.

ION-EXCHANGE EQUILIBRIA AND KINETICS Rigid inorganic exchangers contain water-filled pores and the ions are bound in specific positions. A better model describing elastic resinous exchangers immersed in water is a two-phase system. A swollen grain of the resin resembles an aqueous electrolyte solution. Both the functional groups and counter-ions are dissolved in absorbed water, and the counter-ions are not fixed to individual functional groups, but are present as a cloud containing mutually repulsive ions distributed throughout the whole resin volume. This cloud is attracted by the oppositely charged bound functional groups. The counter-ion cloud extends partly to the grain surface and thus an electric double-layer is formed. If the grain is placed in an electrolyte solution containing the same type of counter-ion, A-, the penetration of the electrolyte into the grain is not complete and an equilibrium is reached. Initially, this can be expressed by the Donnan equation: where a is the activity of the counter-ion A- or co-ion B' in the resin (subscript r ) and in the solution (subscript s). If the ion exchanger (anion exchanger) is in the A- form, there is a higher concentration of A- and a lower concentration of B' in the ion exchanger phase relative to the solution. If A- begins to diffuse out of the ion exchanger, following References p.86

78

FUNDAMENTALS OF ION-EXCHANGE CHROMATOGRAPHY

the concentration gradient, and B' into it from the solution, a net positive charge will result on the grain and a net negative charge in the solution and this Donnan potential will stop any further diffusion. This Donnan equilibria will affect not only the ion-exchange process, but also the swelling of the resin. If the grain in the N form is placed in an electrolyte solution containing different counter-ions, M, the Donnan potential also limits the entry of ions of the same charge, but the re-distribution of different counter-ions between both phases begins and continues until equilibrium is reached. Because electrical neutrality between the grain and the solution must be maintained, the exchange is strictly stoichiometric. According to Samuelson, the selectivity coefficient. k g ,describing the equilibria can be defined by the expression

where M and N are the ions being exchanged and rn and n are the absolute values of their charges. The brackets represent the concentration in the external solution (subscript s) or in the resin phase (subscript r). If the selectivity is calculated from experimental data, the total amount of M and N, including their concentration due to co-ions, must be considered in the expressions [MI, and [N],. For an understanding of ion-exchange chromatography, the rate at which equilibrium is reached is very important and must be considered in addition to the equilibrium itself. Therefore, the basic principles of ion exchange kinetics are now mentioned briefly in accordance with the general theory of Boyd et al. Let us consider the exchange of N' (present in the exchanger) for M' (present in the external solutionlcf., Fig. 6.4):this can be expressed by the equation

NR

+ M'+ M R + IV'

where R represents the resin. Now, assume that the exchanger is suspended in a mixed solution or fixed in a chromatographic column through which the solution is flowing. The exchange process can be divided into five individual steps: (1) the transport of M' from the solution t o the surface of a bead of ion exchanger; ( 2 ) the diffusion of M'through the matrix to the functional group; (3) the chemical exchange inside the particle expressed by the above equation; (4) the diffusion of the exchanged N' from the functional group to the surface of the exchanger; ( 5 ) the transport of N' into the external solution. It is clear that the slowest of these processes should be rate controlling. These individual steps will now be discussed briefly. The bead of exchanger, in spite of the fact that it is situated in a mixed liquid, is surrounded by a still, thin layer of solution. This layer is not mixed with the external solution (or the mixing is not perfect) and it moves with the particle. This relatively stable Nernst film is formed for thermodynamic reasons and the ions can move through it only by diffusion, so for steps (1) and (5) the rate of diffusion through this film is therefore limiting. For steps ( 2 ) and (4), the rate of diffusion through the gel of the swollen ion-exchanger particle is limiting. The process of actual ion exchange, step (3), in the swollen gel of exchanger can be considered to be instantaneous, because the ions are not firmly bound to the functional groups but are .aftrac,ted to themonly in a dynamic equilibrium. Only in special cases does the velocity of

EQUILIBRIA AND KINETICS

79

M‘

Fig. 6.4. Schematic representation of the fundamental terms in ionexchange kinetics. M’ and N’ = exchanging ions; P = ionexchange particle; F = Nernst film; S = external solution;@= functional group;f= rate of diffusion of ions through the film; p = rate of diffusion of ions through the particle.

the chemical reaction in step (3) play a role. From this discussion, it can be seen that there are two main rate-limiting steps in ion-exchange kinetics: “film diffusion” and “particle diffusion”. Film diffusion is enhanced by small particles and particle diffusion by large particles, which can be explained by the fact that film diffusion is inversely proportional to the diameter of the ion-exchanger particle, whereas particle diffusion is inversely proportional to the square of the diameter. Film diffusion is rate limiting at low concentrations of exchanging ions, and particle diffusion at high concentrations, which is understandable when it is realized that higher concentrations of ions in solution increase the rate of film diffusion but do not affect particle diffusion. The exchangeable ions have to jump from the electrostatic field of one charged functional group to another, which requires a considerable activation energy. The film diffusion velocity is obviously independent of resin cross-linking. However, when particle diffusion is rate determining, the exchange rate is decreased substantially by an increase in cross-linking. An increase in temperature causes a greater increase in the rate of diffusion within the particle in comparison with the increase in film diffusion and therefore the rate-controlling influence of film diffusion is enhanced. Kinetic data can be expressed in two ways: (1) by the time necessary for complete ion exchange under defined conditions (the time necessary to reach equilibrium), or (2) by the time of half-exchange. Both methods have been used in the literature, and the data published by Kressman are given as an example. He measured the time for half-exchange on a strongly acidic sulphonated cation exchanger (polystyrene type of resin) in the NH‘4 fprm under comparave conditions and found the follywing values: Na+, 1.25 min; N(C2H5)4,3.0 min; N(CH3)4, 1.75 min; and CbH5-N(CH3)2CH2-C6H5, 1 week. These results illustrate the influence of ionic size. The relationshp between full exchange and References p.86

80

FUNDAMENTALS OF ION-EXCHANGE CHROMATOGRAPHY

the time required for halfexchange was illustrated by Reichenberg and Wall, who measured the kinetics on a similar type of exchanger (X17): complete exchange: half-exchange:

Na+/H+, 2-10 min; Na+/H+, few seconds t o 1-2 min.

These effects have been studied mostly on strongly acidic resins, and strongly basic resins have not been studied to the same extent. The kinetics in weakly acidic and weakly basic exchangers depends on the degree of ionization of their groups and if this is h g h enough, the parameters are comparable with strongly acidic and strongly basic exchangers. When there is a low dissociation of functional groups, the rate ofexchange is decreased and in every case controlled by particle diffusion. The exchange of H'in weakly acidic cation exchangers and of O H in weakly basic anion exchangers is always very slow and is also controlled by particle diffusion, even in very dilute solutions. In most chromatographic columns, particle diffusion limits the rate of exchange. An increased flow-rate through the column will decrease the thickness of the Nernst film and thus will favour mass-transfer contiol. The theoretical treatment of ion-exchange kinetics is very complicated. When the mobilities of exchanging ions are different (pw f p w in Fig. 6.4), an electrical potential gradient is formed which affects the diffusion of both types of counter-ions. The faster ion will be held back, while the slower ion will be accelerated. However, the condition of electrical neutrality must be maintained. The ion leaving the exchanger plays the most important part in determining the rate ( p ~in( Fig. 6.4). The reader who requires more detailed information concerning ion-exchange kinetics should consult more specialized publications, e.g., Helfferich (1966).

COLUMN OPERATION AND ION-EXCHANGE CHROMATOGRAPHY A very efficient means of achieving ion exchange is to pack the beads into a column and then slowly filter the electrolyte through it. The solution entering the column is called the influent and that leaving the column the effluent. By the term capacity of the column is meant the total number of exchangeable groups (in milliequivalents) in the column. When the column is being saturated with a new electrolyte, a small part of the sorbed substance reaches the bottom of the column before the total capacity of the column is exhausted. The break-through capacity of the column is defined by the sorption ability up to the appearance of the sorbed substance in the effluent, and depends on the particle size of the sorbent, flow-rate and composition of the influent. When the sorption process is continued after the break-through, the concentration of the sorbed substance in the effluent eventually reaches that of the influent. This process is usually expressed by break-through curves, the slopes of which define the quality of the sorption and show the chromatographic efficiency of the column under the operating conditions. The steeper the break-through curve, the better is the sorption process. When the influent contains two or more ions, the ion exchanger in the column can act as a chromatographic agent. As the ions differ in their affinity for the exchanger, they are sorbed to different extents, or they are sorbed completely at the top of column and then eluted gradually using different conditions. Ion-exchange chromatography is the dynamic

COLUMN OPERATION AND ION-EXCHANGE CHROMATOGRAPHY

81

replacement of zones of bound ions by ions newly entering the column, accompanied by their separation. The separation depends not only on the difference in exchange potential (in dissociation constants) between the substances being separated, but also on their adsorptivity on the network of the exchanger. All three general types of chromatography can be realized on ion exchangers: frontal analysis, displacement chromatography and elution chromatography. Frontal analysis is the most simple technique. A solution containing the counter-ions to be separated, e.g., A, B and C, is pumped t o the top of the column, which is in the M form (M having lowest affinity for the exchanger). At first, only M ions are found in the effluent, indicating the exchange A tM, B + M and C + M. Then A ions emerge, these having the least affinity in the influent mixture, which indicates that the capacity of the column is exhausted. The column is now in the A form and the exchange is limited to B + A and C + A. A ions can be isolated in a pure form from the effluent only during this stage. After some time, A ions begin to be accompanied by B ions, the effluent having the composition A + B, which indicates that the column is in the B form, and ion exchange is limited to the process C + B. Finally, the whole column is in the C form and no separation takes place, the effluent now having the same composition, A, B and C, as the influent. Frontal analysis in ion exchange has little practical value for preparative purposes and today it also has little importance for analytical use. Displacement chromatography differs from frontal analysis in the experimental procedure required. Let us suppose the same mixture of counter-ions, A, B and C, is applied to the column of exchanger in the M form. The relative affinities are the same. However, only a limited amount of solution with the mixture to be resolved is applied to the top of the column, then a new solution containing the counter-ions Y,having the highest affinity for the exchanger, is used as the influent. During the flow through the column, the ions are arranged in the sequence of their affinities. M is eluted first, followed by A, which is displaced by B, and the following C displaces B. All three types of ions are forced down the column by Y ions, the function of which may be compared with a piston. The yield of pure components separated by this procedure is not high, as mixing occurs across the zones of separation giving an effluent composition of A, A + B, B, B + C, C, C + Y and Y. Displacement chromatography has limited analytical applications, but is sometimes used for preparative purposes as the column capacity is better exploited in comparison with elution chromatography, described below. The most important method is elution chromatography, in which a much smaller amount of material can be applied to the column compared with displacement chromatography. However, the separation of components is often complete and this procedure is therefore very valuable for many analytical applications and for those preparative purposes where the complete or very effective separation of components is required. In order to describe the principle of the method, let us assume that the same mixture of counter-ions, A, B and C, is used and that the column is in the M form, M having the lowest affinity for the exchanger. A dilute solution containing a small amount of A, B and C is applied to the column, so that all the ions are sorbed in a zone at the top of the column. Then an eluting solution containing the counter-ion M, at a low concentration, is used as the influent, which slowly releases the ions A, B and C from their positions at the top of the column and washes them down. Because they have higher affinities than the ion M for the References p.86

82

FUNDAMENTALS OF ION-EXCHANGE CHROMATOGRAPHY

exchanger, a relatively large amount of the eluting solution is required in order to effect a short movement of zones A , B and C. The important fact here is that during elution, the movement of ions A, B and C is governed solely by ion-exchange equilibria:

RA + M =+RM + A

* RM + B RC + M + RM + C

RB + M

where R = resin. This depends on the different affinities of A, B and C, the mobilities of these ions being independent. Therefore, they can emerge in the effluent as individual peaks separated by pure eluting solution. Because of the importance of this technique, elution ion-exchange chromatography will be discussed further in this section. Martin and Synge were the first to apply the idea of a theoretical plate (cfi, Chapter 3 ) to column chromatography and Mayer and Tompkins were the first to extend it to ion-exchange columns. It was found that the particle size has the most important influence on the efficiency of ion-exchange chromatography. If column elution is slow enough to reach equilibrium, then the height equivalent of a theoretical plate (HETP) is approximately equal to the diameter of the particle. Such low values are seldom obtained in practice, as channelling in the column (due to irregularities in the packing) causes this value to be several times higher. Glueckauf (1955a, b) derived formulae from which the plate height can be calculated under non-equilibrium conditions, such conditions usually being found in practice. The HETP, H , is divided into three terms: (1) H particle size = 1.64 r (corresponds to the conditions of equilibrium);

(2)

particle diffusion

-

D,

(D”

0.142.r’. F .

Ds

.’

where r = particle radius, D, = volume distribution coefficient (amount of sorbed solute per ml of the column divided by the amount of solute per ml of solution), F = linear flowrate of solution in the column above the resin bed (cmlsec or (ml of solution/cmZ of column)/sec), E = void fraction of column and Ds and DL are diffusion constants (cm2/ sec) for the solute in the resin and in the interstitial volume, respectively. The HETP increases under non-equilibrium conditions, the value being given by: = Hparticle size + Hparticle diffusion -k Hfiilm diffusion

Fig. 6.5, according to Glueckauf(l955a), shows the factors that control the HETP according to the operating conditions. Usually, ion-exchange chromatography is carried out in the particle diffusion area of this graph. With a moderate flow-rate and a high distribution coefficient, film diffusion is the main factor. At very slow flow-rates, equilibnun: is reached. Because the distribution coefficient can be changed by varying the concentration of the eluting solution, according to this theory it is possible to operate in any part of the diagram. Sharp peaks with an acceptable rate of elution are obtained when operating in the vicinity of point B in the diagram.

ION EXCLUSION, ION RETARDATION AND THE ION-SIEVE PROCESSES

83

I

I

380 r (LEVEL)

300 r 200 r

-

100 r

-

-

- % m

Q

FILM DIFFUSION

- 0

- Ic - -8

-

lor

3.3 r EFFECTIVE EQUILIBRIUM

2 r

7

-6 LONGITUDINAL DIFFUSION

-7

-1

0

+1

2

3

4

5

6

7

Fig. 6.5. Theoretical plate height as a function of operating conditions (after Glueckauf, 1955a). r = radius of resin beads; F = linear flow velocity (cmlsec); E = void fraction of the column; D = distribution coefficient (amount of solute per gram of resin divided by amount of solute per cubic centimetre of the solution); a = distribution coefficient (total solute per cubic centimetre of the column divided by dissolved solute per cubic centimetre of the solution).

According to the theoretical plate concept, it is possible t o predict the length of column necessary for a particular separation. Selected examples of such calculations for inorganic ions have been given in monographs (Helfferich, 1962a; Samuelson; Walton, 1967). These calculations are valid if it is assumed that there is a linear exchange isotherm, small and nearly equal amounts of separating substances, not exceeding a few per cent of the column capacity, and perfect packing of the column. For most laboratory separations, the necessary data for these calculations are not available. Therefore, orienting experiments based on published data must substitute for a mathematical treatment and consequently the examples given in the Chapters 32 and 34-37 in this book are very important.

ION EXCLUSION, ION RETARDATION, THE ION-SIEVE PROCESS AND PARTITION CHROMATOGRAPHY ON ION EXCHANGERS The principle of ion exclusion can be explained simply by considering the Donnan equilibria that affect the ion-exchange process. Let us suppose that one bead of cation exchanger in the K' form comes into contact with a solution of a salt. For simplification, let us consider a salt containing the same cation, e.g., KC1, and in addition some non-ionic soluble substance (e.g., ethylene glycol). The non-electrolyte diffuses into the bead without hindrance, but the diffusion of both ions of the salt is greatly affected. Because of the References p.86

84

FUNDAMENTALS OF ION-EXCHANGE CHROMATOGRAPHY

hgh concentration of K‘ originally present in the bead, the entry of additional ions of the same charge is limited and this factor also influences the penetration of C1-. In order to maintain a neutral charge, the total number of C1- ions penetrating must equal the number of K’ ions that penetrate the bead. After equilibrium has been reached, the relative concentrations of salt and non-electrolyte in the bead therefore differ from the relative ccncentration of both components in the solution. This shift is repeated within the chromatography of the mixture of both components on a column of ion exchanger and results in their separation. The peak of salt is excluded before the peak of non-electrolyte and therefore this process is called “ion exclusion”. In effect, ion retardation is the opposite of ion exclusion. Let us consider the same example as above (potassium chloride and ethylene glycol), the only difference in this case being the use of a special amphoteric ion exchanger called a “snake-cage’’ resin, which contains two types of functional groups, both cationic and anionic, rotating on the same matrix. These groups are internally neutralized and when the internal salt linkages have been split by the penetration of a salt solution, the functional groups are capable of binding both cations and anions. In the example given above, the K‘ ions of the salt are bound to acidic groups and the C1- ions to basic groups. After equilibrium has been reached, the salt is concentrated in the grains of the ion exchanger. Molecules of the glycol penetrate the grain freely and diffuse out without being affected. The chromatographic effect appears when the mixture of salt and non-electrolyte flows slowly through a column prepared from this type of resin. The glycol peak in our example is eluted first because the salt is bound. However, the ions of the salt are in dynamic equilibrium with the functional groups and the flow of water through the column causes this equilibrium to be slowly shifted in the direction of flow. After the resin has been thoroughly washed with water, the futictional groups will again form “inner salts”, and the result of this process is the retardation of the electrolytes, which are then eluted as a second separate peak. Therefore this process is called “ion retardation”. There are two main advantages that ion-exclusion and ion-retardation chromatography possess over conventional ion-exchange methods: (1) electrolytes and non-electrolytes can be separated and (2) the column does not require regeneration after use. Simple elution of both peaks is sufficient, the column then being ready for the next separation. The disadvantage of ion exclusion is the dilution of non-electrolyte. In some instances, ion exchangers can be used to effect chromatography based on other principles. Sometimes this is regarded as a special form of partition chromatography ( c t , Reichenberg). The use of ion sieves permits the separation of large ions from small ions; a suitable degree of cross-linking allows the penetration of the small ions, while the large ions are excluded. The same principle is valid for the separation of non-electrolytes. For example, D-glucose can be separated from methanol using X8 resin. In other cases, Van der Waals forces play a role, ex., a mixture of acetic acid and n-butyric acid can be separated on X5 sulphonated polystyrene resin with water as eluent, the n-butyric acid being retarded. Several separations are known that indicate complicated polar interactions, which influence the chromatographic process, between solute molecules and the functional groups of the exchanger.

LICAND-EXCHANGE CHROMATOGRAPHY

85

LIGAND-EXCHANGE CHROMATOGRAPHY The complex-forming ability of some ions plays an important role in chromatography, because it often improves the selectivity of ion exchange. The interaction of ions with buffers is assumed to occur in this case and the methods describirig the exchange of ions in complex form are treated briefly in Chapter 10 (p.267). In this section, another method will be mentioned, namely the so-called ligand-exchange or ligand chromatography. In this case, a metal ion capable of forming complexes (e.g., Ag+, Cuz+,Ni2+ or Fe3+)is attached firmly to the exchanger and the ligands are exchanged (i.e., the molecules coordinated to the metal ion). Sjostrom attached Fe3+ to a cation exchanger and P-diketones were sorbed selectively on such a column. According to Helfferich (1961, 1962b), amines are sorbed on a cationexchange column loaded with a nickel-ammonia complex and displace ammonia, which can be used in the next step as an eluting agent. Wuster et al. sorbed esters of unsaturated acids on a resin loaded with silver (which forms complexes with n-electrons of double bonds; the so-called argentation chromatography should be mentioned here, cl: , Morns and Nichols). Latterel and Walton and Shinomura e t al. separated amines, and Hill er af. compared the selectivities of the complex-forming Niz+ bound to organic resins and to inorganic exchangers (zirconium phosphate). Seigel and Degens used the commercial chelating resin Chelex 100 (Cu”) for the isolation of amino acids from sea-water and thus extended the use of ion-exchange chromatography to solutions of high ionic strength (saline, brines, etc.). Buist and O’Brien separated peptides from amino acids in urine by the same procedure. Goldstein isolated nucleotides, nucleosides and nucleic acid bases by this method.

ION EXCHANGE IN NON-AQUEOUS SOLUTIONS True ion exchange is also possible in organic solvents and in mixed solutions and alcohols, acetone and other solvents have been used in the pure form and in mixtures with water. The necessary condition is the partial dissociation of the solute. The variation of swelling due t o changes in solvent composition often makes chromatography very difficult, especially when stepwise or gradient elution is used. The resin requires preliminary conditioning with the solvent before application of the sample. The distribution of organic solvents between the resin phase and the outer solution is complicated and depends on the polarity of the organic solvent, its concentration in the solution, the ionic form of the exchanger and the composition of the matrix. London forces between the solvent and the matrix often play an important role, and the chemical similarity of the network and the solvent facilitates penetration and swelling. In some cases, the ion-exchange potentials of particular ions are increased in mixtures with water and organic solvents, but the rate of exchange is slower in non-aqueous systems and diminishes with decreasing polarity of the solvent. Under these conditions, gel diffusion is usually the main factor that determines the rate of exchange. Macroreticular resins are very useful for this type of chromatography owing to the presence of macro-pores inside the matrix; the separation is much faster. References p.86

86

FUNDAMENTALS OF ION-EXCHANGE CHROMATOGRAPHY

The capacity of the exchanger is not usually exhausted in non-aqueous or mixed solutions, but in some instances neutral adsorption of electrolytes causes a large uptake of ions. It is also possible to achieve some special ion exchanges in water-free ammonia. Generally, chromatography in non-aqueous or mixed solutions is used very seldom compared with chromatography in aqueous solutions. In special instances it may offer some advantage, but usually there are difficulties. It is not possible t o give a general approach for specific problems of t h ~ type. s Samuelson published a short survey of this type of chromatography.

REFERENCES Boyd, G. E., Adamson, A. W. and Myers, Jr., L. S., J. Amer. Chem. SOC.,69 (1947) 2836. Buist, N. M. R. and O'Brien, D.,J. Chromatogr., 29 (1967) 398. Dorfner, K., lonenaustauscher: Eigenschaften und Anwendungen, De Gruyter, Berlin, 1963a. Dorfner, K., Ionenaustauschchromatographie, Akademie-Verlag. Berlin, 1963b. Raschka, H. A. and Barnard, J. R., Chelates in Analytical Chemistry, Marcel Dekker, New York, 1967. Genge, J. A. R., in D. R. Browning (Editor), Chromatography, McGraw-Hill, London, 1969. Glueckauf, E., Ion Exchange and Its Application, Society of Chemical Industry, London, 1955a. Glueckauf, E., Trans. Faraday SOC.,51 (1955b) 34. Goldstein, G., Anal. Biochem., 20 (1967) 441. Griesbach, R., Ionenaustauscher in Einzeldarstellungen, Akademie-Verlag, Berlin, 1957. Helfferich, F. G., Nature (London), 189 (1961) 1001. Helfferich, F. G., Ion Exchange, McGraw-Hill, New York, 1962a. Helfferich, F. G., J. Amer. Chem. Soc., 84 (1962b) 3237 and 3242. Helfferich, F. G-, in J. A. Marinsky (Editor), Ion Exchange, Marcel Dekker, New York, 1966, p. 65. Hering, R., Chelatbildende lonenaustauscher, Akademie-Verlag, Berlin, 1967. Hill, A. G., Sedgley, D. and Walton, H. F., Anal. Chim. Acfa, 33 (1965) 84. Inczedy, J., Analytical Applications of Ion Exchangers, Pergamon Press, New York, 1966. Kressman, T. R. E.,J. Phys. Chem., 56 (1952) 118. Kunin, R., in E. Heftmann (Editor), Chromatography, Reinhold, New York, 1961, p. 315. Kunin, R. and Myers, R. J., Ion Exchange Resins, Wiley, New York, 1952. Latterel, J. J.and Walton, H. P., Anal. Chim. A c f a , 32 (1965) 101. Marinsky, J. A. (Editor), Ion Exchange, Marcel Dekker, New York, 1966. Martin, A. J. P.andSynge, R. L. M., Biochem. J . , 35 (1941) 1358. Mayer, S. W. and Tompkins, E. R., J. Amer. Chem. SOC.,69 (1947) 2866. Mikeg, O., in 0. MikeS (Editor), Laboratory Handbook of Chromatographic Methods, Van Nostrand, London, 1964, p. 247. Morns, C. J. 0. R. and Morris, P.,Separation Methods in Biochemistry, Pitman, London, 1964. Morns, L. I. and Nichols, B. W., in E. Heftmann (Editor), Chromatography, Reinhold, New York, 2nd ed., 1967, p. 466. Nachod, F. C.,Zon Exchange, Academic Press, New York, 1949. Novotn);, J., FEBS Lett., 14 (1971) 7. Novotn);, J., FranBk, F. and Sorm, F., Eur. J. Biochem., 16 (1970) 278. Osborn, G. H., Synthetic Ion Exchangers, Chapman and Hall, London, 2nd ed., 1961. Paterson, R., A n Introduction to Ion Exchange, Heyden and Sons, Philadelphia, 1970. Pepper, K. W., Reichenberg, D. and Halle, D. K., J. Chem. SOC.,(1952) 3219. Porath, J. and Fryklund, L., Nature (London), 226 (1970) 1169. Reichenberg, D., in C. Calmon and T. R. E. Kressman (Editors), Ion Exchangers in Organic and Biochemistry, Interscience, New York, 1957, p. 178. Reichenberg, D. and Wall, W. F., J. Chem. SOC.,(1956) 3364.

REFERENCES Reuter, H., Kunstharzionenaustauscher (Syrnposiumbericht) , Akademie-Verlag, Berlin, 1970. Saldadze, K. M., Pachkov, A. S. and Titov, V. S., Ionoobmennye Vysokomolekularnye Soedineniya, Goskhim. Izdat., Moscow, 1960. Samuelson, O., Ion Exchange Separation in Analytical Chemistry, Wiley, New York, 1963. Seigel, A. and Degens, E. T., Science, 151 (1966) 1098. Shinomura, K., Dickson, L. and Walton, H. F., Anal, Chim. Acta, 37 (1967) 102. Sjostrom, E., Trans. Chalmers Univ. Technol., Gothenburg, 136 (1953) 7. Skogseid, A., Dissertation, Norges Techniske Hogskole, Trondheim, 1946. Walton, H. F., in E. Heftmann (Editor), Chromatography, Reinhold, New York, 2nd ed., 1967, pp. 287 and 325. Walton, H. F.,Anal. Chem., 40 (1968) 51R. Walton, H. F.,Anal. Chem.,42 (1970) 86R. Walton, H. F., Anal. Chem.,44 (1972) 256R. Wuster, C. F., Copenhauer, J . H. and Shafer, P. R., J. Amer. Oil Chem. s o c . , 4 0 (1963) 513.

87