Chapter 6 Ion-Interaction Chromatography

165

Chapter 6 Ion-Interaction Chromatography 6.1

INTRODUCTION

Hydrophilic ionic solutes, such as the inorganic anions and cations of interest in IC, show little or no retention on lipophilic stationary phases when typical reversed-phase eluents are used. However, retention and subsequent separation of such ionic solutes on these stationary phases can be achieved by the addition to the eluent of a lipophilic reagent ion having the opposite charge sign to that of the solute ion. This added reagent ion, and the chromatographic process itself, have been described by a variety of names, some of which are listed in Table 6.1. Most of these names impIy some sort of mechanism for the process and may therefore be misleading. Throughout this book, the terms ion-interaction chromatography and ion-interaction reagent (IIR) will be used, since these are quite general terms. In this Chapter, we will examine some of the mechanisms which have been proposed for ion-interaction chromatography and we will then consider the types of stationary phases and eluents which are used with this technique. Specific applications of ion-interaction chromatography to the separation of inorganic anions and cations will then be discussed.

TABLE6.1 ALTERNATIVE NAMES USED TO DESCRIBE ION-INTERACTION CHROMATOGRAPHY AND THE REAGENT ION ADDED TO THE ELUENT [11 Chromatographic process

Reagent ion

Reference

Ion-pair chromatography Paired-ion chromatography Surfactant chromatography Dynamic ion-exchangechromatography Ion-interactionchromatography Hetaeric chromatography Mobile phase ion chromatography (MPIC)

Pairing ion PIC reagent Surfactantion Ion-pairing reagent Ion-interaction reagent Hetaeron Pairing reagent

2 3 4 5

6 7

7

Chapter 6

166

6.2

MECHANISM

6.2.1

Trends in solute retention in ion-interaction chromatography

A convenient way to highlight the trends in solute retention is to compare the retention of a solute on a chromatographic system comprising a lipophilic stationary phase and an eluent consisting of an IIR dissolved in a mixture of water and one or more organic solvents with the retention of the same solute under the same chromatographic conditions, except using an eluent which does not contain the IIR. When this comparison is made, the following trends are observed: (i)

The retention of neutral solutes is not altered significantly when the IIR is added to the eluent. (ii) The retention of solutes having the same charge as the IIR is decreased when the IIR is added to the eluent. (iii) The retention of solutes having the opposite charge to the IIR is increased when the IIR is added to the eluent.

In addition, the following effects on retention are observed when the composition of the eluent is altered: (iv) The retention of solutes having the opposite charge to the IIR is increased when the concentration of IIR in the eluent is increased. (v) The retention of solutes having the opposite charge to the 11R is increased when the lipophilicity of the IIR is increased. (vi) Retention of all solutes decreases when the percentage of modifier in the eluent is increased, and vice versa. Any mechanism suggested for ion-interaction chromatography must necessarily explain these trends in retention behaviour. A large volume of literature has been devoted to the study of such mechanisms and a detailed discussion of this literature is beyond the scope of this book. Moreover, the most of these studies have been devoted to mechanisms for the retention of orgunic ionic species, such as carboxylic acids and organic bases. A summary of the most commonly suggested mechanisms [l] will be presented below and this will be followed by an evaluation of these mechanisms in terms of their applicability to the retention of inorganic ions. 6.2.2

The ion-pair model

In this model [7- 1 I], an ion-pair is envisaged to form between the solute ion and the IIR. This occurs in the aqueous-organic eluent and the resultant neutral ion-pair can then be adsorbed onto the lipophilic stationary phase in the same manner that any neutral molecule with lipophilic character is retained in reversed-phase chromatography. Retention therefore results solely as a consequence of reactions taking place in the eluent. The degree of retention of the ion-pair is dependent on its lipophilicity, which in turn depends on the lipophilicity of the IIR itself. Neutral solute molecules are unaffected by

167

Ion-InteractionChromatography

Bulk eluent

Fig. 6.2 Schematic illustration of (a) the ion-pair, (b) the dynamic ion-exchange and (c) the ioninteraction models for the retention of anionic solutes in the presence of a lipophilic cationic IIR. The solute and the IIR are labelled on the diagram. The large, hatched box represents the lipophilic stationary phase, the black circle with the negative charge represents the counter-anion of the IIR, whilst the white circle with the positive charge represents the counter-cationof the solute. Adapted from [ 11.

the presence of the IIR in the eluent and interact with the stationary phase in the conventional reversed-phase manner. An increase in the percentage of organic solvent in the eluent decreases the interaction of the ion-pairs with the stationary phase and therefore reduces their retention. The ion-pair model is illustrated schematically in Fig.

168

Chaprer6

6.l(a), using a positively charged IIR and a negatively charged solute as an example. 6.2.3 The dynamic ion-exchange model The dynamic ion-exchange model [12-151 proposes that a dynamic equilibrium is established between IIR in the eluent and IIR adsorbed onto the stationary phase, as follows:

where the subscripts E and S refer to the eluent and stationary phases and the superscript on the IIR indicates that it may carry either a positive or negative charge. The adsorbed 1IR imparts a charge to the stationary phase, causing it to behave as an ion-exchanger. The total concentration of IIR adsorbed onto the stationary phase is dependent on the percentage of organic solvent in the eluent, with higher percentages of solvent giving lower concentrations of IIR on the stationary phase. In addition, the more lipophilic the IIR, or the higher is its concentration, then the greater is its adsorption onto the stationary phase. Thus, for a given eluent composition, the concentration of adsorbed IIR (and hence the "ion-exchange'' capacity of the stationary phase) remains constant. However, constant interchange of IIR occurs between the eluent and stationary phase, so the stationary phase can be considered to be a dynamic ion-exchanger. Introduction of a solute with opposite charge to the IIR results in retention by a conventional ion-exchange mechanism. The competing ion in this ion-exchange process may be the counter-ion of the IIR, or another ionic species deliberately added to the eluent. Since the retention times will be dependent on the ion-exchange capacity of the column, they are also dependent on the lipophilicity of the IIR and the percentage of organic solvent in the eluent. Solutes having the same charge as the IIR are repelled from the charged stationary phase surface and show decreased retention times in comparison to those observed in the absence of IIR, whilst retention times for neutral solutes are unaffected by the IIR. Fig. 6.l(b) gives a schematic representation of the dynamic ion-exchange model, again using a positively charged IIR and a negatively charged solute as an example. 6.2.4 The ion-interaction model

The ion-interaction model [ I . 6, 16-19] can be viewed as intermediate between the two previous models in that it incorporates both the electrostatic effects which are the basis of the ion-pair model and the adsorptive effects which form the basis of the dynamic ion-exchange model. The lipophilic IIR ions are considered to form a dynamic equilibrium between the eluent and stationary phases, as depicted in eqn. (6.1). This results in the formation of an electrical double-layer at the stationary phase surface. The adsorbed IIR ions are expected to be spaced evenly over the stationary phase due to repulsion effects, which leaves much of the stationary phase surface unaltered by the IIR. The adsorbed IIR ions constitute a primary layer of charge, to which is attracted a diffuse, secondary layer of oppositely charged ions. This secondary layer of

Ion-InteractionChromutogrqhy

169

charge consists chiefly of the counter-ions of the IIR. The amount of charge in both the primary and secondary charged layers is dependent on the amount of adsorbed IIR, which in turn depends on the lipophilicity of the IIR, the IIR concentration, and the percentage of organic solvent in the eluent. The double-layer is shown schematically in the top frame of Fig. 6.l(c). Transfer of solutes through the double-layer to the stationary phase surface is a function of electrostatic effects and of the solvophobic effects responsible for retention in reversed-phase chromatography. Neutral solutes can pass unimpeded through the double layer, so their retention is relatively unaffected by the presence of IIR in the eluent. A solute having opposite charge to the IIR can compete for a position in the secondary charged layer, from which it will tend to move into the primary layer as a result of electrostatic attraction and, if applicable, reversed-phase solvophobic effects. The presence of such a solute in the primary layer causes a decrease in the total charge of this layer, so to maintain charge balance, a further IIR ion must enter the primary layer. This means that the adsorption of a solute ion having opposite charge to the IIR will be accompanied by the adsorption of an IIR ion. The overall result is that solute retention involves a pair of ions (that is, the solute and IIR ions), but not necessarily an ion-pair. This process leads to increased retention of the solute compared to the situation in which the IIR is absent from the eluent. The lower frame of Fig. 6.l(c) depicts this process for a positively charged IIR and a negatively charged solute. Solutes having the same charge as the IIR will show decreased retention due to electrostatic repulsion from the primary charged layer. 6.2.5 Evaluation of mechanistic models in retention of inorganic ions

Many studies have examined the applicability of the above models to the retention of organic species [e.g. 20-231, but we will consider here only the case of inorganic ions. Such species are very hydrophilic and, in most cases, are unlikely to form ion-pairs in aqueous-organic solutions. Moreover, conductance measurements would be expected to reveal the formation of ion-pairs and such measurements have failed to provide supporting evidence for significant ion-pair formation [6,241. Furthermore, the ionpair model would require that the neutral ion-pairs formed by different solute ions should have varying degrees of lipophilicity in order for them to be separated. These differences can be expected to be very slight for a series of inorganic ions (e.g. C1-, Br-, N a - , NOS-and S04*-),yet the ensuing discussion in this chapter will show that these species are separated readily by ion-interaction chromatography. Despite these shortcomings, there is a persistent trend in the literature to discuss the retention of inorganic ions in this form of chromatography in terms of interactions occurring between the solute and IIR in the eluentphse. The dynamic ion-exchange model generally provides an accurate prediction of the retention order of solutes, since this usually follows the established ion-exchange selectivity order discussed earlier in Chapter 2. In addition, the role of the counter-ion of the IIR is also predicted correctly if this counter-ion is considered to act as an ionexchange competing ion. Nevertheless, there are some shortcomings to the dynamic ionexchange model. Once such shortcoming can be seen by comparing the elution behaviour of solutes in an ion-interaction system in which a particular competing ion is

170

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Ion-Interaction Chromatography

17 1

used to that of a conventional fixed-site ion-exchange system in which the same competing ion is employed. In the latter system, there will be a stoichiometric exchange of solute and competing ions at the ion-exchange site, so that the elution of a solute ion will always be accompanied by a decrease in the concentration of competing ion. This behaviour is not always observed in ion-interaction chromatography, where increases in the concentration of the IJR and its counter-ion often occur [25]. A more detailed discussion of this aspect and its utility for detection purposes will be found in Section 12.3.2. A study of the retention of inorganic anions in ion-interaction chromatography showed results which were in general agreement with the dynamic ion-exchange retention model, but some significant deviations from the predicted dependences indicated that the actual mechanism was more complex [26]. The ion-interaction model, and the formation of an electrical double-layer at the stationary phase surface, gives the most consistent agreement with experimental measurement. Consideration of the double-layer in terms of the Stern-Gouy-Chapman theory enables the effect on solute retention of the ionic strength of the eluent to be predicted accurately for lipophilic organic solute ions [ 161. This approach also permits the effect on solute retention of the concentration of IIR to be predicted. Similarly, electrostatic surface potential calculations, coupled with a Langmuir isotherm for adsorption of the IIR, predicts solute retention behaviour which is in good agreement with experimental results [17, 181. Moreover, studies concerned specifically with the retention of inorganic anions [27] and cations [28] in ion-interaction chromatographic systems have concluded that the ion-interaction model is the most appropriate.

6.3

STATIONARY PHASES AND ELUENTS

6.3.1 Stationary phases Ion-interaction chromatography has been performed successfully on a wide range of stationary phases, including neutral polystyrene divinylbenzene (PS-DVB) polymers 1e.g. 29, 301 and bonded silica materials with c18 [e.g. 311, c8 [32], phenyl [33] and cyano [34] groups as the chemically bound functionality. Each of these stationary phases gives satisfactory retention of ionic solutes, provided the eluent composition is such that an appropriate amount of the IIR is adsorbed. The choice between stationary phases is usually based on such considerations as chromatographic efficiency [35], pH stability [36] and particle size 1371, rather than on differences in chromatographic selectivity. However, it has been noted 1381 that the elution order for solutes can vary when the nature of the stationary phase used to support the IIR is altered. This point is illustrated in Fig. 6.2, which shows chromatograms for inorganic anions, obtained on three different stationary phases. It can be noted that the elution position of sulfate differs markedly between the c18 and PS-DVB stationary phases. Also apparent is the improved chromatographic efficiencies of the silica-based stationary phases compared to that of the PS-DVB material. Further factors to be considered sin the selection of a stationary phase for ioninteraction chromatography are specific interactions existing between the stationary phase and either the IIR or the solutes, and the role of residual silanol groups on silica-

172

Chapter 6

TABLE 6.2 TYPICAL REAGENTS USED AS IIRs IN DYNAMIC COATING ION-INTERACTION CHROMATOGRAPHY OF ANIONS AND CATIONS IIR

Detection mode$

References

Tetramethylammonium Tetrapmpylammonium Tetrabutylammonium Tetrapentylammonium Hexylammonium

spec C, Indirect Spec, Amp C, Spec,Amp spec c,spec, Indirect spec c,spec, Indirect spec spec, ICP C, Spec,Amp spec Spec. Amp Indirect spec Indirect spec Indirect spec Indirect spec Indirect spec Indirect Fluor

38,42 36,43,44 45-47 17, 24, 29 48 49-5 1 41,52-54 26, 31, 53, 55 56 57 58.59 59, 60 59, 60 61 62-64 65

spec RI, ICP spec Spec, PCR. RI PCR Indirect spec Spec,Indirect Amp

66

octylammonium

Hexadecyltrimethy l u m cetyl~thyl~nium Tricaprylylmethylammonium Dodecyltriethylammonium Benzyltributylammonium Naphthylmethy lmbutylammonium Naphthylmethy l t r i p r o p y l a m m o n i u m Methylpyridinium Iron(II)-l,I@phenanthroline Ruthenium(II)-l,lO-phenanthroline on -ations Butanesulfonate Pentanesulfonate Hexanesulfonate Octanesulfonate Dodecylsulfate

Naphthalenesulfonate Diethyldithidamate a

6, 67 66 28, 68,69 70 71 72,73

Spec = spectmphotometry, Amp = axnperomeay, C = conductivity, Fluor = fluorescence, ICP = inductively coupled plasma atomic emission, PCR = post-column reaction, RI = refractive index.

based stationary phases. Some IIRs (e.g. cetylpyridinium ions) and solutes (e.g. iodide) show particularly strong adsorption to PS-DVB stationary phases and this has been attributed to the occurrence of K-x interactions with the aromatic moiety of the polymer [35]. Residual silanol groups on silica-based packings have been shown to act as weak cation-exchange sites and this behaviour exerts an influence on the ion-interaction separation of both anions and cations on these stationary phases [29]. Scrutiny of the literature reveals that the majority of ion-interaction separations are performed on conventional Cis silica-based reversed-phase materials or on neutral PSDVB polymers (such as Hamilton PRP-I, Rohm & Haas XAD-2 and Dionex MPIC columns).

173

Ion-Interaction Chromatography

TABLE6.3 EFFECT OF THE ALKYL CHAIN LENGTH OF THE IIR ON RETENTION TIMES OF ANIONS [37] Solute

Retention ti&

Chloride Bromide Fluoride Iodide Nitrate

(min)

Hexylamine salicylate

Octylamine salicylate

DeCyl& Salicylae

n.r.b n.r. n.r. n.r. n.r.

2.0 f 0.3 2.0 f 0.3 2.3 f 0.3 2.5 f 0.3 2.5 f 0.3

7.0 f 0.3 7.3 f 0.4 8.0 f 0.4

6.5 f 0.3

8.3 f 0.4

A Lichrosphere RP-18column (5 p n particle size) was used. The eluent concentration was 5 mM and the flow-rate was 2.0 mumin. b n.r. = not retained.

a

6.3.2 Type of ion-interaction reagent

Requirements of the IIR The most important component of the eluent in ion-interaction chromatography is the IIR itself. The prime requirements of the IIR are as follows: (i) (ii) (iii) (iv)

An appropriate charge, which is unaffected by eluent pH. Suitable lipophilicity to permit adsorption onto non-polar stationary phases. Compatibility with other eluent components. Compatibility with the desired detection system.

"Dynamic coating" ion-interaction chromatography Anion separations are normally performed using strong base cations, such as tetraalkylammonium ions, as the IIR whilst cation separations are usually performed using strong acid anions, such as aliphatic sulfonate ions. Table 6.2 lists some IIRs which are used for ion-interaction separations. In each case, the IIR is present at a constant, specified concentration in the eluent in order to maintain a desired concentration of IIR on the stationary phase. That is, the coating of IIR is in dynamic equilibrium, as shown in eqn. (6.1). and the column can be said to be "dynamically coated" with IIR. Table 6.2 shows that the IIRs used for dynamic coating ion-interaction chromatography vary from moderately lipophilic (e.g. tetrabutylammonium ions) to very lipophilic (e.g. hexadecyltrimethylammonium ions). The lipophilicity of the IIR governs the degree of adsorption of the IIR onto the stationary phase, which in turn governs the effective ion-exchange capacity of the column and hence the retention times of solute ions. This point is illustrated in Table 6.3, which lists retention times for

r

L

co

f

i

0.032AU

0.01 AU

i

n Pr NI

M

1

I

0

I

2

I

I

4 6 Time (minl (a1

I

8

I

10

1

11

I

0

I

4

I

8 Time (minl (bl

I

12

1

16

Fig. 6.3 Separation of cations by dynamically coated ion-interaction chromatography. (a) A C1g column was used with 10 mM octanesulfonate and 45 mM tartrate at pH 3.4 as eluent. Reprinted from [5] with permission. (b) A 5pm Supelco C1g column was used with an eluent formed from a linear gradient of 0.05-0.40 mM a-hydroxyisobutyric acid at pH 4.2, containing 30 mM octanesulfonate and 7.5% methanol. Reprinted from [68] with permission. Post-column reaction detection was used in each case.

B

o\

Ion-Interaction Chromatography

175

anions obtained using IIRs with differing alkyl chain lengths (and therefore differing lipophilicity). Some of the detection modes available for use with the various IIRs are also listed in Table 6.2 and a discussion of detection for this mode of chromatography will be presented in Part I11 of this book. It is interesting to note that inorganic complexes, such as the 1,lo-phenanthroline complexes of iron(II1) and ruthenium(I1) can be employed as IIRs. Pietrzyk and co-workers [62, 64, 65, 741 have shown that these complexes are adsorbed readily onto PS-DVB stationary phases and their absorbance or fluorescence can be used for indirect detection of anions. Some typical chromatograms obtained for the separation of anions by dynamic coating ion-interaction chromatography were presented in Fig. 6.2, and typical separations of cations are shown in Fig. 6.3. These chromatograms illustrate the excellent chromatographic efficiency which can achieved using ion-interaction as the separation mode. Many of the species listed in Table 6.2 are surfactants which will form micelles if the IIR concentration exceeds the critical micelle concentration for that particular species. These micelles can be adsorbed onto the stationary phase in the same manner as other IIRs. However, it has been shown that the retention times of anionic solutes in micellar eluents decreases as the concentration of the micellar IIR is increased [53]. This behaviour is opposite to that normally observed and has been attributed to interaction of the solute anions with micelles in the eluent, which reduces the electrostatic interaction of these solutes with adsorbed micelles and thereby reduces their retention. The elution order observed for anions in this system is similar to that for conventional anionexchangers, as illustrated in Fig. 6.4.

"Permanent coating" ion-interaction chromatography A quite distinct alternative to the dynamic coating method can also be used. In this approach, a very lipophilic IIR is used to initially equilibrate the stationary phase and is then removed from the eluent in the actual separation step [5, 35, 401. The equilibration process establishes a very strongly bound coating of IIR on the stationary phase and this coating persists for long periods of subsequent usage. For this reason, the method is known as "permanent coating" ion-interaction chromatography. Since the stationary phase has now been converted into an ion-exchanger by virtue of the adsorbed IIR, the eluents used in the separation step are identical to those employed with conventional fixed-site ion-exchange materials (see Chapter 4). Permanent coating ion-interaction chromatography has a number of attractive features when compared with conventional ion-exchange chromatography. These include:

The ion-exchange capacity of the column can be varied over a wide range by altering the composition of the equilibrating solution. Parameters which may be varied are the lipophilicity and concentration of the IIR and the percentage of organic solvent in the equilibrating solution. (ii) The adsorbed layer of IIR can be removed or renewed as desired. Removal of the adsorbed coating can be accomplished by washing the column with an organic solvent, such as methanol or acetonitrile. (iii) The same column can be converted into an anion-exchanger or a cationexchanger through the use of an appropriate IIR.

(i)

176

r~

0

Chapter6

2

I

6

I

I

10 14 Time (min)

1

18

1

22

Fig. 6.4 Separation of anions by dynamic coating ion-interaction chromatography using a micellar eluent. A Spherisorb c18 column was used with 0.01 M cetyltrimethylammonium chloride (buffered at pH 6.8) and acetonitrile (35%) as eluent. Detection was by UV absorption at 205 nm. Reprinted from [53] with permission.

(iv) The high chromatographic efficiency of reversed-phase packing materials is retained when these packings are converted into permanently coated ionexchangers. Table 6.4 lists some of the IIRs used for permanent coating ion-interaction separations of anions and cations. It will be noted that cetyltrimethylammonium is listed as an IIR in Table 6.4 for the permanent coating method and also appears in Table 6.2 for the dynamic coating method. However, when used for permanent coating, cetyltrimethylammonium is not present in the eluent during the separation step. Permanent coating of the column is usually achieved by passing a solution (approximately M)of the IIR in dilute (5%) methanol or acetonitrile through the column for about 20 min. The purpose of the organic solvent is to wet the surface of the lipophilic stationary phase in order to improve binding of the IIR. This coating procedure typically results in the immobilization of about 50 mg of IIR onto a 25 cm column [401, giving an ion-exchange capacity similar to that for fixed-site ionexchangers used in IC. These coated columns have been found to be stable for at least one week [5].

177

Ion-Interaction Chromatography TABLE 6.4

REAGENTS USED AS IIRs IN PERMANENT COATING ION-INTERACTION CHROMATOGRAPHYOF INORGANIC ANIONS IIR

Anions

Cetyltrimethylammonium Cetylpyridinium Tridodecylmethy l a i m Tetraoctylammonium Trioctylmethylammonium Methyl Green

structure

References

CH3(m2)15(m3)3N+ ~3(cH2)15N+c5HS ~3t~3(cH2)1113N+ [CHdCH2)714N+ CH3[CH3(m2)713N+

75-79 37,40. 80-84 5, 85 5, 82 5 86.87

Cations Eicosanesulfate

As mentioned earlier, the eluents used in permanently coated ion-interaction chromatography are the same as those employed in IC using ion-exchange columns. Thus, aromatic carboxylate ions, such as phthalate [77, 881, hydroxybenzoate [86], salicylate [81] and aimesate [80] are commonly used for anion separations, and tartrate has been used for cations [5]. Fig. 6.5 shows typical chromatograms for the separation of inorganic anions and cations using the permanent coating ion-interaction method. 6.3.3 Role of the counter-ion of the IIR The counter-ion of the IIR fills a very important role in dynamic coating ioninteraction chromatography of anionic solutes. This counter-ion usually acts as an ionexchange competing anion and is responsible for the elution (and in many cases also the detection) of the solute anions. Typical counter-ions are hydroxide [38], fluoride [29], chloride [53], perchlorate [49], bromide [24, 441, phthalate [23, 891, citrate [26] and salicylate [90]. The nature of the counter-ion determines the type of separation which is achieved and the following strengths of counter-ions in reducing the retention of anionic solutes has been reported for a PRP-1 column using a quaternary ammonium salt as the IIR [24,91]:

The counter-ion of the IIR also influences the detection modes which are applicable to a particular separation. This occurs in exactly the same manner as applies in ionexchange chromatography with fixed-site exchangers. Thus, counter ions such as citrate, phthalate and hydroxide are suitable for conductivity detection; hydroxide, fluoride and chloride are suitable for direct spectrophotometric detection; and phthalate is suitable for

I

CI-

02-

I

0

I

L

cu

k:

I

8

I

I

12 16 Time (min) (a I

I

20

I

2L

r

0

I

I

50

I

I

I

I

150 100 Time Is) (b)

I

I

200

I

1

250

Fig. 6.5 Separation of (a) inorganic anions and (b) inorganic cations using permanent coating ion-interaction chromatography. (a) A Hamilton PRP1 column coated with cetyltrimethylammonium bromide was used with 24% methanol - 1 mM potassium hydrogen phthalate - 20 mM Tris buffer (pH 9.3) as eluent. Detection was by indirect spectrophotometry at 282 nm. Reprinted from [84] with permission. (b) A C18 column coated with C20H41S04Na was used with 75 mM tartrate at pH 3.4 as eluent. Detection was by spectrophotometry at 530 nm after post-column reaction. Reprinted from [5 ] with permission.

179

Ion-InteractionChromatography Tetrabutylammonium salicylate (0.4 mM) C18

20

Octylamine (5 .mM) salicylate (5 mM) C18

Cetrimide (1 mM) citrate (4 mM) C18

Cetrimide (0.1 C ) phosphate (0.1 M)

CN

Tetrapentylammonium fluoride (1 mM) PRP-1

I s20;-

Fig. 6.6 Typical retention times for anions in dynamic coating ion-interaction chromatography using c18. CN and PRP-1stationary phases. Data taken from [31,34,37,90,91].

indirect spectrophotometric detection. Fig. 6.6 shows some typical retention times for anionic solutes in dynamic coating ion-interaction chromatography. It is not essential that the counter-ion of the IIR serves as the ion-exchange competing anion. An alternative approach is to use a separate eluent component, such as phosphate [92], citrate [26, 661, oxalate 1661 or phthalate [27], for this purpose. This method is sometimes used to assist in the elution of strongly retained ions. The nature of the counter-ion of the IIR is of less importance in ion-interaction chromatography of cations. The reason for this is that the elution of solute cations is usually accomplished with the aid of a complexing ligand, such as a-hydroxyisobutyric acid 1681, which is added to the eluent. 6.3.4 Summary of eluent and stationary phase effects

The discussion thus far has indicated a number of parameters which affect the adsorption of the IIR onto the stationary phase in ion-interaction chromatography. These parameters are summarized below, together with some other factors which influence the retention of solutes:

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Chapter 6

Fig. 6.7 Dependence of the capacity factor of inorganic anions on the concentration of IIR in the eluent. A Partisil ODs-3 column was used with an eluent containing 1.5 mM phthalate and the indicated concentrationsof tetrabutylanumniurniodide. Reprinted from [27] with permission.

(i) (ii) (iii) (iv) (v) (vi) (vii)

The nature of the stationary phase. The lipophilicity of the IIR. The concentration of the IIR in the eluent. The ionic strength of the eluent. The nature of the competing ion in the eluent. The concentration of the competing ion in the eluent. The eluent pH.

The first four of these factors will determine the surface concentration of the IIR on the stationary phase, and hence the surface charge density and the effective ionexchange capacity. The higher the surface concentration of IIR, the greater is the retention of solutes having a charge sign opposite to that of the IIR. Thus, retention times will increase as the lipophilicity of the IIR is increased and as the percentage of modifier in the eluent is decreased. We can also note that solute retention generally increases with the concentration of IIR in the eluent, but there is a threshold concentration above which solute retention decreases with further increases in the concentration of IIR. This retention pattern is illustrated in Fig. 6.7 and the reasons underlying this behaviour will be explored fully in Section 6.4.1 below. At this stage, it will be sufficient to note that the stationary phase surface becomes saturated with IIR and any further addition of IIR to the eluent results in decreased retention because of the increased concentration of the IIR counter-ion. The nature and concentration of the eluent competing ion (whether this is the counter-ion of the IIR or an ion which is added separately) will determine the retention times and elution order for solute ions. Increases in the concentration of the eluent

Ion-InteractionChromatography

181

competing ion will result in decreased solute retention, in the same manner as observed for ion-exchange separations. Finally, the eluent pH will influence the charges on the competing ion and the solutes, provided that these species are weak acids or bases. An example of this effect is the influence of pH in an ion-interaction chromatographic system using tetrabutylammonium as the IIR and phthalate as the competing anion. Increases in eluent pH over the range 4.0-6.0 cause a decrease in solute retention as a result of increased ionization of phthalate, leading to the formation of a strong, divalent competing anion. 6.3.5

Guidelines for eluent selection in ion-interaction chromatography

It is apparent that selection of the correct eluent composition in ion-interaction chromatography requires consideration of all of the factors discussed in the preceding section. This is especially true of the dynamic coating approach, in which small changes in eluent composition can often result in large variations in retention times. Guidelines for eluent selection in this mode of chromatography are available [8], and are summarized below.

Anion separations The most commonly used IIRs are tetraalkylammonium salts. The more lipophilic IIRs (i.e. those with longer alkyl substituents) are best suited to the separation of hydrophilic anions, such as F,0, NOa-, Br and NO3-, whilst the less lipophilic IIRs are best suited to the separation of hydrophobic anions, such as aromatic sulfonates or sulfates. The counter-anion of the IIR must be selected on the basis of the desired ionexchange competing effects and the detection mode which is to be employed. For example, counter-anions such as phthalate, salicylate or Br- will result in shorter solute retention times than F- and OH, but the use of OH- as the counter-anion is necessary if suppressed conductivity detection is to be employed. The degree of retention which is achieved can then be manipulated by varying the type and amount of organic modifier added to the eluent. Further changes in solute retention times can be accomplished by addition to the eluent of an additional competing anion (e.g. S042-, C032-or Ct), sometimes referred to as an "inorganic modifier", or by varying the eluent pH when the solutes are weak acid anions. Cation separations The major factors to be considered in the separation of cations by ion-interaction chromatography are the type of IIR used, the nature of the eluent ligand, and the eluent pH. Aliphatic sulfonic acids are the most commonly used IIRs and the lipophilicity of the IIR (as determined.by the length of the alkyl chain) exerts a strong effect on solute retention. Elution of solute cations is achieved predominantly by complexation with the eluent ligand, so the conditional formation constants for the solutes are of prime importance. These conditional formation constants are determined by the nature and concentration of the ligand and by the eluent pH. Ligands such as citrate, tartrate, oxalate and a-hydroxyisobutyric acid are suitable, with each of these species showing increased complexation as the eluent pH is raised. This effect occurs only until

182

Chapter6

ionization of the ligand is complete, beyond which point further increases in pH do not significantly alter retention times. 6.4

6.4.1

RETENTION MODELS FOR DYNAMIC COATING IONINTERACTION CHROMATOGRAPHY

Model for anion retention

The following retention model for ion-interaction chromatography under dynamic coating conditions was proposed originally by Iskandarani and Pietrzyk [24] and later modified by Xianren and Baeyens [27]. The model is based on adsorption of the IIR onto the stationary phase. We will consider the situation where a lipophilic stationary phase is equilibrated with an eluent consisting of an IIR (which will be assumed to be a quaternary ammonium salt, designated as Q'C-) and a competing anion, A-:

where A, is the number of free adsorption sites on the stationary phase and the subscripts m and r refer to the mobile (eluent) and stationary phases, respectively. The equilibrium constant for eqn. (6.3) will be denoted by K1 and is given by:

The ion-exchange equilibrium between the competing anion (A') in the eluent and the counter-ion, C-, is given by: (6.5)

and the equilibrium constant, K2,for this equation can be written:

If a solute anion, X-, is now introduced into the chromatographic system, we can write an ion-exchange equilibrium as follows:

and the equilibrium constant, K3, for this equation can be written:

ton-Interaction Chromatography

183 (6.8)

The sorption capacity, KO,for the stationary phase is a measure of the total number of sites that can be occupied in the retention process. We can write a mass balance equation which accounts for all occupied and free sites, as follows:

Now the capacity factor, kx’,for solute X- can be written as: (6.10)

where q is the phase ratio. Combining eqns. (6.3)-(6.9) with elimination of Ar, (QA), and (QB), and substitution of the solution for (QX), into eqn. (6.10) gives:

which can be rearranged to give:

Eqn. (6.12) predicts that the reciprocal of the capacity factor is linearly related to [C-],, [A-lm and [X-lm, and inversely related to [Q+]m.

Effect of [AA‘],,, and [X-1, Fig. 6.8 provides a plot of the reciprocal of capacity factor versus the concentration of competing anion (i.e. [A-lm) in the eluent. In this example, the competing anion was phthalate and each of the parameters [Q+]m, [C-], and [X-], was held constant. The predicted linear relationship between the reciprocal of capacity factor and [A-]m is observed. Fig. 6.9 shows a plot of the reciprocal of capacity factor versus the amount of sample injected (i.e. [X-],), with the parameters [Q+Im,[C-], and [A-]m being held constant. Again, the predicted linear relationship is observed. It is important to note that the amounts of injected sample shown in Fig. 6.9 are relatively high and when smaller amounts of sample are injected, the reciprocal of capacity factor does not alter appreciably when the sample size is varied [24].

184

Chupter 6

30

1

0.0

: 1

I

I

2

I

[KHP] lmM1

I

3

I

1

I

Fig. 6.8 Dependence of the capacity factor of inorganic anions on the concentration of the competing anion bhthalate) added to the eluent. A Partisil ODs-3 column was used with an eluent containing 1 mM tetrabutylammonium iodide and the indicated concentrations of potassium hydrogenphthalate. Reprinted from [27] with permission.

Effect of the concentration of IIR in the eluent Eqn. (6.12)'also allows us to examine the effect of increasing the concentration of IIR in the eluent [27]. Increasing the concentration of the IIR (i.e. Q+C-)will increase simultaneously both [Q+Im and [C-],. Two opposing effects are predicted from eqn. (6.12). The f i s t is an increase in analyte retention due to increased adsorption of IIR onto the stationary phase, so we expect the reciprocal of the solute capacity factor to be linearly related to the reciprocal of [Q+]m. The second is a decrease in analyte retention where the reciprocal of the capacity factor is expected to be linearly related to [C-1,. The observed changes in retention which accompany an increase in the eluent concentration of the IIR were illustrated in Fig. 6.7. We can now use eqn. (6.12) to rationalize this behaviour, but first this equation can be rewritten by using eqn. (6.4) to obtain the following expression for [C-],: (6.13)

which can be substituted into eqn. (6.12) to give:

Ion-Interaction Chromatography

185

0.180.16

-

Amount injected (mg)

Fig. 6.9 Dependence of the capacity factor of inorganic anions on the concentration of the analyte. A PRP-1 column was used with 1 mM tetrapentylammoniumfluoride in 1:3 acetoniaile-wateras eluent. Reprinted from [91] with permission. When [A-]m and [X-1, are constant, eqn. (6.14) can be simplified to: (6.15) where [Q+]m can be considered to be the amount of IIR not adsorbed at equilibrium, (QC), is the amount of IIR adsorbed at equilibrium and A, is the number of free sites at equilibrium. We will now consider the effects of increasing the eluent concentration of the IIR. At low IIR concentrations, [Q+]m is low, Ar is high and (QC), is small compared to A,. From eqn. (6.15), this gives:

1 a-

, or kx a

I

kx

[Q'lm

(6.16)

[Q'Im

This relationship is evident in the early part of Fig. 6.7. When half of the available adsorption sites on the stationary phase are occupied, then A, and (QC), are equal, so that: (6.17) This means that the slope of the plot of kx' versus the concentration of IIR is half that observed at lower eluent concentrations of IIR. We can see that this slope will decrease progressively as more IIR is adsorbed. This process continues until a

186

Chapter 6

maximum in the plot is attained, as in Fig. 6.7. At this point, A, is very small in comparison to (QC),, such that: (6.18)

Considering the relative magnitudes of A, and (QC),, the slope of the plot of capacity factor versus the concentration of IIR will be very small, or even zero. Further increases in the concentration of IIR will not result in further adsorption of Q+ onto the stationary phase since all of the available adsorption sites have been exhausted. However, [C-1, will continue to rise, so that the solute capacity factor will fall in accordance with eqn. (6.12).

6.4.2 Model for cation retention In order to develop a retention model for cations in dynamic coating ioninteraction chromatography, we recognize that the eluent composition used in this case usually differs from that employed for a typical anion separation. Eluents for anion separations contain a cationic IIR and its counter-anion, together with an added competing anion. On the other hand, eluents for cation separations contain an anionic IIR and its counter-cation, together with an added ligand which assists in solute elution by complexation effects. This difference between anion and cation eluents also exists in ion-exchange separations (see Chapter 4) and arises because of the strong ion-exchange affinities of many cations. We can therefore consider the situation where a lipophilic stationary phase is equilibrated with an eluent comprising an IIR (which will be assumed to be an aliphatic sulfonic acid salt, designated as P-C+)and a ligand, L-. Using the same approach adopted above for anions. we can write:

A,

+ P, + C,+

% (PC),

(6.19)

where A, is again the number of free adsorption sites on the stationary phase and the subscripts r and m denote the stationary and eluent phases, respectively. The equilibrium constant for eqn. (6.19) will be denoted by Kq and is given by: (6.20)

When a solute cation, X+, is introduced, the following ion-exchange equilibrium exists:

(PC), +

x;

% (PX), +

c,+

and the equilibrium constant (Ks)for this equation can be written:

(6.21)

Ion-Interaction Chromatography

187

(6.22)

The solute cation will also participate in complexation reactions with the eluent ligand, L-,so that the solute X will exist as both the free ion (i.e. X+) and as a complex. If we define ax as the fraction of the total concentration of solute cation existing as the free ion, X+, then we have:

ax =

R'Im -

[X'IT

(6.23)

where [X+]T is the total concentration of solute X in the eluent, regardless of whether it is present in the free or complexed forms. We again write a mass balance equation in terms of the sorption capacity, KO, of the stationary phase:

KO = A,

+ (PC), + (PX),

(6.24)

The capacity factor kx' of solute X+ can be written in terms of the phase ratio, q,

as: (6.25)

Combining eqns. (6.19) - (6.25) gives: (6.26)

Eqn. (6.26) represents rhe simplest case in which the solute cation and the countercation of the IIR are both singly charged. The form of this equation will therefore alter when the solute cation has a multiple charge. Nevertheless, from eqn. (6.26) we can predict that the capacity factor for a solute cation will be directly proportional to ax and [P-lm, and inversely proportional to [C'], and [X+]m. Few published data are available to investigate the validity of these relationships for inorganic cations, despite the fact that this separation mode is commonly employed for transition metal and lanthanide ions. However, a study of the retention behaviour of transition metal cations on a reversedphase column using octanesulfonate as the IIR and oxalate as the eluent ligand has been reported [93]. This work shows that there is a linear relationship between log kx' and log ax when all other variables are held constant (in accordance with eqn. (6.26)), and that a plot of log kx' versus log [Pa], (illustrated in Fig. 6.10), again with all other variables held constant, has the same general shape as that observed for anions. That is, an initial proportionality exists between log kx' and log [P-lm, but the curve reaches a

188

Chapter6

-0.2 O.O\

I

-0.4 -2h

I

-2.5

I

-2.6

I

-2.7 log

rw

I

-2.8

1

-29

1

-3.0

Fig. 6.10. Dependence of the capacity factor of inorganic cations on the concentration of IIR in the eluent. A Waters pBondapak Clg column was used with an eluent containing 2.5 m M oxalic acid at pH 3.4 and the indicated concentrations of octanesulfonate. Reprinted from [93] with

permission.

maximum due to saturation of the available adsorption sites with P- and the increased concentration of the counter-cation, C+. in the eluent. These results are in general agreement with eqn. (6.26). 6.5

APPLICATIONS

Ion-interaction chromatography has found extensive application in the separation of inorganic anions and cations. The technique offers some advantages over the use of fixed-site ion-exchangers in that there is a wide range of eluent variables which can be used to manipulate the retention of solutes. For this reason, ion-interaction chromatography is often applied to the resolution of difficult mixtures of solutes. Numerous examples of such applications can be found in Part V of this book, however some representative applications are listed in Table 6.5 in order to illustrate the scope of the technique. As a general observation, it can be said that ion-interaction chromatography finds its strongest usage in the separation of transition metal and lanthanide cations, for which it is undoubtedly the method of choice. These species may be separated as simple, hydrated metal ions, or as anionic complexes, using a suitable ligand. The first of these approaches has been developed extensively by Cassidy and co-workers and was illustrated in Fig. 6.3(b), which shows an excellent separation of lanthanides. The

189

ton-Interaction Chromarography

second approach is illustrated in Fig. 6.11, which shows the separation of anionic metal cyan0 complexes. Both of these methods have been applied to a wide variety of complex sample matrices.

TABLE6.5 APPLICATIONS OF ION-INTERACTION SEPARATIONSIN IC Solute(s)

Sample

Stationary phasea

IIRb

Det'n Ref methodc

9 8

TBA sulfate TBAphosphate

DSpec ICP

94 95

ISpec DSpec ISpec C ISpec DSpec Amp DSpec

75

Anions Alkylbenzene sulfonates A s O ~ ~organo-, arsenic compounds Carboxylic acids CI-, NO^-, ~043-, ~ 0 4 2 EDTA, ~2042-, citrate F-, C104~-, BF4-, F,cI-, NO^, ~ 0 4 2 IIIO~-.BIO.~-, NO~-,N%-, I-, SCN, Fe(CN)64N&; Br-, NO3N@-,NO3P043-, Cl-, Bf, NOg',

Detergents Shale oil

c18

Lysimeter solns Plants, soils Reactor water Plating baths Tapwater salt Serum various

CIMA chlorided TBA hydroxide PRP- 1 8chlorided MPIC-NS1 TBA hydroxide TBA phthalate C18 Ouylamine c18 HDTMA chloride ClS CTMA citrate PS-DVB

Foods PRP- 1 Meat,vegetables Ci8 C18 Fruit juices

TPAbromide TBAphosphate TBAsalicylate

DSpec DSpec C

98

Pharmaceuticals Urine Batteries

CIS c8

c18

TBA sulfate TBA sulfate OctylamineTSA

DSpec

SCN-

47 92

Leach liquor Ambient air

Cis MPIC-NS1 MPIC-NS1 C18

Octanesulfonate Hexanesulfonate TBA hydroxide TBAhydroxide

PCR C C DSpec

68

c18

Hexanesulfonate

PCR

103

I-,so~~s&-. Iso42-

CIS cl8

Amp

ISpec

96 80 30

97 50 54 55

99 87

51

Cations

Rare earths Ethanolamines Au(I), Au(III) cyanides CN- complexes of transition metals Transition metals a

Plating baths Gold process solutions Brass, urine

MPIC-NS1, PRP-1 and PS-DVB are all styrene-divinylbenzenestationary phases. TBA = tetrabutylammonium, CIMA = cetyltrimethylammonium,CP = cetylpyridinium, HDTMA = hexadecyltrimethylamnium, TPA = tetrapentylammonium, TSA = toluenesulfonate. DSpec = direct spectrophotometry, ISpec =indirect spectrophotometry, C = conductivity, Amp = ampemmeay, PCR = post-column reaction, ICP = inductively coupled plasma. Permanent coating ion-interaction chromatography.

100 101 102

190

Chapter 6

k Pd(ll1

PliIII

0I

10 I

20

Time imin

30

I

LO

Fig. 6.11 Ion-interaction separation of metal-cyano complexes. A Waters Nova Pak C18 column was used with 23:77 acetonitrile-water containing 5 mM Waters Low UV PIC A as eluent. Detection was by direct spectrophotometry at 214 nm. Reprinted from [38] with permission.

6.6 1 2 3 4 5 6

7 8 9 10 11 12 13

14

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193