Chapter 7 Ion-Exclusion Chromatography

195

Chapter 7 Ion-Exclusion Chromatography 7.1

INTRODUCTION

Ion-exclusion chromatography, first introduc d by Wheaton and Bauman in 1953 [l], involves the use of strong anion- or cation-exchange resins for the separation of ionic solutes from weakly ionized or neutral solutes. In this mode of chromatography, the charge sign on the ion-exchange resin used is the same as that of the weakly ionized solutes. That is, solutes with a partial negative charge (such as carboxylic acids) are separated on a cation-exchange resin having anionic sulfonate functional.groups, whereas solutes with a partial positive charge (such as weak bases) are separated on an anionexchange resin having cationic quaternary ammonium functional groups. This is the opposite situation to that occumng in ion-exchange chromatography. As with other IC separation techniques, ion-exclusion chromatography has been described by a variety of names, some of which are listed in Table 7.1. Each of these names implies a mechanism for the separation process and as we will see in the ensuing discussion, the actual mechanism of the process is not clearly defined, but is certainly quite complex. We shall therefore continue to use the term "ion-exclusion chromatography" to describe the technique, whilst recognizing that this title is probably somewhat inaccurate.

7.1.1 Basic principles Ion-exclusion chromatography finds application in the separation of a wide range of small, neutral or partially ionized molecules. Applying the definition of IC used in this text, we will limit discussion of the technique to include its application to certain TABLE 7.1 ALTERNATIVENAMES FOR ION-EXCLUSION CHROMATOGRAPHY Name

Reference

Ion-exclusion chromatography Ion-chromatography exclusion (ICE) Ion-exclusion partition chromatography Donnan exclusion chromatography Ion-moderatedpartition chromatography

1 2 3 4 5

1%

Chapter 7

Fig. 7.2 Schematic representation of ion-exclusionchromatography for (a) acidic solutes, such as acetic acid and HCI, and (b) basic solutes, such as N H 3 and NaOH.

solute types only; namely, carboxylic acids, inorganic weak acid anions, weak organic bases, and water. It may appear that this restricted group of solutes could diminish the importance of ion-exclusion chromatography in comparison to ion-exchange and ioninteraction chromatography, but it will be demonstrated later in this chapter that ionexclusion chromatography is of major importance in IC. The principles of ion-exclusion chromatography can be illustrated in a schematic manner by considering the chromatographic system to be comprised of three distinct phases. The first of these is the flowing eluent, which passes between the beads of ionexchange resin (i.e. through the interstitial volume). The second zone is the polymeric network of the resin material itself, together with its bound ionic functionalities, whilst the third zone is liquid occluded inside the pores of the resin bead. The polymeric resin can be considered as a semi-permeable, ion-exchange membrane which separates the flowing eluent from the stationary occluded liquid inside the resin [ I , 31. The manner in which solutes are separated in ion-exclusion chromatography is illustrated in Fig. 7.1. We fiist consider the behaviour of two solutes, hydrochloric acid and acetic acid, on a cation-exchange resin using water as the eluent. From Fig. 7.l(a), we see that Cl- cannot penetrate into the occluded liquid phase because it is repelled by the anionic functional groups on the resin, in accordance with the Donnan exclusion effect. The C1-ions therefore remain in the flowing eluent phase and are not retained by the column. On the other hand, the acetic acid is only weakly ionized and exists predominantly as neutral acetic acid molecules, with only a small percentage present as acetate anion. The ionized and neutral acetic acid molecules are in dynamic equilibrium with each other, so that the effective negative charge on the acetic acid is therefore determined by the proportions existing in each form. In a water eluent, this effective charge is quite small and because of this, acetic acid can penetrate the negatively charged resin zone and move into the occluded liquid phase. This results in some degree of retention of acetic acid, so that it is eluted somewhat later than hydrochloric acid. As an historical note, this particular separation was that originally reported in the first publication on ion-exclusion chromatography [ 11. In a similar manner, an anion-exchange resin can be used to separate a weak base (ammonia) from a strong base (NaOH), again using water as eluent. This is illustrated in Fig. 7.l(b), which shows that Na+ is repelled by the cationic functional groups on the

Ion-ExclusionChromatography

197

resin phase and is unretained. On the other hand, ammonia, by virtue of its low degree of ionization and hence its low overall charge, can penetrate into the occluded liquid phase and is therefore retained. Fig. 7.1 suggests that retention of solutes in ion-exclusion chromatography is influenced solely by the charge on the solute. That is, all fully ionized solutes can be expected to be unretained and so be eluted together at the void volume of the column, and the retention of partially ionized solutes can be expected to increase as the degree of ionization decreases. These predictions are not fully supported in practice and it can be shown that other factors also play a role in solute retention. These factors will be discussed in Section 7.4, but at this stage we will make the assumption that solute charge is the dominant parameter in determining retention. 7.2

STATIONARY PHASES

Ion-exclusion chromatography was first performed on large particle size, high capacity, fully functionalized polystyrene-divinylbenzenepolymers. Modem stationary phases are essentially the same materials, but differ in some important respects. Some of the stationary phase parameters which can exert an influence on solute retention are: (i) (ii) (iii) (iv)

Particle size. Ion-exchangecapacity. Resin structure. Degree of resin cross-linking.

As with other chromatographic techniques, the separation efficiency is strongly influenced by the particle size of the column packing material. Modem ion-exclusion chromatography is generally performed on 5 or 10 pn particles, however some of the resins used are relatively soft and the use of small diameter particles means that eluent flow-rates must be kept low to avoid compression of the resin bed. The ion-exchange capacity of a typical ion-exclusion packing material is generally greater than that of packings used for ion-exchange separations in IC. These high capacity materials are preferred so that the number of functional groups on the resin is sufficient to exert an appropriate Donnan exclusion effect. The resin structure is also important, with microporous or gel-type resins being most commonly used (for a description of this type of resin, see Section 3.3). However, there has been a recent trend towards macroporous materials of greater rigidity [ 6 ] . Early studies [3] showed that the degree of cross-linking of the resin exerts a considerable effect on solute retention. Highly cross-linked materials (e.g. those with 812% divinylbenzene) show stronger Donnan exclusion effects than resins of lower crosslinking. This means that both strongly and weakly ionized species show more penetration into thk occluded liquid phase on softer resins of low cross-linking (e.g. those with 2% divinylbenzene). Most commercial ion-exclusion resins have approximately 8% cross-linking, with materials of lower cross-linking being used for the separation of those solutes likely to show higher degrees of ionization under the experimental conditions used (e.g. acids in the pKa range 2-4). Ion-exclusion columns

Chapter 7

198

TABLE 7.2 CHARACTERISTICS OF SOME TYPICAL ION-EXCLUSION COLUMNS Column

Cross-

linking

(a> Aminex A-5

Aminex HPX-87H Brownlee Polypore H anion-exclusion Dionex HPICE-AS1 Dionex HPICE-AS2 Dionex HPICE-AS3 Dionex HPICE-ASS Dowex 50W-X2 Dowex 50W-X4 Dowex 50W-X12 Hamilton PRP-X300 Hitachi 2613 cation-exchanger Hitachi 2632 anion-exchanger Interaction ORH-801 Interaction ION-310 Sarasep WA 1 SP-Sephadex C-25 TSK-gel SCX Waters Fast Fruit Juice Waters Ion-exclusion Wescan 269-006 exclusion Wescan 269-038 exclusion Wescan 269-051 Yokagawa Elecmc YEW SCS 5-252

8 8

Particle size (vm)

13

-b

5-8 10 10 25 25 7 38-75 75- 180 38-63 7 18 18 8 8 10 50-100

20 15 15 8 8 9 30

7 7 10 10 10 6

.b

8 8 4 6 2 4 12 -b

8 8 8 8 8

5

Ion-exchange capacity (mequiv/g)

Refa

1.7 1.7

7 8,9 10, 11 12-14 15 16 17 18, 19 20-22 3 6, 23 24-27 28 30-33 34 35 36, 37 38-41 42 43-45 46 47,48 49 50,51

-b

3-5 3-5 3-5 1.5

1.1 0.6 2.1 0.17 4.5 -b -b -b

3.0 4.5 4.2 5.0 5.0 1.4 1.4 1.8 4.5

a These references include some publications in which the indicated columns have been utilized.

Data unavailable. are usually large in comparison to conventional IC columns because a considerable volume of resin material is necessary to provide sufficient occluded liquid phase to permit the separation of solutes of similar size and charge. A typical column would be 30 cm in length, with an internal diameter of 7 mm or more. The characteristics of some representative ion-exclusion packing materials are listed in Table 7.2. Cation-exchange resins are generally used in the H+ form, whilst anion-exchangers are used in the OH- form.

lon-Exchion Chromatography

7.3

199

ELUENTS

7.3.1 Water eluents The eluents used in ion-exclusion chromatography are often very simple in composition. Most of the early work was performed using deionized water as eluent [2, 18, 22,521 and the degree of ionization of the solutes (and hence their retention times) is therefore determined by their pKa or pKb values. The limitations of water as an eluent are that stronger acids or bases show too great a degree of ionization to be retained and the peak shape obtained for solutes which are retained is often poor. This is illustrated in Fig. 7.2(a) for carboxylic acid solutes. In view of the problems observed with peak shape, water is rarely used as an eluent in modem ion-exclusion chromatography, except for the separation of very weakly ionized compounds. An example of such a separation is given in Fig. 7.2(b).

7.3.2

Acid eluents

Following from the original suggestion by Turkelson and Richards [22], it is now common for dilute solutions of strong mineral acids to be employed for the elution of anionic solutes, or dilute solutions of strong bases to be employed for the elution of cationic solutes. In this way, ion-exclusion chromatography can be extended to the separation of relatively strong acids and bases by limiting their degree of ionization. The most commonly used eluents are formed from strong acids, such as sulfuric acid [e.g. 5,7, 10.54, 551, hydrochloric acid [e.g. 13,56-591 and aliphatic sulfonic acids [8,43,45, 60,611. When sulfuric acid is used as the eluent, detection of eluted solutes is generally accomplished by monitoring UV absorbance at low wavelength (200-220 nm). Fig. 7.2(c) shows a typical chromatogram obtained with this eluent. On the other hand, hydrochloric acid is most often used with conductivity detection, after the eluent is passed through a suitable suppressor (see Section 9.5). Aliphatic (and aromatic) sulfonic acids can also be employed for conductivity detection, but because of the relatively low background conductance of these eluents, suppression is not necessary. Weak acids may also be utilized as eluents in ion-exclusion chromatography. Examples include phosphoric acid [62-641, tridecafluoroheptanoic acid (perfluorobutyric acid) 1651, carbonic acid [66, 671, n-butyric acid [19] and benzoic acid [53]. It is interesting to note that eluents of the same pH, when used on the same stationary phase, produce virtually identical chromatograms, regardless of the nature of the acid used. The choice between different eluent acids is therefore governed primarily by the detection method which is to be used. We can also note in passing that organic modifiers, such as methanol, acetonitrile or acetone, are sometimes added to the eluents used in ion-exclusion chromatography. The function of these modifiers is related to the participation of solute adsorption effects in the retention process. This factor is discussed more fully in Section 7.4.3. Some typical capacity factors for carboxylic acids, obtained with various eluents and column types, are illustrated in Fig. 7.3. General trends for the retention of carboxylic acids in ion-exclusion chromatography have been reported [3] and are summarized below:

P

CO32-

loxalic

I Formic

Ja

,Lactic Propionic Ektyric ,Fumoric

Valcric I

0

1

l

10

l

l

l

l

20 30 Time lminl la)

l

l

60

I

1

50

-

0 6 8 12 16 20 Time fminl fb)

I

0

I

2

I

4

I

I

6 8 Time fmin) Icl

I

10

I

12

1-

11

_i!

U

0

6

8121620

T i m (mid Id1

Fig. 72 Ion-exclusion chromatograms with various eluents. (a) A 5 pm TSK cation-exchange resin was used with water as eluent. Reprinted from [53] with permission. (b) A Dionex HPICE-AS1column was used with water as eluent. Reprinted from [12] with permission. (c) An Interaction ORH-801 column was used with 0.01 N H2SO4 as eluent. Chromatogram courtesy of Interaction (d) A Dionex HPICE-AS1 column was used with 50 mM mannitol as eluent. Reprinted from [12] with permission.

P u

Interaction ORH-801 10 mN H2SO4 35 o c oxalic aconitic maleic oxaloacetic a=ketoglutaric citric isocitric pyruvic tartaric. ascorbic malic lactic succjnic formic acetic

-

1

L

0

c

0

m

2-i

kpropionic

4

Loxalic

aconitic oxaloacetic rnaleic a detoalutaric citric isocitric pyruvic tartaric ascorbic rnalic succinic lactic

-

formic acetic Dropionic

kfumaric

I

'

I

Q

4

Interaction ORH-801 10 mN H2SO4 65 OC

Bio-Rad HPX-87H 4 rnN H2SO4 5OoC

-

oxalic maleic oxaloacetic B.-ke tog Iuta r ic

FCp:I:U' "v c \tartaric \;tl$p

a-ketoglutaric malgic -L isocitric

i

-succinic /formic 'lactic

-

Benson OA850 1 mN H s O 4 25 OC

-acetic adipic propionic

Dionex HPICE-AS5 Perfluorobutyric acid IDH 2.8)

E

pyruvic

-

-0

oxalic

-1

tartaric orrnic

acetic

-adipic

L

rnalonic ascorbic lactic rnaleic malic

Fig. 7.3 Typical capacity factors for organic acids using ion-exclusion chromatography. Data courtesy of Dionex, Benson, Interaction and Bio-Rad.

I4

Chapter 7

202

(i)

Members of a homologous series, such as the aliphatic carboxylic acids, are eluted in order of increasing molecular weight, decreasing acid strength (i.e. increasing pKa) and decreasing water solubility. Thus. the elution order of low molecular weight carboxylic acids is formic c acetic c propionic. (ii) Dibasic acids are generally eluted earlier than monobasic acids of the same carbon number. For example, oxalic acid is eluted earlier than acetic acid, and malonic acid earlier than propionic acid. (iii) Carboxylic acids with branched structures are eluted earlier than the corresponding straight chain isomer. For example, iso-butyric acid is eluted earlier than n-butyric acid. (iv) A double bond serves to increase the retention of an acid. For example, acrylic acid is eluted after propionic acid.

7.3.3

Complexing eluents

The retention and detection properties of some solutes can be enhanced if a complexing agent is added to the eluent. An example of this approach is the use of a mannitol eluent for the determination of boric acid [12], in which the mannitol serves to complex the boric acid to form a species which is more easily detectable by conductivity measurements than is boric acid alone. Fig. 7.2(d) shows a typical chromatogram obtained with this approach. A further application of complexing eluents is the use of a tetraborate eluent in the determination of formaldehyde [68, 691.

7.4 7.4.1

FACTORS INFLUENCING RETENTION IN ION-EXCLUSION CHROMATOGRAPHY Degree of ionization of the solute

The degree to which the solute is ionized is the most significant factor which determines solute retention. As the solute becomes more ionized, the Donnan exclusion effect increases in magnitude and this leads to decreased retention. When only the Donnan exclusion effect is considered. solute retention (expressed as the retention volume, VR)is given by:

where Vo is the interstitial volume of eluent (i.e. the volume of eluent flowing between the particles of stationary phase), Vi is the internal volume of eluent (i.e. the volume of occluded liquid inside the pores of the stationary phase) and DA is the distribution coefficient for the solute between the interstitial eluent and the occluded liquid. The value of DA is dependent on the degree of ionization of the solute. Fully ionized solutes have DA = 0, due to the total exclusion of such solutes in accordance with the Donnan effect. The retention volume of fully ionized solutes is therefore given by:

Ion-Exclusion Chromatography

203

and we expect all solutes of this type to be eluted at the same retention volume. On the other hand, neutral solutes have DA = 1, since these solutes can distribute freely between the interstitial eluent and the occluded liquid, without influence from the Donnan effect. The retention volume for neutral solutes is therefore given by:

and again we expect all neutral solutes to be eluted at the same retention volume. Solutes which are ionized partially will be eluted at retention volumes intermediate between the two extremes given by eqns. (7.2) and (7.3), with the observed retention volume for a particular solute being dependent on the acid or base dissociation constant of that solute. The above equations are identical in nature to those which are used to describe retention in size-exclusion (gel permeation) chromatography, in which retention volumes fall between the two extremes determined by total exclusion of solutes, and total penetration of solutes.

Dependence of solute retention on pK, The effect of solute charge on retention in ion-exclusion chromatography has been examined for acidic solutes on a strong cation-exchanger (8% cross-linked) in the H+ form, using water as the eluent [27]. Under these conditions, the degree of ionization of the solute is determined solely by the acid dissociation constant (pKa) of the solute. A plot of retention volume versus pKa1 for a range of solutes is given in Fig. 7.4. The retention behaviour depicted in Fig 7.4 is in close accordance with the predictions made above in eqns. (7.1) - (7.3). Strong acids, such as HNO3, H2SO4, HCl, etc., are completely excluded from the stationary phase and are eluted at the same retention volume (12.8 ml). This volume corresponds to the volume of interstitial eluent present in the chromatographic column (Vo). Solutes which exist as neutral species in the water eluent, such as methanol, HCN and H2CO3, are eluted together at a retention volume of 28.5 ml (Vo + Vi). Solutes which are ionized partially in water, such as H3B03, HCOOH and CH3COOH, are eluted at retention volumes between 12.8 and 28.5 ml. There is a strong correlation between pKa1 and VR. Substitution of the measured values of VR,Vo and Vi into eqn. (7.1) permits the calculation of values of DA for each solute. These values are listed in Table 7.3. Inspection of the data in Table 7.3 shows that some values of DA exceed the theoretical maximum of 1.0. This behaviour is evident for propionic acid and H2S and it can be seen that these solutes show anomalous retention volumes in Fig. 7.4. The reasons underlying this will be discussed further below. A similar study of solute retention volumes has been undertaken using a highly cross-linked (30%)cation-exchange column, with acidic eluents [50]. The results of this study are presented in Fig. 7.5, which shows some of the same characteristics as Fig. 7.4, in that a fully ionized solute (H2SO4) defines the lower limit of retention volume, with neutral solutes (methanol and H2CO3) defining the theoretical upper limit of retention volume. A straight line is drawn through the points for oxalic acid, HF and H2CO3, as was done for Fig. 7.4, and it can be seen that many of the solutes tested do not conform to the retention behaviour evident from Fig. 7.4. That is, there is poor correlation

204

Ckpter 7

p Methanol

151-

E-

-vo-

0

5c

"25

I

m (Y

0

Y

n

-5

104

I

.*#.I

-10

Retention volume (mll

Fig. 7.4 Relationshipbetween retention volumes and first dissociationconstants (pKal) for acids on a stationary phase with 8% cross-linking, using water as the eluent. Reprinted from 1271 with permission. TABLE 7.3 DISTRIBUTION COEFFICIENTS FOR ACIDS, CALCULATED FROM THE RETENTION DATA SHOWN IN FIG. 7.4 [27] Acid

DA

0 0 0 0 0 0 0.01 0.06 0.08 0.09 0.11

Acid

DA

0.36

0.43 0.65 0.81 1.10 1.00 1.00 1

.oo

0.98

1.02 1.40

Ion-Excluswn Chromatography

-Vo

10

-

__z(t___

I I

205

Vi

Methanol

I I

Monocarboxylic acid

I

-

5Dicarboxylic acid

0

Y

a.

0-

I

I

I I I

''W O L

I I I

I

I

I

I

Fig. 7.5 Relationship between retention volume and first dissociation constant (PKal) for carboxylic acids on a stationary phase with 30% cross-linking,using 1 mM H a 0 4 as eluent. Reprinted from [50] with permission.

between pKa1 and retention volume. This behaviour indicates that retention of many solutes is influenced by parameters other than the degree of ionization of the solute.

Dependence of solute retention on eluent pH When changes in the eluent pH produce a change in the degree of ionization of the solute, we can expect this to cause a change in the retention time of that solute. This behaviour is illustrated in Fig. 7.6,for both mono- and dicarboxylic acids on a highly cross-linked cation-exchanger. Each of the solutes shown has at least one fully ionized carboxylate group at pH 6 and is therefore eluted at a retention volume of Vo (i.e. k' = 0) at this pH. For lower pH values, the expected decrease in retention volume (and hence k') with increasing pH is evident. 7.4.2 Molecular size of the solute

The results presented in the preceding Section show that for some solutes, DA (and hence the retention volume) is somewhat less than that predicted by consideration of charge alone. This behaviour is evident in Fig. 7.5 for the C3' and C4' dicarboxylic acids. It has been suggested by a number of authors [4,36,70-741that size-exclusion effects may contribute to the retention process in ion-exclusion chromatography by restricting the

Chapter 7

206

4r

PH (a) Fig. 7.6 Effect of eluent pH on the capacity factors of (a) monocarboxylic acids and (b) dicarboxylic acids. A 30% cross-linked stationary phase was used with 1 m M Na2HP04 (pH adjusted with oxatic acid) as eluent. Reprinted from [50] with permission.

access of larger solute molecules to the occluded liquid in the pores of the stationary phase. Size-exclusion effects should result in the following retention characteristics: Retention volumes for large, partially ionized solutes should be smaller than expected on the basis of solute charge. (ii) Large, neutral molecules can be expected to show DA values which are less than the theoretical value of 1 .O. (iii) Large, neutral solutes which are eluted at retention volumes less than (Vo + Vj) should be eluted in order of decreasing molecular size. (i)

The retention volumes of the C3' and C4' dicarboxylic acids in Fig. 7.5 are in accordance with (i) above and the retention characteristics described in (ii) and (iii) have been confirmed for neutral lactones [70] and oligosaccharides [ 5 ] . From these results, we can conclude that size-exclusion effects make some contribution to the retention of large solutes.

7.4.3 Hydrophobic interactions between the solute and stationary phase The retention behaviour of the C6'- Cg' dicarboxylic acids and the C3 - C5 monocarboxylic acids in Fig. 7.5 cannot be explained on the basis of solute size and charge. All of these solutes show retention volumes which are larger than those

Ion-Exclusion Chromatography

207

predicted on the basis of solute charge and they are eluted in order of increcising molecular weight (which is the opposite of that expected from size exclusion effects). It is clear that the retention of these solutes is influenced by a third factor, in addition to solute charge and size-exclusion effects. The anomalous retention behaviour described above can be attributed to hydrophobic adsorption of the solutes onto the neutral, unfunctionalized regions of the polymeric stationary phase [50, 53, 751. We have noted previously in the discussion of resin-based ion-exchange columns that many organic molecules and ions show strong reversed-phase interactions with styrene-divinylbenzene packing materials. A plot of the logarithm of capacity factor versus the number of carbon atoms is close to linear for many solute types eluted by ion-exclusion chromatography [5]. This behaviour is similar to that observed in reversed-phase HPLC and gives strong support to the proposal that reversed-phase, hydrophobic interactions play a part in the ion-exclusion retention process. Hydrophobic adsorption effects can be expected to increase in magnitude as the alkyl chain length of the solute is increased, leading to larger retention volumes. This behaviour is evident from Fig. 7.5. We can also note that the size-exclusion effect discussed above will be in competition to the hydrophobic adsorption effect. That is, an increase in alkyl chain length of the solute will cause a decrease in retention under the size-exclusion effect and an increase in retention under the hydrophobic adsorption effect (provided the solute charge is constant). This competition can be used to explain the shape of the retention plot for dicarboxylic acids in Fig. 7.5, where size-exclusion is dominant for the C3' and C i acids, whereas hydrophobic adsorption dominates for the Cg' - C{ acids.

Use of organic modifiers in the eluent The existence of hydrophobic adsorption effects creates the possibility for manipulation of solute retention by adding typical reversed-phase organic modifiers, such as methanol or acetonitrile, to the eluent. A decrease in the retention volume for some solutes can be anticipated and this behaviour has been demonstrated by Tanaka and Fritz [53] using benzoic acid as the eluent. Fig. 7.7(a) shows the effect of the addition of methanol to the eluent and it can be seen that small solute molecules, such as CH3COOH and HCOOH, show little or nn change in retention with increasing methanol, whereas larger solutes, such as valeric acid, show decreased retention. It is evident that the chromatographic resolution of a mixture of all the solutes in Fig. 7.7(a) will decrease with increasing modifier content. A further, opposing effect can result from the addition of an organic modifier to the eluent. When the added modifier has a low dielectric constant (e.g. dioxane), the decreased dielectric constant of the eluent causes an increase in the pKa of the solutes [24]. That is, the solutes become weaker acids and their ionization is therefore suppressed, leading to increased retention. This effect is illustrated in Fig. 7.7(b) for some condensed phosphates, which can be separated only when the organic modifier content in the eluent is high. It can be seen that chromatographic resolution of these solutes increases as dioxane is added. This same effect has been reported for the retention of NH4+ on a strong anion-exchange column [28].

LO

-

PZOf

9 0105~,0,3Methanol concn. % ( v l v ) (a1

Dioxane concn. % (vlv)

(b)

Fig. 7.7 Effect of organic modifiers on solute retention in ion-exclusion chromatography. (a) A 5 pm TSK cation-exchange resin (H+ form) was used with an eluent comprising 0.5 mM benzoic acid and the indicated concentrationsof methanol. Reprinted from [53]with permission. (b) An 18 pm Hitachi 2613 cation-exchangeresin (H+form) was used with an eluent comprising water and the indicated concentrationsof dioxane. Reprinted fmm [24] with permission.

n

88 w

Ion-Exclusion Chromatography

209

Many ion-exclusion columns have definite limits to the amount of organic modifier which can be added to the eluent without causing column damage. It is therefore essential that column specifications be consulted before organic modifiers are used to alter solute retention. In conclusion. we note that the ability to manipulate retention in ion-exclusion chromatography using organic modifiers opens up the possibility of gradient elution. This has been achieved using a sulfonated macroporous resin with a methanol gradient [76], and is illustrated in Fig. 7.8.

7.4.4 Ion-exchange capacity of the stationary phase The effect of the ion-exchange capacity of sulfonated resins on the retention of carboxylic acids has been studied by Lee and Lord 1761. They have synthesized a range of sulfonated macroporous PS-DVB resins in which the degree of functionalization ranges from 0-91% and the retention behaviour of carboxylic acids on these resins is shown in Fig. 7.9. It can be seen that there is an increase in retention for most solutes when the ionexchange capacity is increased from 0 (i.e. for the unfunctionalized resin) to 0.20 mequiv/g (i.e. for a partially functionalized resin). This trend is surprising since an increase in the negative charge density due to sulfonate groups would be expected to

Formic Propionic

I

0

I

4

I

8

Time ( m i d

1

12

Fig. 7.8 Gradient elution in ion-exclusion chromatography. A Hamilton PRP-X300 column was used with an eluent consisting of a linear gradient (over 5 min) of 6-641methanol in 1 mN H2SO4. Detection was by UV absorbance at 210 nrn Reprinted from [76] with permission.

210

Chapter 7

3

k' 2 1

0 0.0 0.2

0.4 0.6 0.8 1.0 1.2 Exchange capacity (mequiv/g)

Fig. 7.9 Effect of stationary phase ion-exchange capacity on retention of carboxylic acids in ionexclusion chromatography. The eluent was 1 m N H2SO4. S = succinic acid, A = acetic acid, L = lactic acid, C = citric acid, M = malic acid, T = tartaric acid. Reprinted from [76] with permission.

cause a decrease in the retention of the partially ionized solute acids. A suggested explanation is that hydrogen bonding between the neutral solute molecules (which are present in far grcatcr numbers than the ionized solute molecules) and the sulfonic acid groups on the resin may contribute towards solute retention [76]. The presence of some sulfonate groups on the resin could then lead to the observed increase in retention. However, further increases in ion-exchange capacity (beyond 0.20 mequiv/g) show predictable behaviour in that solute retention decreases as the surface charge on the resin increases. It can be concludcd from these results that there is an optimal ion-exchange capacity for each resin type, so that the use of fully functionalized materials is thereforc not always advantageous. 7.4.5

Ionic form of the ion-exchange resin

In the discussion thus far, all of the parameters affecting retention have been related to ion-exclusion chromatography performed either on cation-exchange resins in the Ii+ form, or on anion-exchange resins in the OH- form. We now turn to the effects which arise when the ionic form of the resin is varied. It has been demonstrated [50J that the retention of carboxylic acids on cation-exchangers decreases in the following sequence of ionic forms:

Ion-ExclusionChromatography

211

This sequence is the reverse of that found for the hydrated radii of these cations, except for NH4+, which has the same radius as K+. It has been suggested that the presence of a bound cation of large radius serves to decrease the available hydrophobic surface area of the resin and to alter the values of both Vo and Vi for the column, all of which can result in reduced solute retention [50]. Some ion-exclusion separations are possible only if the column is in a particular form. This is especially true of the separation of monosaccharides on a calcium-form cation-exchanger, for which ligand-exchange involving interaction between calcium ion and the non-bonding orbitals of the sugar oxygen is thought to occur [5].

7.4.6

Temperature

Temperature can affect retention in ion-exclusion chromatography either by alteration of the chromatographic efficiency in the same manner as observed in most forms of chromatography, or by influencing the degree of ionization of the solute. The first of these effects is evidenced by somewhat reduced retention volumes, improved peak shapes and better separations at elevated temperature due to faster mass-transfer characteristics. In addition, the lower solvent viscosity at higher temperatures permits the use of faster flow-rates on gel-type stationary phases which are subject to pressure limitations. The effects of temperature on solute ionization often vary from solute to solute, as can be seen in the first two columns of Fig. 7.3, which differ only in the temperature used. Some solutes show changes in form at elevated temperature and may therefore be eluted at different retention volumes at different temperatures. An example of this behaviour is the increased retention of partially ionized aldonic acids at higher temperature. due to their conversion into neutral lactones [70]. Increased temperature can also cause a change in the dielectric constant of the eluent, especially when an organic modifier is present. This, in turn, will affect the PKa of the solute and hence its retention. An example of the effects of temperature on retention is illustrated in Fig. 7.10, for phosphate, phosphite and hypophosphite ions eluted with an aqueous acetone eluent [26].

7.4.7

Summary

From the discussion thus far in Section 7.4, we can appreciate that numerous factors play a part in the retention process in ion-exclusion chromatography. These factors are listed below in approximate order of importance. The relative influences of these factors have been determined by examining retention data for carboxylic acids, and may therefore differ for other solutes. The degree of ionization of the solute (which is determined by the pKa of the solute, the eluent pH and the organic modifier content of the eluent). (ii) Hydrophobic (reversed-phase) interactions between the solute and the stationary phase (which are dktermined by the nature of the solute and the organic modifier content of the eluent).

(i)

212

Chapter 7

"0.5 Or

i tu H PO32-

o*o10

0

10 20 30 40 50 60 70 80 Column temp. ('C)

Fig. 7.10 Effect of temperature on the retention of inorganic phosphates using ion-exclusion chromatography. A Hitachi 2613 stationary phase was used with 4050 (v/v) acetone-water as eluent. Reprinted from [26] with permission.

(iii) (iv) (v) (vi) (vii)

The molecular size of the solute. The degree of cross-linking of the stationary phase. The temperature at which the separation is performed. The ion-exchange capacity of the stationary phase. Hydrogen-bonding (normal-phase) interactions between the solute and the stationary phase. (viii) The ionic form of the stationary phase.

7.5

RETENTION MODEL FOR ION-EXCLUSION CHROMATOGRAPHY

The fact that many parameters influence retention in ion-exclusion chromatography makes it difficult, if not impossible, to develop a retention model unless certain simplifying assumptions are made. The most important of these assumptions is that the retention process is dominated by a Donnan exclusion equilibrium mechanism. That is, none of the additional retention processes discussed in Section 7.4 above plays any significant role in solute retention. Using this assumption, Glod and Kemula [77] have reported the following derivation of a retention model. We consider a weak acid, HA, which dissociates according to:

Zon-Exclusion Chromatography

HA % H+ + A-

213 (7.4)

Both HA and A- may exist in both the mobile phase and the stationary (resin) phases (designated by the subscripts m and r, respectively). In a thermodynamic Donnan equilibrium, the chemical potentials of the acid on both sides of the membrane are equal, and if activity effects are neglected, the equilibriumcondition assumes the form:

The dissociation constant of the acid, HA, in both phases is given by: (7.6) The electroneutrality conditions in both phases can be written: (7.7) (7.8) where the Concentration of dissociated functional groups in the stationary phase is given by [S03-lr. Now the concentration, c. of the sample at the peak maximum can be written in terms of the total concentration of all forms of the acid, to give:

The distributioncoefficient, DA.is given by: (7.10) From eqns. (7.6), (7.7) and (7.9) we can write: (7.1 1)

The value of [A-Im can be obtained from eqns. (7.6)-(7.9). if we make the following assumption: c cc

[SO;],

This enables us to write the following expression for DA:

(7.12)

Chapter 7

214

1.0 0.8

-

-

- 2.8 -

0

-

- 2.L -

0.6DA 0.L

- 2.0

-

-

"R

- 1.6 -

c

- 1.2

0.2-

-

-

- 0.8

0l

l

l

l

l

t

l

l

l

l

l

l

l

l

l

Fig. 7.11 Plot of the distribution coefficient, DA,as function of log (C/Ka). The solid line is calculated from eqn. (7.14), whilst the points represent experimental values obtained for 33 solutes. LiChrosorb KAT was used as the stationary phase, with water as eluent. Reprinted from [77]with

permission.

DA

=

2c

+ K, - JKZ + 8K,c 2~ - 2Ka

(7.13)

which can be rewritten as:

(7.14)

2-

G

Ka

-2

Eqn. (7.14) shows that DA (and hence VR) depends only on one experimental parameter, the ratio C/Ka. A plot of the theoretical relationship between DA and log (C/Ka) is shown in Fig. 7.11, together with experimental points obtained for 33 solutes (mineral acids, carboxylic acids and nitrophenols). Good agreement is obtained between theory and experiment. The above retention model has been recently extended to remove the requirement to calculate the sample concentration at the peak maximum (c), and to consider the case where a buffer is added to the eluent [78]. An iterative, numerical procedure is required in order to calculate DA for different solute and eluent conditions. Since this is a lengthy and complex process, the extended model will not be considered here.

Ion-Exclusion Chromatography

n5

1

I

0

215

I

5

I

1

15 ‘firnc (min)

I

20

I

25

Fig. 7.12 Analysis of human urine using ion-exclusion chromatography. An Interaction ORH801 column was used with an eluent comprising 10 mN H2SO4 containing 10% methanol. Detection was by spectrophotometryat 254 nm. Solute identities: 1 = oxalic acid, 2 = oxaloacetic acid, 3 = a-ketoisovaleric acid, 4 = ascorbic acid and a-keto-P-methyl-n-valeric acid, 5 = pphenylpyruvic acid, 6 = uric acid, 7 = a-ketobutyric acid, 8 = homoprotocatechuic acid, 9 = unknown, 10 = unknown, 11 = hydroxypheylacetic acid, 12 = p-hydroxyphenyllactic acid, 13 = homovanillic acid. Reprinted from 1321 with permission.

7.6

APPLICATIONS OF ION-EXCLUSION CHROMATOGRAPHY

Ion-exclusion chromatography has many applications in IC, but for the purposes of illustration, we will consider here only three of the more important applications. These are the determination of carboxylic acids, weakly ionized inorganic compounds, and water. Further applications may be found in the Tables comprising Part V of this book. 7.6.1

Carboxylic acids

The separation of carboxylic acids is the most common application of ion-exclusion chromatography. This mode of chromatography is undoubtedly the method of choice for these solutes. When coupled with direct spectrophotometric detection at low wavelength, ion-exclusion chromatography yields excellent separations and relatively

216

Chqpter 7

TABLE 7.4 SOME APPLICATIONS OF THE DETERMINATION OF CARBOXYLIC ACIDS BY IONEXCLUSION CHROMATOGRAPHY Sample Acid rain Antarcticice Blood coffee Fhit juice Milk

Warrnaceuticals Plasma Plating baths Ringers solution Solder fluxes Sugar cane juice Urine Urine Wine Wine a

COlUmn

Dionex HPICE-AS2 Aminex HPX-87H Dionex HPICE-AS1 Dionex HPICE-AS1 Bio-Rad AG5OW-X2 Aminex HPX-87H Dionex HPICE-AS1 Dionex HPICE-AS1 Wescan 269-006 Dionex HPICE-AS1 Waters Ion-Exclusion Aminex HPX-87H Interaction ORH-801 Aminex HPX-87H Intaaction ORH-801 Dionex HPICE-AS1

EluenP 2.0 mM HCl 5.0 mM MSA 10 mM HCl 10 mM HCl 0.75 mv1n-BA 10 mM H2SO4 25 mM 0.5 mMH2CO3 3.2mMHN03 5.4 mM H2CO-J 1.O mM OSA 5 mM H2SO4 10 mM H2SO4 25 mM H2SO4 10 mM H2SO4 2.0 mM OSA

Detection

Detection Ref

methdb

limit

C

0.03 ppm 15 8 79 lppm 56 0.5 ppm 83 1 ppm 5 1 ng 84 2ppm 67 0.5 ppm 89 0.1 ppm 16 50ppb 90 5PPm 9 0.3 ppm 32 0.5 ppm 54 0.5 ppm 30 1PPm 60

Spec (200nm) 6-9 ppb

C C C Spec (210 nm)

RI C C C C

RI

Spec (254 nm) Spec (200nm)

Spec (210 nm) C

MSA = methane sulfonic acid, n-BA = n-butyric acid, OSA = octane sulfoNc acid. C = conductivity. RI = refractive index, Spec = spectrophotometry.

clean chromatograms for a wide variety of very complex sample matrices. These samples include biological materials, such as urine [54, 791, tissue [13], blood 1801, plasma [67, 80,811, serum [82] and bile [82]; foods and beverages, such as wine [31,60, 76, 831, coffee [56], milk [5] and cane juice 191; and pharmaceuticals, such as Ringers solution [16, 661, tablets [67] and intravenous solutions [84, 851. Fig. 7.12 shows a chromatogram for a urine sample, obtained without sample pretreatment, and illustrates the relatively clean chromatograms which can be achieved for the above complex samples. Industrial and environmental applications of carboxylic acid determinations are also common and include samples such as acid rain [ 151, diesel exhaust [86], plating baths [87-891 and sewage [Sl]. Table 7.4 lists some of the chromatographic conditions employed in these separations.

7.6.2 Weak inorganic acids and bases Ion-exclusion chromatography has found increasing usage for the determination of weakly ionized inorganic species. It is especially attractive as an adjunct to ion-exchange chromatography since the selectivities obtained by these two techniques are quite different (see Sections 2.1.4 and 7.4). Solutes such as fluoride [43, 911, carbonate [39],

Ion-ErcluswnChromarography

217

A s (V)

32-

[O.Ol AU

-

Anions

L rill

0

1

2

Time (min)

3

0

6

12 18

Time (min) (b)

Fig. 7.13 Determination of inorganic species by ion-exclusion chromatography. (a) A Brownlee Polypore high-speed anionexclusion column was used with 6 mM H2SO4 as eluent. Detection was by amperometry using a Pt electrode at +0.4V versus AdAgCl. The sample was peppers in vinegar. Reprinted from [l 11 with permission. (b) An Aminex HPX-87H column was used with 10 mM H3PO4 as eluent. Detection was by spectrophotomeay at 200 nm. Reprinted from [62]

with permission.

cyanide [58], borate [42]. sulfite [47], phosphates [24], nitrite 1921, arsenite 1621. arsenate [62] and ammonium [92] have been determined using this approach. Interference from strongly ionized species is minimal because these solutes are unretained and appear at the column void volume. Ion-exclusion chromatography can therefore readily separate weakly ionized solutes in samples containing high concentrations of ionic species, e.g. seawater and wastewater. Table 7.5 lists some of the applications of this technique in inorganic analysis. Fig 7.13 shows typical chromatograms obtained in two important applications, namely the determination of sulfite (Fig. 7.13(a)) and inorganic arsenic ions (Fig. 7.13(b)). The fact that all strongly ionized solutes are eluted at the void volume in ionexclusion chromatography opens up the possibility of a two-dimensional chromatographic system in which these solutes are collected and then separated on an ion-exchange system. This type of chromatographic system will be discussed in Section 15.4.

218

Chapter 7

TABLE7.5 SOME EXAMPLES OF THE DETERMINATION OF INORGANIC SPECIES BY IONEXCLUSION CHROMATOGRAPHY

Solute(s)a

Sample

column

Eluentb

Demc Ref

Plating baths Mineral water Seawater Fluxes Mouthwash Wastewater Biological fluids Water Water Disinfectant Effluents

Dionex HPICE-AS 1 Aminex HPX-87H Aminex HPX-87H Waters ion-exclusion Wescan 269-006 Cation-exchange resin Dionex HPICE-AS 1 TSK-gel SCX Waters Fruit Juice Waters Fruit juice Dionex HPICE-AS 1 TSK-gel SCX

1 mMH2SO4 lOmMH3P04 5mMH3po4 1.0 mM OSA 3mMH2SO4 40% M e o w 2 0 Water 1 mM benzoic acid 1.25 mM H2SO4 1 mMH2SO4 50mMmannitol 0.1 M fructose

Amp 87 Amp 62 spec 63 C 43 93 C Coul 91 C 82 C 39 42 RI 94 RI 12 C C 41

Water Foods Process water Process water Process water Process water

Bio-Rad AG50W-X8 Wescan ion-exclusion Hitachi 2632 Anion-exchange resin Cation-exchange resin Hitachi 3613

58 C Amp 47 C 28 Coul 92 Spec 92 Spec 95

Hitachi 2613

1 mM HCl 5mMH2SO4 Water 10%MeOH/H20 10%MeOH/H20 0.1 mMH2SO45% MeOH Dioxane-water

Wescan 269-05 1 Dionex HPICE-AS I

5mMH2SO4 5.4mMH2C03

Coul

24

Amp

49 16

C

a DMSO = dimethylsulfoxide.

OSA = mane sulfonic acid. Amp = amperornetry, Spec = spectrophotomeay,C = conductivity, Caul= coulomeay, RI = refractive index

7.6.3

Water

One of the more significant recent developments in ion-exclusion chromatography is the application of the technique to the determination of water. This determination is a very important and frequently encountered analytical problem. Water, being a small, neutral molecule, can be expected to show retention on an ion-exclusion column, provided a suitable non-aqueous eluent is employed. Stevens et al. [20] showed that water can be separated from other sample components by ion-exclusion chromatography on a short column packed with Aminex 50W-X4resin (H+) form, using an eluent comprising methanol and a small amount of HCI, H2SO4 or p-toluenesulfonic acid. Detection was achieved by conductivity measurements, with the water showing decreased conductance relative to that of the eluent. Fig. 7.14(a) shows the chromatogram

219

Ion-ExclusionChromatography

(0)

lime lminl 0 2 1 8

Fig. 7.14 Determination of water using ion-exclusion chromatography. (a) A short (9 x 21 mm) column packed with Aminex 50W-X4 (H+form) resin was used, with a methanolic eluent containing 1.2 mM HCI. Conductivity detection was used. Reprinted from [20] with permission. (b) A 150 x 2.1 mm I.D. column packed with Aminex Q-150s resin in the H+ form was used with 1.0 mM cinnamaldehyde in methanol as eluent. Detection was by spectrophotometry at 300 nm. The sample was 0.184% H20 in dichloroethane. Reprinted from [97] with permission.

obtained. The main drawback with this method was variability in the detector response as the concentration of water in the sample was altered. Fritz and co-workers [96, 971 have reported an alternative ion-exclusion method in which the water is separated using a cation-exchange column in either the Li+ or H+ form, with cinnamaldehyde in methanol as the eluent. Detection is accomplished by spectrophotometric monitoring of the equilibrium existing between cinnamaldehyde and cinnamaldehyde dimethylacetal:

2CH30H + cinnamaldehyde % H 2 0

+ cinnamaldehyde dimethylacetal

(7.15)

This reaction does not occur to any appreciable extent until an acid catalyst is present. The catalyst may be the hydrogen-form cation-exchange resin in the column, or

Chaprer 7

220

an acid can be added to the eluent. After catalysis, the above equilibrium lies well to the right. When water is injected, there is a small shift in the equilibrium towards the formation of cinnamaldehyde. This change can be detected spectrophotometrically at 300 nm. Excellent sensitivity is achieved by this method and the analysis can be completed in less than 2 min. Fig. 7.14(b) shows a typical chromatogram obtained using this approach. Both of the methods depicted in Fig. 7.14 have been applied to a wide range of sample types, with good results. 7.7 1 2 3 4 5

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

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