Modes of CEC Separation

Chapter 3

Modes of CEC Separation Christopher M. J O H N S O N , A l a n P. M c K E O W N and M e l v i n R. E U E R B Y

AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leicestershire, LE11 5RH, UnitedKingdom

CONTENTS

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Unmodified packings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M o d i f i e d packings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1 R e v e r s e d - p h a s e C8, C 18, p h e n y l . . . . . . . . . . . . . . . . . . 3.4.2 The use o f m o b i l e phase additives . . . . . . . . . . . . . . . . . . 3.4.3 Ion exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.3.1 SCX . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.3.2 SAX . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.3.3 Mixed mode . . . . . . . . . . . . . . . . . . . . . . . Chiral stationary phases . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Gel C E C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Monoliths . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Size exclusion C E C . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8 Gradient C E C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9 3.10 Selectivity c o m p a r e d with L C . . . . . . . . . . . . . . . . . . . . . . . 3.11 Guidelines for the analysis o f acidic basic and neutral c o m p o u n d s . . . . 3.11.1 Neutral analytes . . . . . . . . . . . . . . . . . . . . . . . . . . 3.11.2 Acidic analytes . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.11.3 Basic analytes . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.12 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.13 A b b r e v i a t i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.14 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 3.2 3.3 3.4

88 89 89 94 95 98 98 98 99

100 100 101

101 102 103

103 104 104 105 105 106 106 106

Chapter 3

88 3.1 INTRODUCTION

The separation scientist with experience gained from a LC background may tend to limit the modes of electrochromatography to reversed phase (RP), normal phase, ion-exchange and, maybe, size-exclusion. Analysts from an electrophoretic background typically use the term "CE" in a much broader sense to include the main modes of capillary zone electrophoresis, micellar electrokinetic chromatography, capillary gel electrophoresis, isoelectric focusing and isotachophoresis. As capillary electrochromatography (CEC) is a hybrid technique between CE and LC, there are actually many modes of operation ranging from those commonly used in CE to those described in LC. This is shown diagrammatically in Table 3.1. The section of the table topped by the heavy bar indicates the "packed CEC" region and it is these areas which are to be covered in this chapter. There is considerable scope for overlap between the described cells, and many further divisions of these artificial boundaries are possible, for example pressure-assisted electrochromatography (PEC) describes a continuum from pure CEC to pure gLC. Many of the modes of CEC illustrated in Table 3.1 are applicable to both gradient and isocratic elution, aqueous and non-aqueous conditions, as well as to chiral and achiral separations and these will be discussed within the appropriate sections. The complex mechanisms responsible for selectivity will not be discussed, rather this chapter will be limited to describing the scope for application of the different CEC modes. Packed CEC columns are prepared from polyimide coated fused-silica capillaries typically with 20 lam to 180 lam internal diameter (i.d.). The packing material is retained as a column in the fused silica between frits that are generated in-situ by controlled sintering of the siliceous packing by heating, although there are examples of fritless CEC columns [ 1]. The columns typically have a packed segment between the inlet frit and a frit prior to the detection window (from 7 cm to 70 cm), with the unpacked region continuing to the outlet vial. However, attempts have been made to carry out UV detection through the pack segment of the capillary [2-4]. Despite the potential advantages of increased efficiency with in-column detection, the decreased sensitivity due to the particle-induced scattering and increased background noise has made this approach less popular.

TABLE 3.1

89

Modes of CEC

3.2 DEFINITIONS

Rather than explain the detailed theory for different modes of CEC which will be dealt with in a later chapter, it was considered appropriate to collate the information as a table where mode descriptions could be compared and contrasted (Table 3.2).

TABLE 3.2 DEFINITIONS OF CEC MODES

Mode

Packing

Basis of separation

CEC gel

Gel e.g. polyacrylamide or agarose Size and charge

CEC monolith

Mouldedrigid porous polymer

Size, charge and partition

CEC packed SEC

Controlledpore size silica

Size*, charge and exclusion

CEC packed unmodified

Silica gel

Size, charge, adsorption & "reverse phase", ion-exchange

CEC modified

Silica modified with C 18, C8, phenyl, SCX, SAX etc.

Size, charge, partition, ion-exchange

PEC

As above

Size, charge, partition, ion-exchange

*In electrodriven size exclusion chromatography elution time is influenced by the exclusion mechanism and for charged molecules by the electromobility.

3.3 UNMODIFIED PACKINGS

Many reviews have been written on the preparation, physico-chemical properties and application of silica in modern separation science [5-7]. RP LC with silica based bonded stationary phases is utilised for the majority of LC separations in laboratories world-wide. Their ubiquity derives from their versatility, in that generally a wide range of both ionic and non-ionic analyte species can be separated with these columns by careful selection of the stationary phase and mobile phase properties. The use of unbonded silica has tended to be limited to techniques such as adsorption chromatography and supercritical fluid chromatography (SFC) [8,9] which are important techniques, but by no means as widespread as their dominant reversedphase LC cousin. A number of reasons can be suggested for this including their biological incompatibility, the use of specialist equipment (in SFC) and the use of

Referencespp. 106-110

90

Chapter 3

toxic non-aqueous solvents (which is actively discouraged in many laboratories from an environmental and disposal cost perspective). The use of unbonded silica with aqueous-organic eluents was reported many years ago in LC, where it was discovered that good peak shape for strong bases could be achieved [ 10]. A number of publications then followed looking at other analytes and included examinations of the separation mechanism [ 10,11 ]. Interest in this mode of LC was short-lived however, as workers realised that using inorganic buffer salts such as phosphates with high aqueous conditions led to severely reduced column life due to the silica dissolution. Recent work at AstraZeneca R&D Chamwood has circumvented this issue by using amine base buffers in conjunction with a high proportion of organic solvent in the mobile phase [12]. As CEC is a relatively new technique there are few reports using unbonded silica as a stationary phase. Reports to date describe the use of unbonded silica with various conditions and additives including non-aqueous solvents [ 13], dynamic coatings using cationic surfactants [14], blending with ODS phases [15] and cyclodextrin selectors [ 16]. Perhaps more interesting from a pharmaceutical perspective are the few preliminary publications detailing separations using various unbonded silicas for strongly basic [ 17-20] and weakly basic analytes [21 ]. It is of interest to note that certain types of silica exhibited the anomalous peak focusing in a similar fashion to the strong cation exchange materials described by Smith & Evans [22]. In recent work within our laboratories, we have evaluated a number of unbonded silicas by CEC specifically for the separation of a range of pharmaceutically relevant basic analytes and mixtures. We purposefully chose strong basic analytes that comprised a wide range of lipophilicities, molecular weights and log P values to robustly test the separation systems. The basic analyte test mixture contained two AstraZeneca R&D compounds, benzylamine, nortriptyline, diphenhydramine and procainamide. The following section describes initial work from an on-going research program to fully appreciate the role that the silica surface, its components and bonding chemistries play in contributing to CEC separations for various analytes. Packing methodology was based on a slurry procedure similar to that described previously [23] and found to be very repeatable once optimised for all phases studied. Initial testing of the capillaries was based on direct comparison of chromatographic parameters such as the magnitude of the electrosmotic flow (EOF) and the selectivity between two components. Figure 3.1 shows four unbonded silicas tested using a test mixture comprising biphenyl, benzamide, benzyl alcohol and thiourea. It was quickly established that thiourea was retained on all unbonded phases, and could not be used as the EOF marker. A number of other analytes was tried, and biphenyl was selected. It can be seen from Figure 3.2 that the purer silicas (HyPURITY and Kromasil) give a lower EOF than the more acidic traditional Hypersil

Modes of CEC

91

mAU 15 10

(A)

2 12 8 4 0

4

6

min

(B) 12 2

4

6

4

8

0 min

(C)

3 12 .

4

2

4

6

.

.

.

.

.

.

.

.

.

10

8

(D)

1 2~

2

4

6

8

12 min

~3

10

min

Fig. 3.1. Separation of the unbonded silica test mixture using in-house packed (A) Hypersil silica, (B) Hypersil BDS silica, (C) Hypersil HyPURITY and (D) Kromasil silica capillaries (100 l.tm i.d., 33.5 cm total length and 25 cm effective). Conditions: 8:2 v/v ACN-50 mM MES, pH 6.1, 20 kV, 20~ 5 kV for 3 sec. injections, 254 nm. Peak identities: 1 - biphenyl, 2 = benzamide, 3 - benzyl alcohol and 4 = thiourea. Adaptation of [20]. Reproduced with the permission of Chromatographia.

silica. These results are explained by the presence of fewer metal cations in the silica phases which will reduce the proportion of acidic silanols, leading to a reduced EOF generating ability. The better peak resolution of the neutral test mixture on the Kromasil material was attributed to its known greater surface area properties and its slightly larger particle diameter (all Hypersil phases dp= 3 lam and surface area of 170 m2/g whereas Kromasil dp= 3.5 lain and surface area of 340 m2/g). Efficiency values could not be compared for these phases, as the sample loading was different due to the use of electrokinetic injection. The traditional Hypersil unbonded silica at pH 7.8 gave baseline separation within 14 minutes of the basic analyte mixture (Fig. 3.2). The poorer chromatographic performance of the procainamide analyte was attributed to its suspected metal chelating properties. The purer HyPURITY unbonded silica gave broader peak shapes with reduced analysis times than the traditional acidic silica, but complete separation of the analytes was not observed. The separation of the bases on Hypersil BDS was observed

References pp. 106-110

Chapter 3

92 mAU 60-

(A)

II

40 IV I ll

0

2

4

VI

6

30

8

10

12

min

II (B)

20 I

IV V

10 VI 2 40

4

S ......

i0

:mi.

II

(C) 30

l/1V

20

1

10

0

2

4

6

8

min

Fig. 3.2. CEC separation of the basic test mixture using (A) Hypersil silica, (B) Hypersil BDS silica and (C) HyPURITY silica capillaries (100 ~tm i.d., 25 cm effective length, 33.5 cm total). Conditions: 6:2:2 v/v/v ACN-H20-50 mM TRIS, pH 7.8, 20 kV, 20~ 5 kV/3 s injections, 210 rim. Injection mixtures in (A) and (C) were equivolume compositions of each base at 1 mg/ml and in (B), equivolume compositions of each base at 0.1 mg/ml. The EOF marked by biphenyl under these conditions and was similar on all phases at approximately 4 minutes. Peak identities: I = AZ compound A, II - Benzylamine, l l l - Nortriptyline, IV Diphenhydramine, V - AZ compound B and VI = Procainamide. Adaptation of [20]. Reproduced with the permission of Chromatographia.

to fall between these two extremes. It is interesting to note that the EOF is similar on all the unbonded phases at pH 7.8, which is not what was observed at pH 6.1. This observation provides that the non-acidic silanol groups are intrinsically involved in EOF generation, which has not been widely appreciated previously.

93

Modes o f C E C

mAU 20 III 10

1 4 ........

S. . . . . . . .

12 . . . .

min

Fig. 3.3. Separation of neutral, acidic and basic components in their ionised form with Hypersil unbonded BDS silica. Peak identities: I- Benzylamine, II- Caffeine and III = p-hydroxybenzoic acid. The arrow denotes the EOF. Conditions = 6:2:2 v/v/v ACN:H20:50 mM MES, pH 6.1, 20 kV, 20~ 214 nm, 8 bar for 15 sec. inj. Adaptation of [20]. Reproduced with the permission of Chromatographia.

The separation mechanism with unbonded silica is extremely complex and attempts to discuss the details have been reported [19,20]. It is postulated that the separation mechanism with unbonded silica is a balance between the analyte's electromobility and an interaction component (comprising of predominantly ion-exchange, adsorption and, surprisingly, a RP character) with the unbonded silica. The ion-exchange properties of the silica are critical as phase purity is thought to dictate which of these factors dominates over the others. For example, with HyPURITY silica, there are reduced interactions with the silica surface due to a lower ion-exchange capacity, so the analyte's electromobility dominates the separation leading to shorter retention times. With Hypersil silica, the ion-exchange interactions dominate, leading to increased retention and different selectivity. A purpose designed CEC stationary phase that gives excellent EOF character yet allows rapid simultaneous acidic, basic and neutral separations under isocratic conditions without tailing is yet to be discovered. Nevertheless, the Hypersil BDS unbonded silica used in this work was taken forward to explore such complex mixture separations as it combines features of both pure and traditional media. As a phase, it possesses a reasonable EOF (as it is based on traditional silica) with a lower number of activated silanol groups on the silica surface (due to the pre-treatment procedure used to remove surface metal contamination). Figure 3.3 shows the separation of benzylamine, caffeine and p-hydroxybenzoic acid in a single chromatographic analysis.

References pp. 106-110

Chapter 3

94

It is important to note that the acidic and basic species are analysed in their ionic form under these conditions and complete separation of the mixture components is observed. Peak shape is seen to be good for all components, with the basic analyte eluting quickly due to its positive electromobility influence. The neutral species elute just after the EOF, whilst the acid is seen to elute slightly later due to its negative electromobility. It is clear that the unbonded Hypersil BDS may be additionally used for the separation of complex mixtures. 3.4 MODIFIED PACKINGS

The packing materials used in CEC are generally derived from LC technology and over 95% of CEC separations and publications are completed using silica based C18 bonded phases [24]. It is thought that the dominance of the C18 bonded phase for separations in CEC is derived from the majority of workers requiring determinations involving non-polar and weakly ionic analytes only. Nevertheless, work using other bonded phases such as C8 and phenyl [25,26], chiral phases [27,28] anion and cationexchange phases [22,29,30] and mixed mode [31 ] media [32] have been published. The hydrophobic selectivity of the stationary phase can be increased in an analogous way to RPLC, by covalently bonding hydrophobic moieties to a silica support material. In CEC this bonding has an influence on the EOF [33]. The total number of free silanol groups is reduced hence reducing the zeta potential and reducing the EOF. The same effects are found when end-capping is performed. The greater the coverage the more the EOF is reduced. Practically, this could make migration times too long for non-ionic species whose elution depends on the EOF alone. The pH of the mobile phase also has a profound influence on the EOF as the pH dictates the number of silanol groups that are ionised. This has been addressed in a number of ways, which are discussed later. A further practical consideration for CEC is bubble formation. This can result from Joule heating in the column or decompression on passing from the packed to open capillary. In addition to bubbles causing spikes as they pass through the detector, there is the danger of a break in the electrical circuit. Once formed, such bubbles are difficult to remove without applying a high extemal pressure to flush the column. Additionally, as the flits that retain the packing material are often made in-column by sintering the packing material, there is a danger of removing the bonded ligand during the hydrothermal process of preparing the frit which may generate undesirable surface active sites. Joule heating is minimised by using low ionic strength buffers, low voltages, organic buffers, low column temperatures and narrow intemal diameters. Decompression is avoided by low voltages or applying an extemal pressure to the capillary. It is

Modes of CEC

95

the latter approach, which is adopted by purpose made instruments as this, allows the use of high voltages with the concomitant rapid analysis times. However CEC may be performed using standard CE equipment by the use of low temperature and limiting voltages to <15kV [34,35]. A further approach to minimise bubble formation and hence allow unpressurised CEC to be performed, has been to use narrow and resilyated outlet frits [36] or incorporate sub-micellar concentrations of sodium dodecyl sulphate into the mobile phase [37,38]. Typically, packing materials designed for LC have been used for CEC [39]. However, there are significant differences in the requirements for packing materials for CEC and LC. Firstly, the EOF does not suffer from the significant increase in back pressures associated with using smaller particles in LC, smaller particles (0.2-3 lam) [40,41 ] and longer columns may be used in CEC and therefore highly efficient separations are possible. Secondly, the requirement for mechanical strength is lessened with electrodriven separations compared with pressure-driven systems. Finally, only extremely small quantities of packing material are required for a CEC column hence packings that would not be economically viable for LC become realistic options for CEC.

3.4.1 Reversed-phase (i.e. C8, C18, phenyl) In the RP CEC of neutral species selectivity is provided primarily by differences in the partition of the analytes between the hydrophobic stationary phase and the more polar mobile phase. There are also contributions from interactions with the silica support, the major one being polar interactions with ionised silanol groups. This is identical to the process in LC, albeit with the advantages of higher efficiencies in CEC resulting from the plug-flow profile. Additional selectivity is introduced in the case of charged species in CEC due to differences in the analytes' electromobilities. Standard C18 LC stationary phases packed into fused silica capillaries were the first phases to be extensively used for CEC [42-47]. Both porous and non-porous particles have been employed in CEC [38,41 ]. These stationary phases have been used in conjunction with familiar buffered aqueous RP mobile phases with organic modifiers such as methanol, acetonitrile and tetrahydrofuran [48]. The relationship between log retention factor (k) and percentage organic for uncharged species has been shown to be linear with eluents containing methanol and acetonitrile [25,26,33,46]. This allows rapid method development in an analogous way to that used in LC. It is important to note that, in addition to the influence on selectivity, changing the percentage or type of modifier has a profound impact on the EOF as a result of changes in the dielectric constant : viscosity ratio [49]. Hence, whilst mobile phases may be isoelutropic the actual retention times, even for neutral species, will differ significantly due to changes in flow rate induced by changes in viscosity (see Fig. 3.4). References pp. 106-110

Chapter 3

96

23

mAU ~ 40

1

,o

4

6

20

_

2

4

mAU ]

_

8

6

10

12

14

min

1 3

5

10

15

20

2

25

30

35

min

Fig. 3.4. Effect of mobile phase selectivity on the CEC separation of barbiturates (1-6). Electrochromatography was performed at 15~ with an applied voltage of 30 kV on a 25 cm, 100 ~tm i.d., 3 ~tm Hypersil Phenyl packed capillary. Sample concentration was 170 ~tg ml 1 of each component with a 15 kV/5s injection. Detection was at 210 nm. a) ACN-50 mM phosphate buffer, pH 4.5-water (4:2:4 v/v/v), b) MeOH-50 mM phosphate buffer, pH 4.5-water (5:2:3 v/v/v). From Euerby et al [26], 9 of Microcolumn Separations, 1999. Reproduced with permission of John Wiley & Sons, Inc.

Typically, acetonitrile yields higher efficiencies and more rapid elution than methanol due to acetonitrile's higher dielectric constant and lower viscosity. The pH of the mobile phase will have a profound effect on acidic and basic analytes and upon the number of ionised silanol groups on the silica surface and hence the EOF. Buffers must be used to ensure reproducible pH conditions. The type and concentration of buffer in the mobile phase is more limited in CEC than in LC as high inorganic buffer concentrations lead to excessive current and hence Joule heating as in CE. Therefore inorganic buffers such as phosphates and borates are used at low concentration (<50 mM) or organic buffers commonly used in CE such as tris(hydroxymethyl)aminomethane (Tris), and 2-(N-Morpholino)ethane-sulfonic acid (MES) are employed. The ionic strength of the mobile phase is also an important factor as increasing the ionic strength of the mobile phase reduces the zeta potential, which results in a decreased EOF and hence increased retention times. The classical stationary phases used for LC possess relatively high levels of metal ions in the silica support material. These give rise to a large number of highly acidic silanol groups. After bonding of the stationary phase the remaining isolated silanols prove troublesome for the analysis of bases. In recent years, a new generation of pure silicas have been manufactured via highly controlled, improved processes. These phases possess lower metal content and hence the acidity of any residual silanol

97

Modes of CEC

mAU

~1

3

2

.

,.

20 10 0

201

1~

3~

2

4 6

2!1

6

r

1

4

5

6

7

8

9

10

11

min

Fig. 3.5. Chromatograms of the optimised separation of the barbiturates (1-6) on three different packing materials. Electrochromatography was performed with an applied voltage of 30 kV on a 25 cm, 100 ktm i.d., 3 ~tm. Sample concentration was 170 ~g ml"1 of each component with a 15 kV/5s injection. Detection was at 210 nm. (a) Hypersil C8, ACN-50 mM MES, pH 6.1-water (5:2:3 v/v/v), 15~ (b) CEC Hypersil, ACN-50 mM MES, pH 6.1-water (4:2:4 v/v/v), 15~ (c) Hypersil Phenyl, MeOH-50 mM phosphate buffer, pH 4.5-water (5:2:3 v/v/v), 60~ * corresponds to an artifact in the mobile phase. From Euerby et al [26], 9 of Microcolumn Separations, 1999. Reproduced with permission of John Wiley & Sons, Inc.

groups is greatly reduced, in turn, improving basic analyte separations in LC [50]. Unfortunately, it is the presence of ionised silanols which generate the EOF essential for the elution of neutral species in CEC, therefore the practical working pH range in CEC is reduced with the new generation silica based packing materials. In addition to generating excessively long retention times with these pure phases at low pH, there is the practical problem of the phase drying out resulting in breakdown of the current. It is for this reason that stationary phases are being developed specifically for CEC. Robust and reproducible methods have been developed with traditional RP materials for neutral and ion suppressed acidic analytes [51,52], in application to pharmaceutical analysis [34,53,54], aromatic compounds [55], phenols in tobacco smoke [56], preservatives in creams [40,41] nucleosides [57,58] and cannabinoids [59]. Typical efficiencies were >100,000 plates/m. The analysis of bases, however, remains a challenge (see later section). The use of other column chemistries such as C8 and phenyl phases allows the selectivity of the stationary phase to be optimised in a similar fashion to that used in LC [60-62], (see Fig. 3.5).

Referencespp. 106-110

Chapter 3

98 3.4.2 The use of mobile phase additives

As the generation of a significant EOF requires the presence of ionised groups on the surface of the stationary phase, classical silicas with high metal contents have been widely used in CEC. Before "new generation" silica RP column chemistries became available, undesirable secondary interactions of basic analytes in LC were observed. The traditional approach to the analysis of bases by LC using stationary phases containing acidic silanols was to use small competing bases added to the mobile phase, such as triethy!amine or, in CE, triethanolamine has been used. Recent work in this area has demonstrated that strong and weak acids and bases, and neutrals can all be eluted in one CEC run by the use of triethylamine phosphate or triethanolamine phosphate [63] (see Fig. 3.6) or hexylamine at pH 2.5 [64,65]. By the use of this approach acids are chromatographed in their ion-suppressed mode whilst strong and weak bases are positively charged. The uncharged species are separated according to differences in lipophilicity by interactions with the RP stationary phase giving rise to significantly different selectivity to that in CE. The selectivity also differs from LC for the charged bases due to additional differences in electrophoretic mobility. An added benefit is the ability to distinguish bases in mixtures of acids, bases and neutrals as the positively charged species elute before the EOF at pH 2.5. The orthogonal separation mechanism provided by this approach is extremely attractive to the pharmaceutical industry. The inclusion of basic additives in the run buffer leads to a reduction in the EOF. This is due to the reduction in the number of free silanol sites on the silica surface. However, above 50 mM the continued reduction in the EOF is less pronounced [63]. In practice, sufficient EOF is generated, even in the presence of mobile phase additives, to elute neutral species in acceptable times. The upper limit on the additive concentration is most frequently due to excessive baseline noise arising from high background absorbance. The inclusion of mobile phase additives leads to a further level of complexity in method development and prohibits coupling to mass spectrometry. However, this approach is a practical solution until better stationary phases are developed. 3.4.3 Ion exchange - SAX, SCX and Mixed Mode

3.4.3.1 SCX In order to extend the practical working pH range in CEC the use of stationary phases containing charged functional groups has been utilised. A strong cation-exchange phase (SCX, which contains a sulphonic acid group) will be negatively charged from pH 2 to pH 9. Hence, a good EOF is maintained throughout this region

99

Modes o f CEC

140 ~ 120 "~ r~

100

g

8o

III

~ 60 b-s 4o ~ 20

EOF

'

'

'

I

'

2

'

'

I

4

'

'

'

I

6

'

'

'

I

8

'

'

'

I

'

'

10

'

I

12

'

'

'

I

'

14

Time (min)

Fig. 3.6. Electrochromatogram of benzylamine (I), caffeine (II) and benzoic acid (III). Efficiency values of 4642, 76331, and 6399 plates per column were obtained respectively for I, II, and nI. Electrochromatography was performed at 15~ with an applied voltage of 25 kV on a 25 cm, 100 ~tm i.d., 3 ~tm Hypersil Phenyl packed capillary. Mobile phase: ACN-50 mM triethanolamine phosphate, pH 2.5-H20 (6:2:2 v/v/v). Sample concentration was 100 ~tg ml 1 of each component, 5 kV/5s injection. Detection at 214 nm. From [63]. Reproduced with permission of The Royal Society of Chemistry

[22]. Considerable excitement was generated when Smith and Evans reported efficiencies in excess of 8 x 106 plates m 1 for the analysis of the basic tricyclic antidepressants. These high efficiencies have also been obtained by other workers for a range of structurally diverse basic compounds. Unfortunately these workers, including Smith and Evans, have experienced severe non-reproducibility of the phase, in that severe tailing and fronting have been unexpectedly observed in the middle of successful runs [46,66,67]. Explorations into the focussing mechanism have been reported [68], but it is yet to be fully characterised and understood. However, the separation of weakly basic aromatics on this phase has been demonstrated [21]. An additional disadvantage of the SCX phase is that it possesses little hydrophobic character and hence is not very selective for the separation of neutral analytes. Harnessing the phenomenon of high peak efficiencies in a repeatable fashion could lead to a new surge in the interest for CEC. 3.4.3.2 SAX

In the case of a strong anion-exchange phase (SAX, which typically contains a quaternary amine), EOF reversal is observed, and an ion-exchange mechanism with negatively charged species will also result [69]. This approach has been successfully applied to the analysis of iodide and iodate [70], and has recently been reported for the References pp. 106-110

1O0

Chapter 3

separation of a range of large proteins using a custom synthesised tentacular anion exchanger [71]. The resolving power of CEC with this phase for protein work was demonstrated with separations of chicken egg-white conalbumin variants in a single four-minute run with isocratic eluents. Other workers have reported the use of anion exchanger columns for lanthanide and inorganic ion analysis [29] and aqueous and non-aqueous chiral derivatised amino acid analysis [72,73]. 3.4. 3.3 Mixed mode

There are two types of mixed mode phases 1) physically distinct particles possessing separate lipophilic and ion exchange chemistries [74] and 2) the lipophilic and ion exchange chemistries on the same silica particle [26,75]. The use of mixed mode phases (which contain charged functional groups and lipophilic spacers or separate alkyl ligands) has yielded rapid, efficient methods for the analysis of neutral and ion suppressed acids. Custom synthesised multilayer mixed-mode phases containing ligands with a sulphonic acid sub-layer and a C18 top-layer have also been reported [32,58]. These phases have been successfully used to separate nucleobases and even strongly acidic analytes such as nucleic acids of various sizes from dinucleotides to t-RNAs. Excellent peak area, and migration time precision can be obtained for neutral and acidic compounds for a given mixed-mode column. A major advantage of using mixed mode phases is that they allow the rapid analysis of ion suppressed acidic and neutral analytes at low pH [26]. With the exception of one recent report by Rozing [76], using a new Zorbax SCX/C18, no success has been reported in analysing basic compounds with SCX mixed mode phases. 3.5 CHIRAL STATIONARY PHASES Chiral CEC will be discussed in detail later in the book but is included here to exemplify the application of the high efficiencies obtained with electro-driven techniques which makes them attractive for chiral analysis where selectivity factors are sometimes small. CE has made use of chiral additives in the electrolyte whilst LC tends to utilise chiral stationary phases. Both options have been explored for chiral CEC [27,28,77]. The small amount of packing material necessary for capillaries allows the use of chiral stationary phases that would be prohibitively expensive for standard LC. Cyclodextrins, proteins, antibiotics and molecular imprinting have all been used to form chiral stationary phases [78-80]. After some less than encouraging peak efficiencies obtained using the chiral CEC approach, much improved chiral resolutions have been achieved using CEC compared to LC or CE [81-83].

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3.6 GEL CEC

Columns packed (filled) with natural and synthetic polymers such as acrylamides, dextrans, glycols, poly(ethylene oxides), poly(ethylene glycols), methacrylates, agaroses and various cellulose derivatives have been widely applied and reviewed for a number of applications in capillary gel electrophoresis [84,85]. The rationale for such a diverse range of polymers is associated with their differences in physicochemical properties which allows separation of a variety of molecules and biopolymers over a wide range of polymer concentrations [86]. Gel CEC is included here simply because many publications describe covalently immobilised polymers or mimetic gels within a fused silica capillary as "electrochromatography" and a distinction between Gel CEC and CGE is therefore difficult to define. Nevertheless, Gel CEC will be covered in greater depth in a later chapter, and it is included here simply because, due to the lack of a clear definition, it could be argued to be the most widely used mode of CEC. 3.7 MONOLITHS The use of hydrothermally formed retaining frits in capillary columns packed with stationary phase particles is an accepted limitation in CEC. The introduction of the frit to hold the packed bed is vital, yet introduces problems such as EOF and flow non-uniformities, compromised frit permeability [87], capillary fragility, increased likelihood of bubble formation [88] and a thermally induced modified frit surface chemistry which can detrimentally alter the chromatography [23]. Practical aspects to be considered include the appreciable effort and skill of the analyst who is required to repeatably manufacture capillaries of a particular phase and redevelop the fritting and packing methodology for each different stationary phase type. The use of continuous bed columns (i.e. monoliths) can address many of these points. Early monolith research focussed on the polymerisation of acrylamides analogous to the approach utilised in CGE [89]. As the area developed, workers tried different chemistries but experienced problems that ranged from shrinkage of the monolith from the capillary wall and / or cracking of the monolith structure - leading to poor chromatography. It is these issues that have generally hampered the development of monolith columns for LC as well as CEC. A monolithic column was recently defined as "A continuous unitary porous structure prepared by in situ polymerisation or consolidation inside the column tubing and, if necessary, the surface is functionalised to convert it into a sorbent with the desired chromatographic binding properties" [90]. Whilst quite general, this definition actually covers a range of methods that can be used to produce continuous bed columns. References pp. 106-110

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The many reported methodologies for producing monolithic columns published to date differ in their manufacturing process and the end-product monolith. Porous moulded organic polymers are perhaps the most reported in CEC and involve in-situ polymerisation of monomer solutions in the presence of a porogen using various initiator techniques such as free radical polymerisation [91] or photopolymerisation [92]. By carefully controlling the polymerisation components and conditions for this methodology, monoliths of defined physicochemical properties may be produced which are robust and exhibit excellent chromatographic properties [93]. Particle-fixed continuous beds are another example involving the immobilisation of stationary phase particles of known characteristics using whole column sintering [94]. Finally, sol-gel technology involving the hydrolysis and polycondensation of precursors in a defined solvent in-situ to produce a hydrogel, which may then be converted to a xerogel upon drying before functionalising has been reported [95]. A derivation on this theme is the use of particle-loaded sol-gels where polycondensation of a mixture of polyalkoxysiloxanes and the stationary phase to produce end frits to effectively retain the phase [96] or to completely immobilise the column have been demonstrated [97,98]. Thus, the monolith approach is highly customisable for a particular need or application. It is an important area in column technology development with the number of publications in CEC, ~tLC and even monolith based standard LC increasing dramatically. Details and specific application examples of the use of these various monolith technologies in CEC will be discussed in a later chapter. 3.8 SIZE EXCLUSION CEC In size exclusion chromatography, selectivity for neutral molecules is based on molecular size and shape. The stationary phase consists of either a polymeric gel or a silica gel with controlled pore size. Larger molecules are excluded from the pores whilst smaller molecules, can enter the pores and are hence eluted later. In CEC of charged molecules additional selectivity is introduced based on electromobility. The mobile phase is used to change the molecular shape and/or charge and to optimise secondary interactions. Capillaries packed with unmodified silica gel with pore sizes between 100 and 10 nm have been evaluated for CEC [99]. In addition to the practical application to polymer analysis, the technique is of theoretical interest as a demonstration of the appreciable intra-particle flow in CEC with associated efficiency gains, which is absent in LC. Li and Remcho [ 100] studied packing media pore sizes from 6 to 400 nm and deduced that only the larger pore diameters (>200 nm) supported pore flow, which led to higher peak efficiencies. For SEC a low pore flow is required to obtain selectivity based on exclusion/inclusion in the pore. Hence low ionic strengths must be used to induce double layer overlap in the pores. The optimisation of the

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pore-to-interstitial flow for size-exclusion has been investigated by Stol et al [101 ]. The implications to RP CEC have also been explored [99,102] and the findings indicate that higher efficiencies and linear flow rates are obtained with larger pore sizes (e.g. 400 nm) and higher buffer strengths which favour high intra-particle flow. In further work, Stol et al [103] verified this work and demonstrated substantial intraparticulate flow with much smaller unbonded packing media (30 nm) in addition to the 400 nm material. The high efficiencies observed for the neutral test probes were attributed to better electrokinetic flow homogeneity due to the large pore size and enhancement of the mass transfer kinetics. A rigorous theoretical treatment of flow characteristics in packed beds and determination of an ideal packed capillary structure for electrokinetic flow was offered by Luo and Andrade [104]. 3.9 GRADIENT CEC AND VOLTAGE ASSISTED CAPILLARY LC Preliminary work has been published by a number of workers using research instrumentation [ 105-108]. There are, at time of publication, few commercially available continuous gradient CEC instruments. It is possible to perform simple step gradients using standard CEC instruments by changing the mobile phase during the run [109,110]. This will be a critical area for the development and acceptance of the technique. Preliminary reports using a prototype gradient CEC system capable of performing capillary LC with voltage assisted flow have been very encouraging [25]. It has been found that a large number of compounds of widely different lipophilicity may be eluted and resolved using isocratic CEC that would have required gradients for LC. This presumably is due to the high peak capacity of CEC and hence higher efficiencies compared to LC. The combination of pressure-driven and electro-driven flow offers a great potential for optimisation of separations. For example, CEC systems using columns with low EOF may not elute all species by electromobility alone, whereas the application of pressure will ensure a sufficient flow to elute all species [76,111 ]. The selectivity and structural information afforded by being able to elute analytes with and without applied voltage is also of practical benefit [47]. Once again the availability of commercial instrumentation will be the key to the success of this technique. 3.10 SELECTIVITY COMPARED WITH LC The potential influence of the nature of the driving force on the chromatographic selectivity for neutral molecules has been investigated using CEC, gLC and pressurised flow electrochromatography. The comparison was performed on the same prototype instrument and the same column to eliminate all other possible influences References pp. 106-110

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on the selectivity. The relationship between k and the percentage of organic modifier present in the mobile phase was used to compare the consequence of the different mechanisms on the separation of non-ionised molecules. The slopes and intercepts of the plots of log k against percentage organic in the mobile phase, obtained by using the different flow generation mechanisms, were statistically evaluated. As expected, a linear relationship was found between log k and the percentage of organic modifier for a series of weak acids and bases analysed in their ion-suppressed mode and for neutral compounds. Under the conditions investigated, the chromatographic selectivity in electro, pressure driven flow and a combination of thereof was shown to be equivalent for the non-ionised molecules studied [25]. Similar findings were reported by another group in that no significant differences could be demonstrated between CEC, PEC and ~tLC modes on the same capillary for separating a range of 27 neutral analytes [ 112]. In contrast other groups have reported the observation that the chromatographic characteristics of porous reverse phase materials depends on the mode of flow generation [113]. Other authors have additionally reported the different elution profiles between PEC and capillary LC for analytes that include basic amines [47]. On a practical basis, numerous workers such as Ross et al [114] have shown that method transfer between LC and CEC for neutral or ion suppressed analytes is straightforward and simple. The obvious benefits of using CEC compared to LC include increased efficiencies and hence enhanced peak capacity and the orthogonal nature of CEC compared to LC when ionic analytes are present. Retention modelling of neutral [ 115] and basic analytes [12,65] taking into account retention factors and electromobilities of the analytes have shown good agreement between predicted and experiment retention times. These results highlight the predictive nature of CEC and the possibility of performing computer optimisation routines. The possibility of using voltage to "fine tune" pressure separations is an attractive technique which will require more attention when commercially available instrumentation becomes available. 3.11

GUIDELINES FOR THE ANALYSIS OF ACIDIC BASIC AND NEUTRAL COMPOUNDS

3.11.1 Neutral analytes

Traditional LC stationary phase, such as Hypersil or Spherisorb ODS1 materials are ideally suited to the analysis of neutral analytes as the phases possess a high content of acidic silanols, hence at pH 7-9 high EOF generation is achieved which facilitates rapid analysis. In marked contrast, many pharmaceutical compounds pos-

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sess an ionizable functionality and hence different approaches must be used for their analysis as discussed in the sections below.

3.11.2 Acidic analytes Ionised acids tend to migrate towards the anode counter to the EOF therefore they are either not loaded onto the column during electrokinetic injection or are not swept towards the detector and hence are not detected. In order to perform CEC of acidic analytes they must be run in their non ionised form i.e. in acidic mobile phase. As a consequence of the low mobile phase pH there is a significantly reduced EOF hence long retention times are observed. It is highly recommended that the use of mixed mode phases such as the SAX/C18 and SCX/C6 are employed when using low pH mobile phases in order to enhance the EOF [ 116].

3.11.3 Basic analytes The analysis of basic analytes on stationary phases with silica support is still problematic in LC due to the possibility of the basic analyte undergoing mixed mode interactions with the stationary phase i.e. hydrophobic and ionic interactions. Residual isolated silanols are responsible for the deleterious ionic interactions, the result of which is excessive peak tailing. The CEC analysis of basic analytes is thought to be problematic because in order to generate a good EOF, an acidic silica is essential. It is these silanols groups which generate the EOF and are responsible for unwanted peak tailing. A number of approaches to improving the peak shape of basic analytes on silica supports for CEC with stationary phase design have been taken from LC. Many of the problems encountered in the analysis of bases by LC are also manifest in CEC. The use of low pHs or reducing the number of acidic silanols leads to extremely low EOFs which may cause excessive retention of concomitantly chromatographed neutral species in addition to the practical consideration of maintaining a "wetted" capillary. These factors have led to a perception that the analysis of bases by CEC is difficult, if not practically impossible. A number of "new" generation silica support materials have also been evaluated. The reduction in EOF is significant leading to excessive retention time for neutral and acidic species. However, pharmaceutical bases can generally be analysed using such phases, as the intrinsic electromobilities of the molecules are sufficient to ensure elution. Unfortunately, there has been little success to date. The best approach to date for the analysis of basic compounds by CEC has been to incorporate a small basic compound such as triethylamine, triethanolamine [63] or hexylamine [64] into the low pH mobile phase. The small bases act in a competitive manner to restrict the access of the basic analytes to the silanol groups on the surface References pp. 106-110

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of the silica. This approach has been shown to work for a number of basic analytes [63-65]. The same approach also allows the simultaneous analysis of acids, bases and neutral, the only drawback being the relative slow EOF. 3.12 CONCLUSIONS The enhanced sample loadability together with the high efficiencies obtainable in CEC have stimulated much interest in this technique. The entire ranges of LC and CE modes are potentially available within CEC. Many of these are only in the initial investigation stages. With the advent of stationary phases specifically designed for CEC and a growing theoretical understanding of the mechanisms involved in CEC, the continued development of the technique is assured. Nevertheless, support from stationary phase manufacturers for custom designed CEC phases and a robust column format are critical for its continued development and acceptance as a mainstream technique. 3.13 ABBREVIATIONS

CEC CGE EOF MES PEC RP SAX SCX SFC TRIS

capillary electrochromatography capillary gel electrophoresis electrosmotic flow 2-(N-morpholino)ethane-sulfonic acid pressure assisted electrochromatography reversed-phase strong anion exchange strong cation exchange supercritical fluid chromatography tris(hydroxymethyl)aminomethane

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