**2.3.5 Macrocyclic antibiotics**

problematic selectors such as CDs and their derivatives.

Proteins provide strong and highly selective interactions, so called affinity interactions, with the analytes. Stereoselectivity is further influenced by the tertiary structure of proteins. Different analytes can selectively bind to different binding sites of proteins. Proteins can be used, depending on their ionization state, for neutral, basic or acidic analytes. However, there are several limitations associated with the use of proteins in CE enantioseparations, mainly low obtainable separation efficiency, adsorption of proteins to the capillary wall and high UV absorption. To eliminate these disturbing effects, capillary coating and/or partial filling techniques and/or indirect detection have to be used [Vespalec & Boček, 2000;

Advanced Chiral Separation 35

Figure 2.28. 3D structure of avidin. A typical arrangement of avidin complex (avidin-biotin).

Various proteins, e.g., bovine serum albumin, 1-acid glycoprotein, ovomucoid, avidin (Figure 2.28), transferring, pepsin, penicillin G acylase, have been successfully employed in CE enantioseparations of amino acids and drugs. [Haginaka, 2000; Hödl et al., 2006; Gotti et al., 2006; Martínez-Gómez et al., 2007]. Although this group of selectors is frequently used in model CE enantioseparations, it is less often applied in pharmaceutical analysis, see e.g., ref. by Martinez-Gomez et al. [Martínez-Gómez et al., 2007] devoted to the **e**nantiomeric quality control of antihistamines in pharmaceuticals by affinity electrokinetic chromatography with human serum albumin as chiral selector. However, no chiral application to biological samples has been referred from 2000, probably because of achieving the goal with less

Macrocyclic antibiotics, introduced by Armstrong et al. [Armstrong et al., 1994b], possess several asymmetric centres and many functional groups and structures (like hydrophobic pocket) allowing multiple interactions (affinity, inclusion, etc.) with the analytes and

18-Crown-6-tetracarboxylic acid (18C6H14) found widespread application as a chiral selector in CE for the chiral separation of amino acids, dipeptides, sympathomimetics and various drugs containing primary amino groups [Kuhn, 1999]. Wang et al. [Wang C.Y. et al., 2003] demonstrated applicability of a new CWE, (S,S)-1,7-bis(4-benzyl-5-hydroxy-2-oxo-3 azapentyl)-1,7-diaza-12-crown-4, for the chiral CE separations. However, there are several limitations with CWEs. The separation electrolyte may not contain cations, such as potassium or ammonium ions, because they compete with the enantiomers for the CWE cavity. This increases claims on the purity and composition of electrolyte systems, and limits some applications. Therefore, according to our best knowledge, there is only one application of CWE to the chiral analysis of biological sample from 2000 [Cho et al., 2004]. 18C6H14 was applied for enantioseparation of gemifloxacin in urine (for detail see Table 3.1).

### **2.3.3 Polysaccharides/oligosacharides**

A variety of linear neutral and charged carbohydrates were found to be applicable as selectors for chiral CE separations. The low UV absorption and often high attainable separation efficiency make these selectors attractive for CE [Riekkola et al., 1997]. The enantiorecognition capability of various neutral oligo- polysacharides (maltodextrins, dextrins, dextrans) is significantly influenced by their higher molecular structures, like helical hydrophobic cavities or pores of polymeric networks [Nishi, H. et al., 1996a]. Two applications to biological samples from 2000 [Bortocan & Bonato, 2004; Nojavan & Fakhari, 2011] show enantioselective separation and determination of primaquine and its metabolite, carboxyprimaquine, in rat liver mitochondrial fraction and plasma samples, and cetirizine and hydroxyzine in human plasma, by maltodextrin as chiral selector (for detail see Table 2.1).

Figure 2.27. Chemical structure of HS-Cys. Reprinted from ref. [Park et al., 2004].

Electrostatic interactions provide additional stereoselectivity effects in the case of ionic polysacharides [Nishi H., 1997] (heparin, dextran sulphate, chondroitin sulphate, hyaluronate, chitosans, aminoglycosides, colominic acid, highly sulfated cyclosophoraoses) suitable for a variety of basic drugs such as -blockers and sympathomimetics [Nishi H., 1997; Nishi, H.S. et al., 1996b; Du et al., 2002; Park et al., 2004]. For example, recently Park et al. [Park et al., 2004] synthesized highly sulfated cyclosophoraoses (HS-Cys) (Figure 2.27) from a family of neutral cyclosophoraoses isolated from *Rhizobium leguminosarum* and applied them as chiral selectors for the resolution of -blocker and sympathomimetic enantiomers. Contrary to the sulfated compounds, the neutral cyclosophoraoses themselves showed no chiral recognition ability for these drugs investigated.

### **2.3.4 Proteins**

34 Chiral Capillary Electrophoresis in Current Pharmaceutical and Biomedical Analysis

18-Crown-6-tetracarboxylic acid (18C6H14) found widespread application as a chiral selector in CE for the chiral separation of amino acids, dipeptides, sympathomimetics and various drugs containing primary amino groups [Kuhn, 1999]. Wang et al. [Wang C.Y. et al., 2003] demonstrated applicability of a new CWE, (S,S)-1,7-bis(4-benzyl-5-hydroxy-2-oxo-3 azapentyl)-1,7-diaza-12-crown-4, for the chiral CE separations. However, there are several limitations with CWEs. The separation electrolyte may not contain cations, such as potassium or ammonium ions, because they compete with the enantiomers for the CWE cavity. This increases claims on the purity and composition of electrolyte systems, and limits some applications. Therefore, according to our best knowledge, there is only one application of CWE to the chiral analysis of biological sample from 2000 [Cho et al., 2004]. 18C6H14 was

A variety of linear neutral and charged carbohydrates were found to be applicable as selectors for chiral CE separations. The low UV absorption and often high attainable separation efficiency make these selectors attractive for CE [Riekkola et al., 1997]. The enantiorecognition capability of various neutral oligo- polysacharides (maltodextrins, dextrins, dextrans) is significantly influenced by their higher molecular structures, like helical hydrophobic cavities or pores of polymeric networks [Nishi, H. et al., 1996a]. Two applications to biological samples from 2000 [Bortocan & Bonato, 2004; Nojavan & Fakhari, 2011] show enantioselective separation and determination of primaquine and its metabolite, carboxyprimaquine, in rat liver mitochondrial fraction and plasma samples, and cetirizine and hydroxyzine in human

applied for enantioseparation of gemifloxacin in urine (for detail see Table 3.1).

plasma, by maltodextrin as chiral selector (for detail see Table 2.1).

showed no chiral recognition ability for these drugs investigated.

Figure 2.27. Chemical structure of HS-Cys. Reprinted from ref. [Park et al., 2004].

Electrostatic interactions provide additional stereoselectivity effects in the case of ionic polysacharides [Nishi H., 1997] (heparin, dextran sulphate, chondroitin sulphate, hyaluronate, chitosans, aminoglycosides, colominic acid, highly sulfated cyclosophoraoses) suitable for a variety of basic drugs such as -blockers and sympathomimetics [Nishi H., 1997; Nishi, H.S. et al., 1996b; Du et al., 2002; Park et al., 2004]. For example, recently Park et al. [Park et al., 2004] synthesized highly sulfated cyclosophoraoses (HS-Cys) (Figure 2.27) from a family of neutral cyclosophoraoses isolated from *Rhizobium leguminosarum* and applied them as chiral selectors for the resolution of -blocker and sympathomimetic enantiomers. Contrary to the sulfated compounds, the neutral cyclosophoraoses themselves

**2.3.3 Polysaccharides/oligosacharides**

Proteins provide strong and highly selective interactions, so called affinity interactions, with the analytes. Stereoselectivity is further influenced by the tertiary structure of proteins. Different analytes can selectively bind to different binding sites of proteins. Proteins can be used, depending on their ionization state, for neutral, basic or acidic analytes. However, there are several limitations associated with the use of proteins in CE enantioseparations, mainly low obtainable separation efficiency, adsorption of proteins to the capillary wall and high UV absorption. To eliminate these disturbing effects, capillary coating and/or partial filling techniques and/or indirect detection have to be used [Vespalec & Boček, 2000; Tanaka & Terabe, 1995; Armstrong et al., 1994a].

Figure 2.28. 3D structure of avidin. A typical arrangement of avidin complex (avidin-biotin). Reprinted from [ks.uiuc.edu].

Various proteins, e.g., bovine serum albumin, 1-acid glycoprotein, ovomucoid, avidin (Figure 2.28), transferring, pepsin, penicillin G acylase, have been successfully employed in CE enantioseparations of amino acids and drugs. [Haginaka, 2000; Hödl et al., 2006; Gotti et al., 2006; Martínez-Gómez et al., 2007]. Although this group of selectors is frequently used in model CE enantioseparations, it is less often applied in pharmaceutical analysis, see e.g., ref. by Martinez-Gomez et al. [Martínez-Gómez et al., 2007] devoted to the **e**nantiomeric quality control of antihistamines in pharmaceuticals by affinity electrokinetic chromatography with human serum albumin as chiral selector. However, no chiral application to biological samples has been referred from 2000, probably because of achieving the goal with less problematic selectors such as CDs and their derivatives.

### **2.3.5 Macrocyclic antibiotics**

Macrocyclic antibiotics, introduced by Armstrong et al. [Armstrong et al., 1994b], possess several asymmetric centres and many functional groups and structures (like hydrophobic pocket) allowing multiple interactions (affinity, inclusion, etc.) with the analytes and

tartrate-boric acid complex chiral selector in situ synthesis and its application in chiral non-

Advanced Chiral Separation 37

Figure 2.30. Chemical structures of SPD–borate complex and analytes. Reprinted from ref.

The limited stability of these selectors, their UV absorption (detection difficulties) and rather slow ligand-exchange kinetics (poor enantioresolution) [Blanco & Valverde, 2003] could be the main reasons for a rare utilization of these selectors in chiral CE. Recently, tartaric acid–Cu(II) complex has been used as a selector for the chiral separation of drugs with amino alcohol structure [Hödl et al., 2007], however, it has not been employed in real bioanalyses so far.

Ion-pairing reagents {e.g., (+)-*S*-Camphor-10-sulfonic acid; 2*R*,3*S*,4*R*,5*S* (−) 2,3,4,6-di-*O*isopropylidene-2-keto-l-gulonic acid; 1*S*,4*R*(+) ketopinic acid, see examples in Figure 2.31}, are applicable in CE usually in non-aqueous medium as the essential interactions (hydrogen bondings and dipole–dipole interactions) are less effective in aqueous medium [Bjornsdottir et al., 1996; Carlsson et al., 2001; Hedeland et al., 2007]. Exceptions are the use of chiral counterions as supporting agents for separations with coselectors (e.g., CDs) in aqueous

Figure 2.31. Chemical structures of ion-pairing reagents. (S)-(+)-ketopinic acid (left), (+)-(S)-

camphor-10-sulfonic acid (right). Reprinted from [sigmaaldrich.com].

from [Kodama et al., 2005].

**2.3.7 Ion-pairing reagents**

organic mixtures [Kodama et al., 2003].

aqueous capillary electrophoresis is presented by Wang et al. [Wang L.J. et al., 2011].

providing extremely high enantiorecognition capability. Since these compounds have strong UV absorption, partial filling methods [Desiderio et al., 1997] and countercurrent approaches [Oswald & Ward, 1999] have been applied to overcome detection problems. Moreover, capillaries have to be coated for the elimination of adsorption of antibiotics at the capillary wall [Wang Z. et al., 2007].

Figure 2.29. Chemical structure of vancomycin.

Several classes of antibiotics have been introduced as chiral selectors showing different enantiorecognition capabilities towards amino acids, drugs, etc.: ansamycins (rifamycin B, rifamycin SV), glycopeptides (vancomycin, ristocetin, teicoplanin, avoparcin), aminoglycosides (streptomycin sulfate, kanamycin sulfate, fradiomycin sulfate), macrolides (erythromycin), polypeptides, [see e.g., Wang Z. et al., 2007; Hou J.G. et al., 2003; Ha P.T.T. et al., 2004b]. For the chemical structure of vancomycin see Figure 2.29.

Lack of recent CE applications to biological samples can be explained by the same arguments used for proteins. On the other hand, applications in this field were accomplished by antibiotics immobilized in CEC stationary phases (see section 2.2.2 and Table 2.1).

### **2.3.6 Ligand-exchange selectors**

Ligand-exchange selectors {usually chelate complexes (hemicomplexes) of amino acids with metal cations, e.g., histidine-copper(II)} can form ternary complexes with appropriate analytes (compounds containing aminocarboxyl or hydroxycarboxyl groups like amino acids and hydroxyl acids) having different stability for the two enantiomers [Gassmann et al., 1985]. Rizkov et al. [Rizkov et al., 2010] demonstrated beta-Amino alcohol selectors for enantioselective separation of amino acids by ligand-exchange capillary zone electrophoresis in a low molecular weight organogel. An approach related to the ligandexchange of metal complexes is the formation of mixed borate–diol ternary complexes [Kodama et al., 2006]. For example, recently the use of (5S)-pinandiol (SPD) as the chiral selector in the presence of borate as a central ion and SDS has been described (**Figure 2.30**) [Kodama et al., 2005]. Three diols (1-phenyl-1,2-ethanediol, 3-phenoxy-1,2-propanediol and 3-benzyloxy-1,2-propanediol) were successfully resolved by this technique. Di-n-amyl L-

providing extremely high enantiorecognition capability. Since these compounds have strong UV absorption, partial filling methods [Desiderio et al., 1997] and countercurrent approaches [Oswald & Ward, 1999] have been applied to overcome detection problems.  On the other hand, applications in this field were accomplished by

Ligand-exchange selectors {usually chelate complexes (hemicomplexes) of amino acids with metal cations, e.g., histidine-copper(II)} can form ternary complexes with appropriate analytes (compounds containing aminocarboxyl or hydroxycarboxyl groups like amino acids and hydroxyl acids) having different stability for the two enantiomers [Gassmann et al., 1985]. Rizkov et al. [Rizkov et al., 2010] demonstrated beta-Amino alcohol selectors for enantioselective separation of amino acids by ligand-exchange capillary zone electrophoresis in a low molecular weight organogel. An approach related to the ligandexchange of metal complexes is the formation of mixed borate–diol ternary complexes [Kodama et al., 2006]. For example, recently the use of (5S)-pinandiol (SPD) as the chiral selector in the presence of borate as a central ion and SDS has been described (**Figure 2.30**) [Kodama et al., 2005]. Three diols (1-phenyl-1,2-ethanediol, 3-phenoxy-1,2-propanediol and 3-benzyloxy-1,2-propanediol) were successfully resolved by this technique. Di-n-amyl L-

et al., 2004b]. For the chemical structure of vancomycin see Figure 2.29.

antibiotics immobilized in CEC stationary phases (see section 2.2.2 and Table 2.1).

capillary wall [Wang Z. et al., 2007].

Figure 2.29. Chemical structure of vancomycin.

HO

HO

NH

CH3

NH

HO

**2.3.6 Ligand-exchange selectors**

tartrate-boric acid complex chiral selector in situ synthesis and its application in chiral nonaqueous capillary electrophoresis is presented by Wang et al. [Wang L.J. et al., 2011].

Figure 2.30. Chemical structures of SPD–borate complex and analytes. Reprinted from ref. from [Kodama et al., 2005].

The limited stability of these selectors, their UV absorption (detection difficulties) and rather slow ligand-exchange kinetics (poor enantioresolution) [Blanco & Valverde, 2003] could be the main reasons for a rare utilization of these selectors in chiral CE. Recently, tartaric acid–Cu(II) complex has been used as a selector for the chiral separation of drugs with amino alcohol structure [Hödl et al., 2007], however, it has not been employed in real bioanalyses so far. &

### **2.3.7 Ion-pairing reagents**

Ion-pairing reagents {e.g., (+)-*S*-Camphor-10-sulfonic acid; 2*R*,3*S*,4*R*,5*S* (−) 2,3,4,6-di-*O*isopropylidene-2-keto-l-gulonic acid; 1*S*,4*R*(+) ketopinic acid, see examples in Figure 2.31}, are applicable in CE usually in non-aqueous medium as the essential interactions (hydrogen bondings and dipole–dipole interactions) are less effective in aqueous medium [Bjornsdottir et al., 1996; Carlsson et al., 2001; Hedeland et al., 2007]. Exceptions are the use of chiral counterions as supporting agents for separations with coselectors (e.g., CDs) in aqueous organic mixtures [Kodama et al., 2003].

Figure 2.31. Chemical structures of ion-pairing reagents. (S)-(+)-ketopinic acid (left), (+)-(S) camphor-10-sulfonic acid (right). Reprinted from [sigmaaldrich.com].

concentration range applicable (no critical micelle concentration) and therefore, higher obtainable signal to noise ratio (S/N). Further, the covalent bonds between surfactant monomers are stable (less background noise from ionized surfactant monomers of low molecular weights in mass spectrometer) and there are no disruptions in micelle formation (wider range of buffer additives applicable, e.g., organic modifiers). Micelle polymers have lower surface activity, association kinetics is fast due to a shallower and faster penetration of the solute into the compact polymeric structure (enhanced efficiency) [Riekkola et al., 1997; Palmer & McCarney, 2004b; Palmer, 2007; Zhao, S. et al., 2007; Akbay et al., 2005; Hou, J. G. et al., 2006; Rizvi et al., 2007]. Many of these properties are beneficial for enhancing chiral, as well as achiral, recognition {a broad range of structurally diverse racemic compounds such as phenylethylamines, -blockers, 2-(2-chlorophenoxy) propionic acid, benzoin derivatives, derivatized amino acids and benzodiazepines} and for improving detection (very pronounced in MS) when compared with traditional micelle systems [Rizvi et al., 2007]. Therefore, the utilization of these selectors tends to rise up as it is apparent from the following recent applications in biological samples (human urine, plasma), such as poly(sodium *N*-undecenoxy carbonyl-L-leucine) sulfate vs. pseudoephedrine [Rizvi et al., 2007], polysodium-*N*-undecenoyl-L,L-leucyl-valinate vs. warfarin [Hou, J. et al., 2007], for

Advanced Chiral Separation 39

Figure 2.33. Structure of monomer and micelle polymer of alkenoxy surfactants. Reprinted

details see Table 2.1.

from ref. [Rizvi et al., 2004].

The ion-pairing mechanism under non-aqueous conditions took part in the enantioseparation of many drugs such as -blockers [Bjornsdottir et al., 1996; Carlsson et al., 2001; Hedeland et al., 2007]. It cooperated also in bioanalytical applications separating enantiomers of salbutamol with heptakis-(2,3-di-O-acetyl-6-O-sulfo)--CD in urine samples under non-aqueous EKC conditions [Servais et al., 2004, 2006] (for detail see Table 2.1).

### **2.3.8 Amphiphilic molecules, micelles, micelle polymers and microemulsions**

Amphiphilic molecules, composed of a polar head group and a hydrophobic tail, can form micelles above their critical micelle concentration (Figure 2.32). Micelles are aggregates (usually spherical) with a surface created by heads and core by tails. A typical recognition of analytes is based on the formation of associates with the micelles (less polar parts of analytes tend to enter the micelle core) having different stability for the two analytes of different hydrophobicity, or in other words, differences in their partition coefficients between the micelle phase and the electrolyte bulk phase. Chiral recognition is given by modifying partition coefficients of chiral analytes due to different steric effects between chiral analytes and chiral core or chiral surface of micelles. Micelle pseudostationary phases were introduced into CE by Terabe et al. [Terabe et al., 1984]. Different classes of charged as well as neutral amphiphilic molecules, namely bile salts, saponines, long-chain *N*-alkyl-lamino acids, *N*-alkanoyl-L-amino acids, *N*-dodecoxycarbonyl amino acids, alkylglycosides, alkylglucosides, were applied as chiral selectors in aqueous solutions for a wide range of substances of a hydrophilic or hydrophobic nature, see the specialized review article [Palmer & McCarney, 2004a]. For example, sodium cholate was used for the enantiomeric separation of mono and diacid benzoporfyrine derivates in serum and microsome samples [Peng et al., 2002] (for details see Table 2.1).

Micelle polymers {e.g., poly- sodium *N*-undecenoxy carbonyl-L-leucinate/isoleucinate (Figure 2.33), 3-[(3-dehydroabietamidopropyl)dimethyl-ammonio]-1-propane-sulfonate, undecyl-L-valine} can offer additional benefits in comparison to conventional micelles, such as elimination of intrinsic micelle equilibrium (enhanced chiral recognition), wide {e.g.,

The ion-pairing mechanism under non-aqueous conditions took part in the enantioseparation of many drugs such as -blockers [Bjornsdottir et al., 1996; Carlsson et al., 2001; Hedeland et al., 2007]. It cooperated also in bioanalytical applications separating enantiomers of salbutamol with heptakis-(2,3-di-O-acetyl-6-O-sulfo)--CD in urine samples under non-aqueous EKC conditions [Servais et al., 2004, 2006] (for detail see Table 2.1).

Amphiphilic molecules, composed of a polar head group and a hydrophobic tail, can form micelles above their critical micelle concentration (Figure 2.32). Micelles are aggregates (usually spherical) with a surface created by heads and core by tails. A typical recognition of analytes is based on the formation of associates with the micelles (less polar parts of analytes tend to enter the micelle core) having different stability for the two analytes of different hydrophobicity, or in other words, differences in their partition coefficients between the micelle phase and the electrolyte bulk phase. Chiral recognition is given by modifying partition coefficients of chiral analytes due to different steric effects between chiral analytes and chiral core or chiral surface of micelles. Micelle pseudostationary phases were introduced into CE by Terabe et al. [Terabe et al., 1984]. Different classes of charged as well as neutral amphiphilic molecules, namely bile salts, saponines, long-chain *N*-alkyl-lamino acids, *N*-alkanoyl-L-amino acids, *N*-dodecoxycarbonyl amino acids, alkylglycosides, alkylglucosides, were applied as chiral selectors in aqueous solutions for a wide range of substances of a hydrophilic or hydrophobic nature, see the specialized review article [Palmer & McCarney, 2004a]. For example, sodium cholate was used for the enantiomeric separation of mono and diacid benzoporfyrine derivates in serum and microsome samples

Figure 2.32. Mechanism of formation of micelle associate with neutral molecule. Reprinted

Micelle polymers {e.g., poly- sodium *N*-undecenoxy carbonyl-L-leucinate/isoleucinate (Figure 2.33), 3-[(3-dehydroabietamidopropyl)dimethyl-ammonio]-1-propane-sulfonate, undecyl-L-valine} can offer additional benefits in comparison to conventional micelles, such as elimination of intrinsic micelle equilibrium (enhanced chiral recognition), wide

**2.3.8 Amphiphilic molecules, micelles, micelle polymers and microemulsions**

[Peng et al., 2002] (for details see Table 2.1).

from ref. [Terabe et al., 1984].

concentration range applicable (no critical micelle concentration) and therefore, higher obtainable signal to noise ratio (S/N). Further, the covalent bonds between surfactant monomers are stable (less background noise from ionized surfactant monomers of low molecular weights in mass spectrometer) and there are no disruptions in micelle formation (wider range of buffer additives applicable, e.g., organic modifiers). Micelle polymers have lower surface activity, association kinetics is fast due to a shallower and faster penetration of the solute into the compact polymeric structure (enhanced efficiency) [Riekkola et al., 1997; Palmer & McCarney, 2004b; Palmer, 2007; Zhao, S. et al., 2007; Akbay et al., 2005; Hou, J. G. et al., 2006; Rizvi et al., 2007]. Many of these properties are beneficial for enhancing chiral, as well as achiral, recognition {a broad range of structurally diverse racemic compounds such as phenylethylamines, -blockers, 2-(2-chlorophenoxy) propionic acid, benzoin derivatives, derivatized amino acids and benzodiazepines} and for improving detection (very pronounced in MS) when compared with traditional micelle systems [Rizvi et al., 2007]. Therefore, the utilization of these selectors tends to rise up as it is apparent from the following recent applications in biological samples (human urine, plasma), such as poly(sodium *N*-undecenoxy carbonyl-L-leucine) sulfate vs. pseudoephedrine [Rizvi et al., 2007], polysodium-*N*-undecenoyl-L,L-leucyl-valinate vs. warfarin [Hou, J. et al., 2007], for details see Table 2.1.

Figure 2.33. Structure of monomer and micelle polymer of alkenoxy surfactants. Reprinted from ref. [Rizvi et al., 2004].

Microemulsions, spherical aggregates of nanometre size consisting of an oil core covered/stabilized by amphiphilic molecules in water solution (Figure 2.36), are optically transparent and thermodynamically stable phases that offer a new alternative to micelles for chiral separations aimed at hydrophobic molecules. Chiral microemulsions can utilize one chiral entity (either surfactant, cosurfactant or oil are chiral) or more than one chiral entity (e.g., both surfactant and cosurfactant are chiral) [Ryan et al., 2009; Kahle & Foley, 2006]. Only a few applications to chiral separations have been described (none yet in biological samples), including chiral oils, chiral amphiphilic molecules, as well as CDs as chiral additives [Marsh et al., 2004; McEvoy et al., 2007]. Kahle and Foley [Kahle & Foley, 2007a; 2007b, 2007c] investigated one-, two- and three-chiral-component microemulsions, composed of the chiral surfactant dodecoxycarbonylvaline, the cosurfactant 2-hexanol and one of the chiral oils dibutyl or diethyl tartrate for MEEKC. Six basic drugs, viz. four ephedrine derivatives and two -blockers, were used as test solutes. For most analytes, enantioselectivity, as well as resolution, were higher with the best dual-chirality system compared with the best one-chiral-component microemulsion due to synergistic effects.

Advanced Chiral Separation 41

Figure 2.36. Schematic representation of an oil-in-water microemulsion droplet. Adapted

Several alternative chiral selectors, compared to those described in sections 2.3.1-2.3.8, have been employed in chiral CE, including calixarenes (macrocyclic compounds consisting of benzene rings linked by methylene groups and chiral substituent, see Figure 2.37), cyclosphoraoses (unbranched cyclic -D-glucans), hemispherodextrins (capped CDs), tergurides (ergot alkaloids), amphiphilic aminosaccharides, cyclopeptides, guanosine gels, aptamers (single-stranded RNA or DNA oligonucleotides 15 to 60 base in length that bind with high affinity to specific molecular targets), e.g., anti-arginine 1-RNA aptamer, dendrimers (repetitively branched cascade molecules, see Figure 2.38), etc. [Van Eeckhaut & Michotte, 2006; Gübitz & Schmid, 2004, 2008; Riekkola et al., 1997; Mokhtari et al., 2011, Chen Y.M. et al. 1998]. These selectors can solve particular/specific problems in chiral

from ref. [Ryan et al., 2009].

**2.3.9 Alternative chiral selectors**

Figure 2.34. Molecular structures of the vesicle-forming amphiphilic molecules. Sodium *N*- [4-*n*-dodecyloxybenzoyl]-L-leucinate (SDLL) and sodium *N*-[4-*n-*dodecyloxybenzoyl]-Lisoleucinate (SDLIL). Reprinted from ref. [Mohanty & Dey, 2006].

The use of vesicle-forming amphiphilic molecules (e.g., sodium N-[4-n-dodecyloxybenzoyl]- L-leucinate) [Mohanty & Dey, 2006] and ionic liquids (i.e., salts in the liquid state, organic salts with low melting points soluble in both polar and nonpolar solvents, e.g., undecenoxycarbonyl-L-pyrrolidinol bromide) [Rizvi & Shamsi, 2006] can be counted among the recent trends in chiral CE with new potentialities for biologically active chiral compounds. For example, two vesicle-forming single-tailed amino acid derivatized surfactants sodium *N*-[4-*n*-dodecyloxybenzoyl]-L-leucinate (SDLL) and sodium *N*-[4-*n*dodecyloxybenzoyl]-L-isoleucinate (SDLIL) (Figure 2.34) have been synthesized and used as a pseudo-stationary phase in MEKC for various model chiral analytes, namely atropisomers (±)-1,1'-bi-2-naphthol, (±)-1,1'-binaphthyl-2,2'-diamine, (±)-1,1'-binaphthyl-2,2' diylhydrogen phosphate and Tröger's base and chiral compound benzoin [Mohanty & Dey, 2006]. Results of these studies have suggested formation of vesicles in aqueous solutions. Microenvironment of the vesicle determined the depth of penetration of the analytes into vesicle, and, in this way, it influenced the separation selectivity. The application of novel ionic liquid-like surfactants and their polymers for chiral separation of acidic analytes in MEKC was reported first by Rizvi and Shamsi in 2006 [Rizvi & Shamsi, 2006]. The two model acidic analytes, (*rac*)-α-bromophenylacetic acid and (*rac*)-2-(2 chlorophenoxy)propanoic acid, were successfully separated with the amino alcohol-derived chiral ionic liquids (given in Figure 2.35) and their polymers at 25 mM surfactant concentration.

Figure 2.35. Amino alcohol-derived chiral ionic liquids for capillary electrophoresis. Reprinted from ref. [Rizvi & Shamsi, 2006].

Figure 2.34. Molecular structures of the vesicle-forming amphiphilic molecules. Sodium *N*- [4-*n*-dodecyloxybenzoyl]-L-leucinate (SDLL) and sodium *N*-[4-*n-*dodecyloxybenzoyl]-L-

The use of vesicle-forming amphiphilic molecules (e.g., sodium N-[4-n-dodecyloxybenzoyl]- L-leucinate) [Mohanty & Dey, 2006] and ionic liquids (i.e., salts in the liquid state, organic salts with low melting points soluble in both polar and nonpolar solvents, e.g., undecenoxycarbonyl-L-pyrrolidinol bromide) [Rizvi & Shamsi, 2006] can be counted among the recent trends in chiral CE with new potentialities for biologically active chiral compounds. For example, two vesicle-forming single-tailed amino acid derivatized surfactants sodium *N*-[4-*n*-dodecyloxybenzoyl]-L-leucinate (SDLL) and sodium *N*-[4-*n*dodecyloxybenzoyl]-L-isoleucinate (SDLIL) (Figure 2.34) have been synthesized and used as a pseudo-stationary phase in MEKC for various model chiral analytes, namely atropisomers (±)-1,1'-bi-2-naphthol, (±)-1,1'-binaphthyl-2,2'-diamine, (±)-1,1'-binaphthyl-2,2' diylhydrogen phosphate and Tröger's base and chiral compound benzoin [Mohanty & Dey, 2006]. Results of these studies have suggested formation of vesicles in aqueous solutions. Microenvironment of the vesicle determined the depth of penetration of the analytes into vesicle, and, in this way, it influenced the separation selectivity. The application of novel ionic liquid-like surfactants and their polymers for chiral separation of acidic analytes in MEKC was reported first by Rizvi and Shamsi in 2006 [Rizvi & Shamsi, 2006]. The two model acidic analytes, (*rac*)-α-bromophenylacetic acid and (*rac*)-2-(2 chlorophenoxy)propanoic acid, were successfully separated with the amino alcohol-derived chiral ionic liquids (given in Figure 2.35) and their polymers at 25 mM surfactant

potentialities

Figure 2.35. Amino alcohol-derived chiral ionic liquids for capillary electrophoresis.

isoleucinate (SDLIL). Reprinted from ref. [Mohanty & Dey, 2006].

concentration.

Reprinted from ref. [Rizvi & Shamsi, 2006].

Microemulsions, spherical aggregates of nanometre size consisting of an oil core covered/stabilized by amphiphilic molecules in water solution (Figure 2.36), are optically transparent and thermodynamically stable phases that offer a new alternative to micelles for chiral separations aimed at hydrophobic molecules. Chiral microemulsions can utilize one chiral entity (either surfactant, cosurfactant or oil are chiral) or more than one chiral entity (e.g., both surfactant and cosurfactant are chiral) [Ryan et al., 2009; Kahle & Foley, 2006]. Only a few applications to chiral separations have been described (none yet in biological samples), including chiral oils, chiral amphiphilic molecules, as well as CDs as chiral additives [Marsh et al., 2004; McEvoy et al., 2007]. Kahle and Foley [Kahle & Foley, 2007a; 2007b, 2007c] investigated one-, two- and three-chiral-component microemulsions, composed of the chiral surfactant dodecoxycarbonylvaline, the cosurfactant 2-hexanol and one of the chiral oils dibutyl or diethyl tartrate for MEEKC. Six basic drugs, viz. four ephedrine derivatives and two -blockers, were used as test solutes. For most analytes,

enantioselectivity, as well as resolution, were higher with the best dual-chirality system compared with the best one-chiral-component microemulsion due to synergistic effects.

Figure 2.36. Schematic representation of an oil-in-water microemulsion droplet. Adapted from ref. [Ryan et al., 2009].

### **2.3.9 Alternative chiral selectors**

Several alternative chiral selectors, compared to those described in sections 2.3.1-2.3.8, have been employed in chiral CE, including calixarenes (macrocyclic compounds consisting of benzene rings linked by methylene groups and chiral substituent, see Figure 2.37), cyclosphoraoses (unbranched cyclic -D-glucans), hemispherodextrins (capped CDs), tergurides (ergot alkaloids), amphiphilic aminosaccharides, cyclopeptides, guanosine gels, aptamers (single-stranded RNA or DNA oligonucleotides 15 to 60 base in length that bind with high affinity to specific molecular targets), e.g., anti-arginine 1-RNA aptamer, dendrimers (repetitively branched cascade molecules, see Figure 2.38), etc. [Van Eeckhaut & Michotte, 2006; Gübitz & Schmid, 2004, 2008; Riekkola et al., 1997; Mokhtari et al., 2011, Chen Y.M. et al. 1998]. These selectors can solve particular/specific problems in chiral

*New chiral selectors.* A sensitive MEKC-MS method using poly(sodium *N*-undecenoxy carbonyl-L-leucine) sulfate was developed for enantioselective analysis of pseudoephedrine in human urine [Rizvi et al., 2007]. The permanent charge of this micelle chiral selector allows for setting separation conditions (electrolyte pH) suitable for obtaining the highest detection sensitivity maintaining sufficient enantioresolution. Other new chiral selectors and new chiral stationary phases can be also found in some applications given for the advanced

Advanced Chiral Separation 43

*Countercurrent migration.* Rudaz et al. [Rudaz et al., 2005] used the countercurrent migration of the negatively charged highly sulfated--CD to achieve a highly effective enantioseparation of amphetamine derivatives in plasma. This strategy allowed using a low concentration (0.15%) of the chiral selector for the complete enantioseparation of seven

Anionic CD, the heptakis(2,6-diacethyl-6-sulfato)--CD, migrating in the opposite direction to the analytes, was highly effective in low concentrations (0.85 mM) for the simultaneous chiral CD-EKC-MS separation of methamphetamine, 3,4-methylenedioxyamphetamine and

Figure 2.39. Electropherograms of extracts of blank plasma and plasma of spiked concentration of 4 M racemic drug. Conditions: detection buffer, 5 mM Ru(bpy)3 2+ in 100 mM (pH 6.5) phosphate buffer; separation buffer, 3 mg/mL of S--CD in 40 mM (pH 4.5) acetate buffer;

The CD-EKC method based on a combination of discontinuous buffer (separation buffer differed from detection solution, essential for chemiluminiscence detection) with low concentration (3 mg/mL) of anionic highly sulfated--CD was used for the baseline

applied voltage, 22 kV; injection time, 5 s. Reprinted from ref. [Fang, L. et al., 2006].

electromigration modes and mechanisms below.

amphetamine derivatives in analysis times of less than 6 min.

amphetamine in clinical human urine samples [Iio et al., 2005].

separations, however, their use is less universal in most cases as they can be expensive and their availability is limited. This could also be the reason why no application in chiral bioanalysis has been demonstrated so far.

Figure 2.37. Chiral calixarene. Reprinted from [rsc.org].

Figure 2.38. The chemical structures of the chiral dendrimers with axial chirality. Reprinted from ref. [Chen, Y.M. et al., 1998].

### **2.4 Applications of chiral separation systems in bioanalysis**

The following examples illustrate successful EKC enantioseparations of drugs in biological samples utilizing some of the progressive chiral principles mentioned in section 2.2. and/or new chiral selectors mentioned in section 2.3. CEC and MCE applications in this field are also included.

separations, however, their use is less universal in most cases as they can be expensive and their availability is limited. This could also be the reason why no application in chiral

Figure 2.38. The chemical structures of the chiral dendrimers with axial chirality. Reprinted

The following examples illustrate successful EKC enantioseparations of drugs in biological samples utilizing some of the progressive chiral principles mentioned in section 2.2. and/or new chiral selectors mentioned in section 2.3. CEC and MCE applications in this field are

**2.4 Applications of chiral separation systems in bioanalysis**

bioanalysis has been demonstrated so far.

Figure 2.37. Chiral calixarene. Reprinted from [rsc.org].

from ref. [Chen, Y.M. et al., 1998].

also included.

*New chiral selectors.* A sensitive MEKC-MS method using poly(sodium *N*-undecenoxy carbonyl-L-leucine) sulfate was developed for enantioselective analysis of pseudoephedrine in human urine [Rizvi et al., 2007]. The permanent charge of this micelle chiral selector allows for setting separation conditions (electrolyte pH) suitable for obtaining the highest detection sensitivity maintaining sufficient enantioresolution. Other new chiral selectors and new chiral stationary phases can be also found in some applications given for the advanced electromigration modes and mechanisms below.

*Countercurrent migration.* Rudaz et al. [Rudaz et al., 2005] used the countercurrent migration of the negatively charged highly sulfated--CD to achieve a highly effective enantioseparation of amphetamine derivatives in plasma. This strategy allowed using a low concentration (0.15%) of the chiral selector for the complete enantioseparation of seven amphetamine derivatives in analysis times of less than 6 min.

Anionic CD, the heptakis(2,6-diacethyl-6-sulfato)--CD, migrating in the opposite direction to the analytes, was highly effective in low concentrations (0.85 mM) for the simultaneous chiral CD-EKC-MS separation of methamphetamine, 3,4-methylenedioxyamphetamine and amphetamine in clinical human urine samples [Iio et al., 2005].

Figure 2.39. Electropherograms of extracts of blank plasma and plasma of spiked concentration of 4 M racemic drug. Conditions: detection buffer, 5 mM Ru(bpy)3 2+ in 100 mM (pH 6.5) phosphate buffer; separation buffer, 3 mg/mL of S--CD in 40 mM (pH 4.5) acetate buffer; applied voltage, 22 kV; injection time, 5 s. Reprinted from ref. [Fang, L. et al., 2006].

The CD-EKC method based on a combination of discontinuous buffer (separation buffer differed from detection solution, essential for chemiluminiscence detection) with low concentration (3 mg/mL) of anionic highly sulfated--CD was used for the baseline

the chiral selector enabling optimum separating conditions for the trace chiral analytes in

Advanced Chiral Separation 45

*Charged chiral pseudophases/carrier systems.* The enantioseparations of cyanobenz[*f*]isoindole serine (CBI) derivatives of glutamate and aspartate in squirrel brain samples were accomplished with a highly sulfated--CD as chiral selector at low pH and reverse polarity

*Mixed selector systems.* Aspartate enantiomers (NDA derivatives) were separated in the samples prepared from tissue of the central nervous system of *A. californica* using mixed

Figure 2.41. Separation of CBI-Ser enantiomers using different running buffers: (a) 100mM borate (pH 9.5), 30mM CD and 60mM SDC; (b) 100mM borate (pH 9.5) and 30mM -CD; (c) 100mM borate (pH 10.0), 60mM SDC. Capillary was 50 m i.d.×50 cm effective length. Voltage applied was 15 kV. Ser enantiomer concentration was 2.0 M. Reprinted from ref.

The CD-MEKC separation of CBI-D/L-serine enantiomers was achieved by using a dual chiral selector system consisting of -CD and chiral micelles formed by deoxycholic acid (SCD) [Zhao, S.L. et al., 2005b]. The essential role of the mixed chiral selector system for the

concentrated biological matrices [Mikuš et al., 2006a], see Figure 2.40.

micelles composed from -CD and SDS [Miao et al., 2005].

[Kirschner et al., 2007].

[Zhao, S.L. et al., 2005b].

enantioseparation of an oppositely charged racemic antiarrhythmic drug, disopyramide, in spiked plasma samples, see Figure 2.39 [Fang, L. et al., 2006].

Naphthalene-2,3-dicarboxaldehyde (NDA) derivatized baclofen was separated in human plasma using the CD-EKC-LIF method based on a highly sulfated--CD [Kavran-Belin et al., 2005]. The anionic CD migrated in the opposite direction toward negatively charged baclofen (pH 9.5) transported by EOF toward the cathode.

The NACE-MS with countercurrent migration of heptakis(2,3-di-*O*-acetyl-6-*O-*sulfo)--CD present in acidified methanol medium was used for the separation and determination of low levels of the enantiomers of a basic chiral drug (salbutamol) in biological samples (human urine) [Servais et al., 2006].

Figure 2.40. Electropherograms showing the effect of the charge of ionizable chiral selector, CE--CD, on the EKC separation of PHM enantiomers in a partially pretreated urine matrix. The separations were carried out with the uncharged CE--CD (a, b) and with the negatively charged CE--CD (c). Separating conditions: (a, b) EKC electrolyte system consisting of a 25mM glycine-acetic acid buffer, pH 3.2, 0.2 methylhydroxyethylcellulose as an EOF suppressor, with a 0 (a) and 15 (b) mg/mL concentration of CE--CD, (c) EKC electrolyte system consisting of a 25mM aminocaproic acid-acetic acid buffer, pH 4.5, 0.2 methylhydroxyethylcellulose as an EOF suppressor, with a 2.5 mg/mL concentration of CE- -CD. Sample and analyte: (a, b) PHM was present in the sample (20-times diluted urine) at a 150 ng/mL concentration. (c) PHM was present in the sample (4-times diluted urine) at a 30 ng/mL concentration. Reprinted from ref. [Mikuš et al., 2006a].

Negatively charged carboxyethyl--CD considerably improved enantioresolution of several oppositely charged H1-antihistamines (pheniramine and its metabolite, dimethindene, dioxopromethazine) in comparison with native CDs [Mikuš et al., 2006a]. Countercurrent migration and enhanced complexation were responsible not only for improved chiral separation of the drugs and the metabolite, but also achiral separation allowing resolution of the drug enantiomers from their metabolic products, as well as from the sample matrix constituents when metabolic study of pheniramine in urine samples was carried out [Marák et al., 2007]. A high effectivity of the separation was demonstrated by a low concentration of

enantioseparation of an oppositely charged racemic antiarrhythmic drug, disopyramide, in

Naphthalene-2,3-dicarboxaldehyde (NDA) derivatized baclofen was separated in human plasma using the CD-EKC-LIF method based on a highly sulfated--CD [Kavran-Belin et al., 2005]. The anionic CD migrated in the opposite direction toward negatively charged

The NACE-MS with countercurrent migration of heptakis(2,3-di-*O*-acetyl-6-*O-*sulfo)--CD present in acidified methanol medium was used for the separation and determination of low levels of the enantiomers of a basic chiral drug (salbutamol) in biological samples (human

Figure 2.40. Electropherograms showing the effect of the charge of ionizable chiral selector, CE--CD, on the EKC separation of PHM enantiomers in a partially pretreated urine matrix. The separations were carried out with the uncharged CE--CD (a, b) and with the negatively charged CE--CD (c). Separating conditions: (a, b) EKC electrolyte system consisting of a 25mM glycine-acetic acid buffer, pH 3.2, 0.2 methylhydroxyethylcellulose as an EOF suppressor, with a 0 (a) and 15 (b) mg/mL concentration of CE--CD, (c) EKC electrolyte system consisting of a 25mM aminocaproic acid-acetic acid buffer, pH 4.5, 0.2 methylhydroxyethylcellulose as an EOF suppressor, with a 2.5 mg/mL concentration of CE- -CD. Sample and analyte: (a, b) PHM was present in the sample (20-times diluted urine) at a 150 ng/mL concentration. (c) PHM was present in the sample (4-times diluted urine) at a

(c)

Negatively charged carboxyethyl--CD considerably improved enantioresolution of several oppositely charged H1-antihistamines (pheniramine and its metabolite, dimethindene, dioxopromethazine) in comparison with native CDs [Mikuš et al., 2006a]. Countercurrent migration and enhanced complexation were responsible not only for improved chiral separation of the drugs and the metabolite, but also achiral separation allowing resolution of the drug enantiomers from their metabolic products, as well as from the sample matrix constituents when metabolic study of pheniramine in urine samples was carried out [Marák et al., 2007]. A high effectivity of the separation was demonstrated by a low concentration of

30 ng/mL concentration. Reprinted from ref. [Mikuš et al., 2006a].

spiked plasma samples, see Figure 2.39 [Fang, L. et al., 2006].

baclofen (pH 9.5) transported by EOF toward the cathode.

urine) [Servais et al., 2006].

the chiral selector enabling optimum separating conditions for the trace chiral analytes in concentrated biological matrices [Mikuš et al., 2006a], see Figure 2.40.

*Charged chiral pseudophases/carrier systems.* The enantioseparations of cyanobenz[*f*]isoindole serine (CBI) derivatives of glutamate and aspartate in squirrel brain samples were accomplished with a highly sulfated--CD as chiral selector at low pH and reverse polarity [Kirschner et al., 2007].

*Mixed selector systems.* Aspartate enantiomers (NDA derivatives) were separated in the samples prepared from tissue of the central nervous system of *A. californica* using mixed micelles composed from -CD and SDS [Miao et al., 2005].

Figure 2.41. Separation of CBI-Ser enantiomers using different running buffers: (a) 100mM borate (pH 9.5), 30mM CD and 60mM SDC; (b) 100mM borate (pH 9.5) and 30mM -CD; (c) 100mM borate (pH 10.0), 60mM SDC. Capillary was 50 m i.d.×50 cm effective length. Voltage applied was 15 kV. Ser enantiomer concentration was 2.0 M. Reprinted from ref. [Zhao, S.L. et al., 2005b].

The CD-MEKC separation of CBI-D/L-serine enantiomers was achieved by using a dual chiral selector system consisting of -CD and chiral micelles formed by deoxycholic acid (SCD) [Zhao, S.L. et al., 2005b]. The essential role of the mixed chiral selector system for the

methyltrimethylammonium with a selectivity of 1.0078 was almost baseline separated in 3.5

Advanced Chiral Separation 47

*CEC.* CEC separations are carried out mainly in packed capillary columns. Immobilization provides an effective solution especially for the chiral selectors with problematic detection properties, such as proteins, macrocyclic antibiotics, etc., that was demonstrated on many model examples, such as vancomycin CSP vs. warfarin and various -blockers (atenolol, metoprolol, pindolol, oxprenolol, alprenolol, propranolol, carteolol, talinolol) vs. MS

Figure 2.43. Electrochromatogram of blank urine sample spiked with 10 g/mL of racemic MRT and 8-OH-M and 20 g/mL of racemic DMR. Both samples were subjected to the SPE procedure. Electrochromatographic conditions: stationary phase composition, vancomycin-CSP, mobile phase, 100 mM ammonium acetate buffer (pH 6)/H2O/MeOH/ACN (5:15:30:50, by vol.). Other CEC conditions: capillary column, 75 m i.d., 33 cm total length, 23 cm packed length, 24.5 cm effective length; applied voltage, 25 kV; capillary temperature, 20°C; pressurized column at both ends with 10 bar; injection by pressure at 10 bar, 0.5 min, followed by a plug of mobile phase at 10 bar, 0.2 min. (1) MRT, (2) 8-OH-M and (3) DMR.

Various new CSPs are also employed in model analyses. A new chiral capillary electrophoresis column coated by carbosilane dendrimers with peripheral Si-Cl groups and beta-cyclodextrin was prepared by Shou et al. [Shou et al., 2008]. The separation characteristics of stability and longevity of coated capillaries modified by carbosilane dendrimers were excellent. The varying of separation efficiency was less than 5%, after running for one month. The optimum conditions contained the running voltage of 12 kV, the UV detector wavelength of 214 nm, the sample injected time of 7 s and the phosphate buffer solution concentration of 40 mM. Chlortrimeton, hydrochloric promethazine and benzedrine were selected as the separation model targets. Especially, G2P columns could be used as separate chlortrimeton enantiomers effectively. Under these conditions, the column

efficiency was 2.5 x 105 plates/m with a resolution of 1.43 and baseline separation.

Other examples of the CEC application are illustrated on biological samples. For example, CEC based on vancomycin CSP offered an effective solution not only for the enantioseparation of

h [Zhao J. & Jorgenson, 1999].

detection [Zheng J. & Shamsi, 2006; Zheng J. et al., 2006].

Reprinted from ref. [Aturki et al., 2007].

enantioresolution in comparison with the capability of individual chiral selectors is demonstrated in Figure 2.41, compare electropherograms (a-c). In addition, an effectivity of the mixed chiral selector system in the enantioseparation of CBI-D/L-serine enantiomers in real biological matrices was demonstrated in this work. In this way, for the first time, peaks corresponding to L-serine and D-serine were completely separated in *Aplysia* ganglian (a sea mollusc widely used as a neuronal model) homogenates.

The evaluation of the enantioselectivity of glycogen-based dual chiral selector systems towards basic drugs in capillary electrophoresis is presented by Chen et al. [Chen J. et al., 2010].

*Flow counterbalanced systems.* Several FCCE modes have been developed and applied for enantioseparations so far, like microfluidic temperature gradient focusing [Balss et al., 2004], electric field gradient focusing [Koegler & Ivory, 1996a, 1996b, Ivory, 2000, Huang, Z. & Ivory, 1999]. Notice that these techniques have a great potential not only in enantioseparations, but also in a sample pretreatment (focusing, see chapter 3).

Figure 2.42. Effect of increasing counterpressure on the separation of (±)-chlorpheniramine in the presence of 2 mg/mL CM-CD). Reproduced from ref. [Chankvetadze et al., 1999].

The effect of increasing counterpressure on the separation of (±)-chlorpheniramine in the presence of 2 mg/mL CM--CD) was studied by Chankvetadze et al. [Chankvetadze et al., 1999] and remarkable results are illustrated in Figure 2.42. Applications of counterpressure techniques for the enantioseparations in complex ionic matrices, however, have not been demonstrated. isotopic model

Synchronous cyclic capillary electrophoresis (SCCE) was proposed by Jorgenson's group [Zhao J. et al., 1999] as a technique which allows overcoming the dispersion problems in FCCE caused by the parabolic counterflow profile. This technique was applied for isotopic and chiral separations. In the third cycle, the chiral compound (-hydroxybenzyl)

enantioresolution in comparison with the capability of individual chiral selectors is demonstrated in Figure 2.41, compare electropherograms (a-c). In addition, an effectivity of the mixed chiral selector system in the enantioseparation of CBI-D/L-serine enantiomers in real biological matrices was demonstrated in this work. In this way, for the first time, peaks corresponding to L-serine and D-serine were completely separated in *Aplysia* ganglian (a sea

The evaluation of the enantioselectivity of glycogen-based dual chiral selector systems towards basic drugs in capillary electrophoresis is presented by Chen et al. [Chen J. et al.,

*Flow counterbalanced systems.* Several FCCE modes have been developed and applied for enantioseparations so far, like microfluidic temperature gradient focusing [Balss et al., 2004], electric field gradient focusing [Koegler & Ivory, 1996a, 1996b, Ivory, 2000, Huang, Z. & Ivory, 1999]. Notice that these techniques have a great potential not only in

Figure 2.42. Effect of increasing counterpressure on the separation of (±)-chlorpheniramine in the presence of 2 mg/mL CM-CD). Reproduced from ref. [Chankvetadze et al., 1999].

The effect of increasing counterpressure on the separation of (±)-chlorpheniramine in the presence of 2 mg/mL CM--CD) was studied by Chankvetadze et al. [Chankvetadze et al., 1999] and remarkable results are illustrated in Figure 2.42. Applications of counterpressure techniques for the enantioseparations in complex ionic matrices, however, have not been demonstrated.

Synchronous cyclic capillary electrophoresis (SCCE) was proposed by Jorgenson's group [Zhao J. et al., 1999] as a technique which allows overcoming the dispersion problems in FCCE caused by the parabolic counterflow profile. This technique was applied for isotopic and chiral separations. In the third cycle, the chiral compound (-hydroxybenzyl)

enantioseparations, but also in a sample pretreatment (focusing, see chapter 3).

mollusc widely used as a neuronal model) homogenates.

2010].

methyltrimethylammonium with a selectivity of 1.0078 was almost baseline separated in 3.5 h [Zhao J. & Jorgenson, 1999].

*CEC.* CEC separations are carried out mainly in packed capillary columns. Immobilization provides an effective solution especially for the chiral selectors with problematic detection properties, such as proteins, macrocyclic antibiotics, etc., that was demonstrated on many model examples, such as vancomycin CSP vs. warfarin and various -blockers (atenolol, metoprolol, pindolol, oxprenolol, alprenolol, propranolol, carteolol, talinolol) vs. MS detection [Zheng J. & Shamsi, 2006; Zheng J. et al., 2006].

Figure 2.43. Electrochromatogram of blank urine sample spiked with 10 g/mL of racemic MRT and 8-OH-M and 20 g/mL of racemic DMR. Both samples were subjected to the SPE procedure. Electrochromatographic conditions: stationary phase composition, vancomycin-CSP, mobile phase, 100 mM ammonium acetate buffer (pH 6)/H2O/MeOH/ACN (5:15:30:50, by vol.). Other CEC conditions: capillary column, 75 m i.d., 33 cm total length, 23 cm packed length, 24.5 cm effective length; applied voltage, 25 kV; capillary temperature, 20°C; pressurized column at both ends with 10 bar; injection by pressure at 10 bar, 0.5 min, followed by a plug of mobile phase at 10 bar, 0.2 min. (1) MRT, (2) 8-OH-M and (3) DMR. Reprinted from ref. [Aturki et al., 2007].

Various new CSPs are also employed in model analyses. A new chiral capillary electrophoresis column coated by carbosilane dendrimers with peripheral Si-Cl groups and beta-cyclodextrin was prepared by Shou et al. [Shou et al., 2008]. The separation characteristics of stability and longevity of coated capillaries modified by carbosilane dendrimers were excellent. The varying of separation efficiency was less than 5%, after running for one month. The optimum conditions contained the running voltage of 12 kV, the UV detector wavelength of 214 nm, the sample injected time of 7 s and the phosphate buffer solution concentration of 40 mM. Chlortrimeton, hydrochloric promethazine and benzedrine were selected as the separation model targets. Especially, G2P columns could be used as separate chlortrimeton enantiomers effectively. Under these conditions, the column efficiency was 2.5 x 105 plates/m with a resolution of 1.43 and baseline separation.

Other examples of the CEC application are illustrated on biological samples. For example, CEC based on vancomycin CSP offered an effective solution not only for the enantioseparation of mirtazapine (MRT) and its metabolites {8-hydroxymirtazapine (8-OH-M) and *N*desmethylmirtazapine (DMR)} in urine samples (pretreated by off-line solid-phase extraction), but also for their reliable UV absorbance detection [Aturki et al., 2007], see Figure 2.43.

Figure 2.44. Separation of mexiletine hydrochloride in human plasma sample by Asp-CD fabricated monolithic column. (A) Plasma sample; (B) free plasma. 1 and 2, the enantiomers' peaks of mexiletine hydrochloride. Mobile phase: phosphate/trolamine buffer, 5mM, pH= 4.5; temperature: 20°C; voltage: −10 kV; injection: −2 kV, 2 s. Reprinted from ref. [Li Y. et al., 2010].

Figure 2.45. Enantiomeric separation of noradrenaline with CM--CD by formation of sandwich complexes with 18-crown-6 at pH 6.5 and 2.5. Separation voltage, 3 kV; detection potential, 1500 mV; injection, 1 kV (3 s); concentration of standard, 10-4 *M*. Reprinted from

Advanced Chiral Separation 49

A combination of an advanced enantioseparation mechanism (dual chiral selector system) with advanced CE format (MCE) was demonstrated by Schwarz and Hauser [Schwarz & Hauser, 2001]. The separation of the enantiomers of noradrenaline in the presence of 18 crown-6 is illustrated in Figure 2.45. The crown ether on its own has a small effect on the migration, but cannot lead to a chiral separation (Figure 2.45b). In combination with the carboxymethylated -cyclodextrin (Figure 2.45c), a chiral separation which is better than the one achieved by only carboxymethylated -cyclodextrin is obtained. The resolution factors (*R*) were determined as 1.30 and 1.35 (5.2 mg/mL CM--CD, pH=6.7) with and without the addition of 12 m*M* 18-crown-6, respectively. Moreover, such an advanced system is capable of also separating multicomponent mixtures of enantiomers, as presented in Figure 2.46.

ref. [Schwarz & Hauser, 2001].

Very attractive applications due to rapid separations and good enantioresolutions are represented by the chiral monolithic based CEC. A monolithic column fabricated with Asp- CD was applied to the separation of mexiletine hydrochloride enantiomers in a human plasma sample. Because of complex matrices, as little as possible trolamine was added to the phosphate buffer to decrease absorption between the sample and the solid-phase. Figure 2.44 shows the electropherogram of mexiletine hydrochloride extracted from the plasma sample. From Figure 2.44, two enantiomers were separated by baseline, the migration times were 3.46 and 3.84 min, respectively. Some components in plasma were also determined, but they did not interfere with the determination of the enantiomers. Thus, the method was applied to the complex matrix sample, and the column had good selectivity.

*MCE.* MCE with (+)-18-crown-6-tetracarboxylic acid as a chiral selector was applied for the countercurrent chiral separation of gemifloxacin in urine samples after an appropriate sample pretreatment (see in 3.2.2) [Cho et al., 2004]. A successful chiral separation was achieved in ca. 3 min, with an extremely small amount of the chiral selector (50 M), and minute amounts of both sample as well as BGE. This demonstrated significant potentialities of electromigration methods in highly effective ultraspeed microscale chiral analyses of drugs in complex matrices.

mirtazapine (MRT) and its metabolites {8-hydroxymirtazapine (8-OH-M) and *N*desmethylmirtazapine (DMR)} in urine samples (pretreated by off-line solid-phase extraction),

Figure 2.44. Separation of mexiletine hydrochloride in human plasma sample by Asp-CD fabricated monolithic column. (A) Plasma sample; (B) free plasma. 1 and 2, the enantiomers' peaks of mexiletine hydrochloride. Mobile phase: phosphate/trolamine buffer, 5mM, pH= 4.5; temperature: 20°C; voltage: −10 kV; injection: −2 kV, 2 s. Reprinted from ref. [Li Y. et al., 2010].

Very attractive applications due to rapid separations and good enantioresolutions are represented by the chiral monolithic based CEC. A monolithic column fabricated with Asp- CD was applied to the separation of mexiletine hydrochloride enantiomers in a human plasma sample. Because of complex matrices, as little as possible trolamine was added to the phosphate buffer to decrease absorption between the sample and the solid-phase. Figure 2.44 shows the electropherogram of mexiletine hydrochloride extracted from the plasma sample. From Figure 2.44, two enantiomers were separated by baseline, the migration times were 3.46 and 3.84 min, respectively. Some components in plasma were also determined, but they did not interfere with the determination of the enantiomers. Thus, the method was

*MCE.* MCE with (+)-18-crown-6-tetracarboxylic acid as a chiral selector was applied for the countercurrent chiral separation of gemifloxacin in urine samples after an appropriate sample pretreatment (see in 3.2.2) [Cho et al., 2004]. A successful chiral separation was achieved in ca. 3 min, with an extremely small amount of the chiral selector (50 M), and minute amounts of both sample as well as BGE. This demonstrated significant potentialities of electromigration methods in highly effective ultraspeed microscale chiral analyses of

applied to the complex matrix sample, and the column had good selectivity.

drugs in complex matrices.

but also for their reliable UV absorbance detection [Aturki et al., 2007], see Figure 2.43.

Figure 2.45. Enantiomeric separation of noradrenaline with CM--CD by formation of sandwich complexes with 18-crown-6 at pH 6.5 and 2.5. Separation voltage, 3 kV; detection potential, 1500 mV; injection, 1 kV (3 s); concentration of standard, 10-4 *M*. Reprinted from ref. [Schwarz & Hauser, 2001].

A combination of an advanced enantioseparation mechanism (dual chiral selector system) with advanced CE format (MCE) was demonstrated by Schwarz and Hauser [Schwarz & Hauser, 2001]. The separation of the enantiomers of noradrenaline in the presence of 18 crown-6 is illustrated in Figure 2.45. The crown ether on its own has a small effect on the migration, but cannot lead to a chiral separation (Figure 2.45b). In combination with the carboxymethylated -cyclodextrin (Figure 2.45c), a chiral separation which is better than the one achieved by only carboxymethylated -cyclodextrin is obtained. The resolution factors (*R*) were determined as 1.30 and 1.35 (5.2 mg/mL CM--CD, pH=6.7) with and without the addition of 12 m*M* 18-crown-6, respectively. Moreover, such an advanced system is capable of also separating multicomponent mixtures of enantiomers, as presented in Figure 2.46.

**3**

**Advanced Sample Preparation**

species advantages

**3.1 On-line sample preparation techniques in capillary electrophoresis - introduction**  The importance of sample preparation is related to high demands on (i) the sensitivity of quantitative determination of trace analytes, (ii) the selectivity of monitoring of analytes in multicomponent sample matrix and (iii) the automatization and miniaturization of analysis

*Sensitivity.* One of the most pronounced limitations of capillary electromigration methods when compared to more traditional liquid-phase separation techniques, such as HPLC, is a poor concentration sensitivity of photometric detectors, which are the most popular among on-capillary CE detectors. The reached LODs are by two orders inferior in comparison to the HPLC technique because of a short optical pathlength and small sample injection volume. Two basic approaches can be distinguished among many efforts that have been made to improve the sensitivity of detection in CE. Either more sensitive detection schemes can displace the UV detection mode [Hernández et al., 2008, 2010; Hempel, 2000] or an increased analyte mass can be cumulated in its zone prior to detection utilizing a proper sample

*Selectivity.* To achieve adequate separation selectivity, the analysis of real/complex samples in CE usually requires efficient sample treatment to remove interfering solutes, inorganic and organic salts, and particulate matter (as it is reviewed in this section). At the same time, the creation of a chiral environment is necessary for the separation of enantiomeric species by the electrokinetic chromatography (EKC) principle accomplished in zone electrophoretic mode simply by the addition of chiral selector molecules. In the same way, chiral selectivity can be implemented in isotachophoretic mode. Chiral separation principles in CE have been described in many review papers [Chankvetadze, 1997; Chankvetadze & Blaschke, 2001; Gübitz & Schmid, 2007, 2008; Preinerstorfer et al., 2009; Fanali, 2002; Fanali et al., 1998b; Ossicini & Fanali, 1997]. In addition to those, capillary electrochromatography (CEC) separation principles cannot be omitted for review, e.g., those by Schurig [Wistuba & Schurig, 2000a, 2000b]. On-line sample preparation and enantioseparation mechanisms should create a harmonized system, especially in chiral EKC and ITP where the chiral selector is not immobilized as it is in CEC, and all possible mutual negative interferences

*Automatization and miniaturization.* As one moves toward small sample volumes, sample handling and preparation steps become more difficult and the concentration step is therefore preferably done on-line instead of off-line. The microfluidic devices, such as microchips, can provide several additional advantages over electromigration techniques performed in capillary format. The heat dissipation is much better in chip format compared

[Mikuš & Maráková, 2010].

preparation, as it is reviewed in this section.

have to be carefully eliminated.

Figure 2.46. Rapid enantiomer MCE separation and amperometric detection of a mixture of adrenaline, noradrenaline and dopamine employing sandwich complexes. Buffer: (a) 50 mM MES, pH=6.7; (b) 20 mM phosphate, pH=3.0, 5.2 mg/mL CM--CD, 12 mM 18-crown-6. Separation voltage, 3 kV; detection potential, (a) 1300 mV, (b) 1500 mV; injection, 1 kV (3 s); concentration of standards, 10-4 M. Peak marks: dopamine hydrochloride, D, adrenaline, A, noradrenaline, NA. Reprinted from ref. [Schwarz & Hauser, 2001].

### **Advanced Sample Preparation**

### **3.1 On-line sample preparation techniques in capillary electrophoresis - introduction**  The importance of sample preparation is related to high demands on (i) the sensitivity of

50 Chiral Capillary Electrophoresis in Current Pharmaceutical and Biomedical Analysis

Figure 2.46. Rapid enantiomer MCE separation and amperometric detection of a mixture of adrenaline, noradrenaline and dopamine employing sandwich complexes. Buffer: (a) 50 mM MES, pH=6.7; (b) 20 mM phosphate, pH=3.0, 5.2 mg/mL CM--CD, 12 mM 18-crown-6. Separation voltage, 3 kV; detection potential, (a) 1300 mV, (b) 1500 mV; injection, 1 kV (3 s); concentration of standards, 10-4 M. Peak marks: dopamine hydrochloride, D, adrenaline, A,

noradrenaline, NA. Reprinted from ref. [Schwarz & Hauser, 2001].

quantitative determination of trace analytes, (ii) the selectivity of monitoring of analytes in multicomponent sample matrix and (iii) the automatization and miniaturization of analysis [Mikuš & Maráková, 2010].

*Sensitivity.* One of the most pronounced limitations of capillary electromigration methods when compared to more traditional liquid-phase separation techniques, such as HPLC, is a poor concentration sensitivity of photometric detectors, which are the most popular among on-capillary CE detectors. The reached LODs are by two orders inferior in comparison to the HPLC technique because of a short optical pathlength and small sample injection volume. Two basic approaches can be distinguished among many efforts that have been made to improve the sensitivity of detection in CE. Either more sensitive detection schemes can displace the UV detection mode [Hernández et al., 2008, 2010; Hempel, 2000] or an increased analyte mass can be cumulated in its zone prior to detection utilizing a proper sample preparation, as it is reviewed in this section.

*Selectivity.* To achieve adequate separation selectivity, the analysis of real/complex samples in CE usually requires efficient sample treatment to remove interfering solutes, inorganic and organic salts, and particulate matter (as it is reviewed in this section). At the same time, the creation of a chiral environment is necessary for the separation of enantiomeric species by the electrokinetic chromatography (EKC) principle accomplished in zone electrophoretic mode simply by the addition of chiral selector molecules. In the same way, chiral selectivity can be implemented in isotachophoretic mode. Chiral separation principles in CE have been described in many review papers [Chankvetadze, 1997; Chankvetadze & Blaschke, 2001; Gübitz & Schmid, 2007, 2008; Preinerstorfer et al., 2009; Fanali, 2002; Fanali et al., 1998b; Ossicini & Fanali, 1997]. In addition to those, capillary electrochromatography (CEC) separation principles cannot be omitted for review, e.g., those by Schurig [Wistuba & Schurig, 2000a, 2000b]. On-line sample preparation and enantioseparation mechanisms should create a harmonized system, especially in chiral EKC and ITP where the chiral selector is not immobilized as it is in CEC, and all possible mutual negative interferences have to be carefully eliminated. demands because CE. utilizing additional

*Automatization and miniaturization.* As one moves toward small sample volumes, sample handling and preparation steps become more difficult and the concentration step is therefore preferably done on-line instead of off-line. The microfluidic devices, such as microchips, can provide several additional advantages over electromigration techniques performed in capillary format. The heat dissipation is much better in chip format compared

**3.2 On-line sample preparation techniques based on electrophoretic principles**

chiral analysis by CE.

preconcentration).

Several reviews have been published recently, focusing on the on-line sample preconcentration CE techniques based on electrophoretic principles leading to the compression of a long sample plug into a narrow band with a high concentration of analytes, so called stacking techniques [Lin C.H. & Kaneta, 2004; Gebauer et al., 2009, 2011; Silva, 2009; Ryan et al., 2009; Malá et al., 2007, 2011; Urbánek et al., 2003]. Among the latest detailed review papers are those by Lin and Kaneta [Lin C.H. & Kaneta, 2004], Simpson et al. [Simpson et al., 2008], Breadmore [Breadmore, 2007], Breadmore et al. [Breadmore et al., 2009] and Malá et al. [Malá et al., 2011], including research articles gathered in the period 2000-2011. The papers provide fundamentals and applications of basic types of on-line sample preconcentration techniques. McKibbin and Terabe [Britz-McKibbin & Terabe, 2003] emphasized on-line preconcentration strategies for trace analysis of metabolites by CE. Ruiz and Marina [Ruiz & Marina, 2006] reviewed sensitive

Advanced Sample Preparation 53

The stacking procedures, described in section 3.2.1, are modifications of the basic zone electrophoretic and/or isotachophoretic and/or isoelectric focusing separation modes (Figure 2.2). The stacking procedures are based on increasing analyte mass in its zone during the electromigration process via electromigration effects, enhancing sensitivity in this way. In all the cases, the key requirements are that there is an electrophoretic component in the preconcentration mechanism and that the analytes concentrate on a boundary through a change in velocity. Then we can recognize (i) field-strength-induced changes in velocity (field-enhanced sample stacking [Kim J.B. & Terabe, 2003; Quirino & Terabe, 2000; Chien & Burgi, 1992; Weiss et al., 2001], isotachophoresis and transient isotachophoresis [Beckers & Boček, 2000; Schwer et al., 1993; Shihabi, 2002]), and (ii) chemically induced changes in velocity (dynamic pH junction [Britz-McKibbin & Chen, 2000; Aebersold & Morrison, 1990; Kim J.B. et al., 2003], sweeping [Kitagawa et al., 2006; Palmer et al., 2001; Quirino & Terabe, 1998; Quirino & Terabe, 1999; Quirino et al., 2000]), see Table 3.2. In addition to these techniques, the counter-flow gradient focusing [Shackman & Ross, 2007], electrocapture [Horáková et al., 2007] and many others can be considered as the techniques based on a combination of field-strength and chemically induced changes in velocity offering new and interesting possibilities in on-line sample preparation (mainly

Some of the stacking techniques (and their combinations) can provide, besides (i) the preconcentration, other benefits, such as (ii) an effective sample purification isolating solute (group of solutes) from undesired matrix constituents [Simpson et al., 2008] or they can be combined with (iii) the chemical reaction of the analyte(s) [Ptolemy et al., 2005, 2006], in this way simplifying the overall analytical procedure. On the other hand, great attention must be paid to the selection of the type of chiral selector as its charge can interfere with a sample

preparation technique that employs electrophoretic principles.

to that in a capillary and therefore higher electric fields can be applied across channels of the microchip. This fact enables, along with a considerably reduced length of channels, significant shortening of separation time. The sample and reagent consumption is markedly reduced in microchannels. Hence, the chiral MCE can provide a unique possibility of ultraspeed enantiomeric separations of microscale sample amounts. On the other hand, the efficiency is often weak due to the shape of the chips and the quality of the injection [Guihen et al., 2009]. Until now, electrophoresis, rather than chromatography, has been the primary principle applied in microchip separations for several reasons as follows: (i) the materials typically used for microchips can barely withstand the high pressures applied for chromatographic separations; (ii) the packing of the chromatographic particles into the channels without voids can be difficult, (iii) the performance of chromatography decreases with decreased column length [Preinerstorfer et al., 2009]. Nevertheless, both electrophoretic [Gong & Hauser, 2006; Piehl et al., 2004; Belder et al., 2006], as well as electrochromatographic, modes [Weng, et al. 2006] are applicable in microchip format. Nowadays, the on-line coupling of sample treatment systems to CE or MCE are of great interest because it allows the automatization of the analytical process (from sample preparation to data treatment), which is a current trend in analytical chemistry.

Thus, the importance of on-line sample preparation is pronounced when ultratrace analytes (ng/mL and less) are determined in minute amounts (l, g and less) of samples with complex matrices (variable in qualitative and quantitative composition). This is a common situation in the analyses of drugs, their metabolites and biomarkers in biological samples where preconcentration of analytes, elimination of matrix interferents, and minimized sample handling is necessary to obtain relevant analytical results. Mikuš and Maráková [Mikuš & Maráková, 2010] recently provided a review on the chiral capillary electrophoresis with on-line sample preparation. The latest panorama of sample preparation methods for animal/human and plant samples given by Chen et al. [Chen Y., 2008] has been composed from almost 500 references, highlighting some promising methods which have fast developed over recent years and giving a somewhat brief introduction on most of the well-developed methods, including on-line stacking methods in CE. Wu [Wu X.Z., 2003] illustrated new approaches to sample preparation for CE and Kataoka [Kataoka, 2003] highlighted their utilization in clinical and pharmaceutical analysis. In addition to these rather general reviews, specialized reviews are included at the beginning of each following subsection. These highlight the role of electromigration effects and interactions in on-line sample preparation, and summarize basic electrophoretic (stacking) and non electrophoretic (mostly chromatographic and extraction) on-line sample preparation techniques aimed at preconcentration and purification of complex samples in chiral pharmaceutical and biomedical research. Application examples are listed in Table 3.1 showing the chiral pharmaceutical and biomedical analyses supported by on-line sample preparation procedures described in this chapter. On the other hand, conventional off-line sample preparation techniques, such as solid-phase extraction, liquid-phase extraction, solid-liquid extraction and dialysis (included in the above mentioned reviews), are not discussed here.

to that in a capillary and therefore higher electric fields can be applied across channels of the microchip. This fact enables, along with a considerably reduced length of channels, significant shortening of separation time. The sample and reagent consumption is markedly reduced in microchannels. Hence, the chiral MCE can provide a unique possibility of ultraspeed enantiomeric separations of microscale sample amounts. On the other hand, the efficiency is often weak due to the shape of the chips and the quality of the injection [Guihen et al., 2009]. Until now, electrophoresis, rather than chromatography, has been the primary principle applied in microchip separations for several reasons as follows: (i) the materials typically used for microchips can barely withstand the high pressures applied for chromatographic separations; (ii) the packing of the chromatographic particles into the channels without voids can be difficult, (iii) the performance of chromatography decreases with decreased column length [Preinerstorfer et al., 2009]. Nevertheless, both electrophoretic [Gong & Hauser, 2006; Piehl et al., 2004; Belder et al., 2006], as well as electrochromatographic, modes [Weng, et al. 2006] are applicable in microchip format. Nowadays, the on-line coupling of sample treatment systems to CE or MCE are of great interest because it allows the automatization of the analytical process (from sample preparation to data treatment), which is a current trend in

Thus, the importance of on-line sample preparation is pronounced when ultratrace analytes (ng/mL and less) are determined in minute amounts (l, g and less) of samples with complex matrices (variable in qualitative and quantitative composition). This is a common situation in the analyses of drugs, their metabolites and biomarkers in biological samples where preconcentration of analytes, elimination of matrix interferents, and minimized sample handling is necessary to obtain relevant analytical results. Mikuš and Maráková [Mikuš & Maráková, 2010] recently provided a review on the chiral capillary electrophoresis with on-line sample preparation. The latest panorama of sample preparation methods for animal/human and plant samples given by Chen et al. [Chen Y., 2008] has been composed from almost 500 references, highlighting some promising methods which have fast developed over recent years and giving a somewhat brief introduction on most of the well-developed methods, including on-line stacking methods in CE. Wu [Wu X.Z., 2003] illustrated new approaches to sample preparation for CE and Kataoka [Kataoka, 2003] highlighted their utilization in clinical and pharmaceutical analysis. In addition to these rather general reviews, specialized reviews are included at the beginning of each following subsection. These highlight the role of electromigration effects and interactions in on-line sample preparation, and summarize basic electrophoretic (stacking) and non electrophoretic (mostly chromatographic and extraction) on-line sample preparation techniques aimed at preconcentration and purification of complex samples in chiral pharmaceutical and biomedical research. Application examples are listed in Table 3.1 showing the chiral pharmaceutical and biomedical analyses supported by on-line sample preparation procedures described in this chapter. On the other hand, conventional off-line sample preparation techniques, such as solid-phase extraction, liquid-phase extraction, solid-liquid extraction and dialysis

(included in the above mentioned reviews), are not discussed here.

analytical chemistry.

### **3.2 On-line sample preparation techniques based on electrophoretic principles**

Several reviews have been published recently, focusing on the on-line sample preconcentration CE techniques based on electrophoretic principles leading to the compression of a long sample plug into a narrow band with a high concentration of analytes, so called stacking techniques [Lin C.H. & Kaneta, 2004; Gebauer et al., 2009, 2011; Silva, 2009; Ryan et al., 2009; Malá et al., 2007, 2011; Urbánek et al., 2003]. Among the latest detailed review papers are those by Lin and Kaneta [Lin C.H. & Kaneta, 2004], Simpson et al. [Simpson et al., 2008], Breadmore [Breadmore, 2007], Breadmore et al. [Breadmore et al., 2009] and Malá et al. [Malá et al., 2011], including research articles gathered in the period 2000-2011. The papers provide fundamentals and applications of basic types of on-line sample preconcentration techniques. McKibbin and Terabe [Britz-McKibbin & Terabe, 2003] emphasized on-line preconcentration strategies for trace analysis of metabolites by CE. Ruiz and Marina [Ruiz & Marina, 2006] reviewed sensitive chiral analysis by CE. to

The stacking procedures, described in section 3.2.1, are modifications of the basic zone electrophoretic and/or isotachophoretic and/or isoelectric focusing separation modes (Figure 2.2). The stacking procedures are based on increasing analyte mass in its zone during the electromigration process via electromigration effects, enhancing sensitivity in this way. In all the cases, the key requirements are that there is an electrophoretic component in the preconcentration mechanism and that the analytes concentrate on a boundary through a change in velocity. Then we can recognize (i) field-strength-induced changes in velocity (field-enhanced sample stacking [Kim J.B. & Terabe, 2003; Quirino & Terabe, 2000; Chien & Burgi, 1992; Weiss et al., 2001], isotachophoresis and transient isotachophoresis [Beckers & Boček, 2000; Schwer et al., 1993; Shihabi, 2002]), and (ii) chemically induced changes in velocity (dynamic pH junction [Britz-McKibbin & Chen, 2000; Aebersold & Morrison, 1990; Kim J.B. et al., 2003], sweeping [Kitagawa et al., 2006; Palmer et al., 2001; Quirino & Terabe, 1998; Quirino & Terabe, 1999; Quirino et al., 2000]), see Table 3.2. In addition to these techniques, the counter-flow gradient focusing [Shackman & Ross, 2007], electrocapture [Horáková et al., 2007] and many others can be considered as the techniques based on a combination of field-strength and chemically induced changes in velocity offering new and interesting possibilities in on-line sample preparation (mainly preconcentration).

Some of the stacking techniques (and their combinations) can provide, besides (i) the preconcentration, other benefits, such as (ii) an effective sample purification isolating solute (group of solutes) from undesired matrix constituents [Simpson et al., 2008] or they can be combined with (iii) the chemical reaction of the analyte(s) [Ptolemy et al., 2005, 2006], in this way simplifying the overall analytical procedure. On the other hand, great attention must be paid to the selection of the type of chiral selector as its charge can interfere with a sample preparation technique that employs electrophoretic principles.

New

Plasma HP-

UV, MS, Spiked real

Grard et al.,

2002

Thormann,

2006

Hedeland

Baldacci &

samples

preconcentration (180x) 3 ng/mL

EH/off, SPE/off clean-up, UV, ESI-MS Metabolic study

adrenoreceptors

antagonists

Lorazepam and its

Urine HP-

micelles SWP/on preconcentration

EK + tITP/on

DAD, 0.2 %

Enantiomeric

S-timolol,

1R,2S-0.033%

pharmaceuticals

purity testing of et al., 2007

ephedrine

LIF, 10-10 M Biomedical

study et al., 2007

Kirschner

preconcentration

metabolites

S-timolol, 1R,2S-

ephedrine solution Infusion Ketopinic acid and

diisoproylidenek

etogulonic acid

(NACE)

S-

preconcentration

derivatization

0.2x10

study

UV, 2 and

Biomedical

Ptolemy et

al., 2005

+ preconcentration, DPJ/on

preconcentration (100x)

EH, SPCD/on derivatization

UV, 0.4-

Biomedical

Ptolemy et

al., 2006a

study

Spiked samples

Balss et al.,

2004

0.6x10-6 M

DAD

(baclofen)

DAD,

Enantiomeric

Marák et al.,

2008

2.5 ng/mL

UV,

0.6x10-9 M model samples

DAD,

Spiked real

samples

3-5 ng/mL

et al., 1999

Fang, H.F.

et al., 2006b

Petersson

pharmaceuticals

Spiked real and

purity testing of

acid), LIF

(glutamic

+ preconcentration

(1200x)

Bacterial OPA/NAC HD + SPCD/on

CBI-amino acids Squirrel brain

samples

Muramic acid and

diaminopimelic acid cultures

Amino acids Bacterial

Advanced Sample Preparation 55

cultures

urine

DL-Glutamic acid,

baclofen

Dexbrompheniramin

Pharmaceutical

CE-

preconcentration (~102)

+ clean-up

preparations

Terbutaline Plasma, water

DM-

preconcentration (7000x)

preconcentration

matrices

derivatives

Ephedrine Urine

e

OPA/NAC,

CD

Analyte Sample Chiral selectora

Fenoprofen and

amino acid derivates

Cimaterol,

clenbuterol,

terbutaline

Amlodipine Plasma and

HPCD

EME/ preconcentration

3 ng/mL Nojavan &

Fakhari, 2010

Choi et al.,

2009

(124x)

SDME/in preconcentration

UV,

Spiked real

samples

0.5 ng/mL

DAD,

Spiked real

Li H. et al.,

2008

Mikuš et al.,

2006a

samples

Spiked real

samples and

metabolic study

0.92 ng/mL

UV, 1.1-4.8

ng/mL

(~103) + clean-up

urine

Human urine (+)-(18-crown-6)-

tetracarboxylic

acid

Trihexyphenidyl Serum CMCD LLE/off clean-up, FESS/on

preconcentration (490x)

preconcentration (~102)

+ clean-up

Human urine CE--CD ITP-EKC; ITP/on

Primary amine

drugs

(amphetamine)

Pheniramine and its

metabolites,

dimethindene,

dioxopromethazine

Pheniramine and its

Human urine CE--CD ITP-EKC; ITP/on

preconcentration (~102)

DAD, 5.2-

Metabolic study

Marák et al.,

2007

6.8 ng/mL

DAD, 9.3-

Pharmacokinetic

Mikuš et al.,

2008a

10.4 ng/mL

DAD,

Spiked real

Denola et al.,

2007

samples

<300

ng/mL

UV, LIF,

Spiked real

Cho et al.,

2004

samples

3-4x10-6 M

study

+ clean-up

preconcentration (~102)

+ clean-up

preconcentration (100-1000x)

Serum -CD PP/off clean-up, LVSS/on

Amlodipine Human urine HP--CD ITP-EKC; ITP/on

metabolites

Metoxamine,

carvedilol,

terbutaline,

metaproterenol

Gemifloxacin Urine, saliva CWE EK clean-up, MCE with

coupled channels/on cleanup

River water vancomycin LVSEP-ASEI/on

Human urine -CD t-ITP and FESI/on

Sample preparation: t

main purposeb

preconcentration (~103)

preconcentration (250x)

ype and

Detection

Application Ref.

and LOD

UV, 0.38-

Spiked real

Wang Z.Y.

et al., 2011

et al., 2011

samples

1 ng/mL Huang L.

2.10 ng/mL

FMOC-carnitine Synthetic

HDM-

CD FI/on derivatization DAD,

5x10-6 M

Enantiomeric

Mardones

model samples

FESS/on preconcentration, derivatization/off LIF, 0.5x10-9 M Spiked model samples Mechref & El Rassi, 1997

DAD,

purity testing in et al., 1999

samples

Fenoxy acid

Water matrices Octyl-

maltopyranoside

CD LLE/off clean-up, FESS/on

preconcentration 25-75

ng/mL

analysis

al., 1998

Forensic Tagliaro et

herbicides

Ephedrine,

Hair

amphetamine and

related compounds

blockers Serum CM

CD

3-carboxyadipic acid Minerals Vancomycin FESS/on preconcentration

Sulindac and its

Advanced Sample Preparation 57

Plasma DM-

UV,

Pharmakokineti

c study et al., 2006

1-3x10-7 M

LIF,

Environmental

Asami &

Imura, 2006

Britz-McKibbin

analysis

Spiked real

samples,

biomedical

et al., 2003b

study

Spiked real

Ibrahim et

al., 2007

samples

2x10-9 M

LIF,

4x10-9 M

preconcentration (500x)

preconcentration

preconcentration -CD, SDS -

metabolites

Glufosinate River water

sample

Flavins Bacterial cell

SWP/on preconcentration

(60x)

extracts,

plasma and

urine

Propiconazol Grape HP-

Triadimenol Methanol

HS-

CD

SRMP/on preconcentration

(10x) 0.8-3.8

ng/mL

matrices

micelles

SPE/off clean-up, SWP/on

DAD,

ng/mL

DAD,

Spiked model

Otsuka et al.,

2003

samples

preconcentration (100x) 90-100

preconcentration (5-25x)

10-50

Spiked real

Huang L.

samples et al., 2008

ng/mL

DAD,

Environmental

Castro-

analysis

Pu

y

ana et al.,

2008

Chen, Y.L.

(1000x) 10

PP/off clean-up, FESI/on UV,

(1

*R*, 2

pseudoephedrine,

(1

*R*, 2

(1

*S*, 2

pseudoephedrine,

(*S*)-(+)-

methamphetamine

Aspartate Tissue samples

CD

Microdialysis/on clean-up,

LIF,

Biomedical

Thompson

et al., 1999

study

9x10-7 M

(Daspartate)

Derivatization/on

from rats

D-serin + other

Tissue

CD, HP-

Microdialysis/on clean-up,

LIF,

Biomedical

O'Brien et al.,

2003

study

270x10-9 M;

LIF (sheath

flow

detection

cell),

21x10-9 M

Amperomet

Pharmacokinetic

Hadwiger

et al., 1996

study

ric

detection,

0.6 ng/mL

UV,

Pálmarsdóttir

et al., 1996

2.5x10-10 M

Isoproterenol Intravenous

M--CD

Microdialysis/on clean-up,

pH-mediated sample

stacking/on preconcentration

dialysed

samples

Bambuterol Plasma SLM/on + MLC/on clean-up,

double stacking/on

preconcentration (40 000x)

SLM/on + MLC/on clean-up,

UV,

Spiked real and

Pálmarsdóttir

et al., 1995

model samples

5x10-9 M

double stacking/on

preconcentration (400x)

Terbutaline, but also

Plasma,

Alkyl- or

hydroxyalkyl--

CDs

synthetic

samples

brompheniramine,

propranolol,

ephedrine

Derivatization/on

CD, HP--CD

homogenates

neurotransmitters

*S*)-

*S*)-ephedrine,

*R*)-

Urine, serum -CD

LLE/on clean-up, FESI/on

DAD, 0.15

Spiked real

Fang, H.F.

et al., 2006a

samples

to 0.25

ng/mL

preconcentration (3800x)

 of

a Or derivatization agent creating diastereomeric products.

b An on- or off-line mode is given behind slash, preconcentration factor or amount of removed interfering compounds are given in brackets

 ITP = isotachophoresis, tITP = transient isotachophoresis, EKC = electrokinetic chromatography, CFGF = counter-flow gradient focusing, CWE = crown ether, NACE = non-aqueous capillary electrophoresis, MCE = electrophoresis on microchip, SPCD = incapillary sample preconcentration with chemical derivatization, OPA/NAC= *ortho*-phthalaldehyde/ *N*-acetyl l-cysteine, S-CD = sulphated--CD, HS--CD = highly sulphated--CD, M--CD = methyl--CD, DM--CD = dimethyl--CD, CM--CD = carboxymethyl--CD, CE--CD = carboxyethyl--CD, HP--CD = hydroxypropyl--CD, HP--CD = hydroxypropyl--CD, HDM-- CD = heptakisdimethyl--CD, EH = enzymatic hydrolysis, HD = hydrodynamic injection, EK = electrokinetic injection, FI= flow injection, DPJ= dynamic pH junction, SRMP=stacking with reverse migrating phase, FESS = field-enhanced sample stacking, LVSS = large volume sample stacking, FESI= field-enhanced sample injection, SPE = solid-phase extraction, LLE = liquid-liquid extraction, LPME = liquid-phase microextraction, LVSEP-ASEI=large volume sample stacking with EOF as a pump plus anion-selective exhaustive injection, PP = protein precipitation, CME = centrifuge microextraction, SDME = single drop microextraction, SLM = supported liquid membrane technique, DAD = diode array detection, UV-ultraviolet (absorbance detection), LIF = laser induced fluorescent detection, MS = mass spectrometry, LOD = limit of detection, SDS = sodium dodecylsulphate, FMOC = 9-fluorenylmethyl chloroformate, EME=electro membrane extraction.

Technique Principle Characteristic features

FESI

LVSS,

NSM,

solution zone is concentrated at the boundar

of a high-conductivit

conductivity ratio of these two zones (c

Gseparation solution / Gsample solution). Water plu

injected prior to the injection of the anal

FESI.

stacked

g is

preconcentration and purificati

NSM ~10-fold; LVSS ~100-fold; FESI >1000-fold

Limitations. Limited applications {charged analytes,

separations only cations or anions in one run, samples with

a low-conductivity matrix (can be overcome b

mediation)}. Requirements {removin

(LVSS), EOF suppression}. Reproducibility with FESI (injecting

amount is biased by electrophoretic mobility, time depletion of

the analyte in the sample solution).

ges. Flexible. Directl

significant matrix ion (acting as a leader). Applicable for low-

conductivity (diluted) samples

ion (inserted in capillar

y applicable to samples with a

with the aid of added leading

y or in sample).  Sample

purification aspect (neutral and oppositel

compounds do not interfere). Preconcentration: more than

Limitations. Preconcentration on

run. Analysis of neutral compounds is not possible.

ly cations or anions in one

three orders.

y char

ged

y as

soluble supernatants of precipitated proteinic samples.

ysis of water

tITP Temporary ITP action bein

Advanced Sample Preparation 59

zone electrophoretic regime. Anal

of each separated zone is ad

determined b

justed to that

y the concentration of the ion in

terminatin

the foregoing neighbour zone (canalyte ion ~ c

leading ion) resulting in the preconcentration of

diluted sample zones.

zones of electrolyte solution. The concentration

(terminating ion) electrophoretic mobility

between the highest (leading ion) and lowest

g replaced later by

Advanta

yte mi

grates

y pH

g of sample matrix

electrokinetical injection (FESI) with simultaneous

on aspect. Preconcentrations:

amount of charged analyte from the sample with

yte in

~

zone. Concentration effect is related to

y separation solution

y

Advantages. Eas

y to perform b

concentrations or electrical conductivities of the sample and

separation solutions. Possibility to inject a selectivel

y simpl

y optimizing

y lar

ge

Analyte from a low-conductivity sample

FESS:

Electrophoretic (stacking)

*N*-acetyl l-cysteine, S-CD =

Tryptophan Urine CD ITP-EKC; ITP/on preconcentration + clean-up (~99%) UV, 1.5 ng/mL Spiked real samples Danková et al., 1999 Isoxyzolylpenicilines

Milk HPCD

LLE/off clean-up, LVSS/on preconcentration DAD, 2 ng/mL Food analysis Zhu et al., 2003 Clenbuterol Acetic acid matrices DMCD

tITP/on preconcentration UV, 10-6 M Spiked model samples Toussaint et al., 2000 Table 3.1. Chiral CE determinations of biologically active compounds in various biological matrices employing advanced (on-line) sample preparation.

a Or derivatization agent creating diastereomeric products.

brackets

b An on- or off-line mode is given behind slash, preconcentration factor or amount of removed interfering compounds are given in

ITP = isotachophoresis, tITP = transient isotachophoresis, EKC = electrokinetic chromatography, CFGF = counter-flow gradient

focusing, CWE = crown ether, NACE = non-aqueous capillary electrophoresis, MCE = electrophoresis on microchip, SPCD = in-

sulphated--CD, HS--CD = highly sulphated--CD, M--CD = methyl--CD, DM--CD = dimethyl--CD, CM--CD =

carboxymethyl--CD, CE--CD = carboxyethyl--CD, HP--CD = hydroxypropyl--CD, HP--CD = hydroxypropyl--CD, HDM--

CD = heptakisdimethyl--CD, EH = enzymatic hydrolysis, HD = hydrodynamic injection, EK = electrokinetic injection, FI= flow

injection, DPJ= dynamic pH junction, SRMP=stacking with reverse migrating phase, FESS = field-enhanced sample stacking, LVSS =

large volume sample stacking, FESI= field-enhanced sample injection, SPE = solid-phase extraction, LLE = liquid-liquid extraction,

LPME = liquid-phase microextraction, LVSEP-ASEI=large volume sample stacking with EOF as a pump plus anion-selective

exhaustive injection, PP = protein precipitation, CME = centrifuge microextraction, SDME = single drop microextraction, SLM =

supported liquid membrane technique, DAD = diode array detection, UV-ultraviolet (absorbance detection), LIF = laser induced

fluorescent detection, MS = mass spectrometry, LOD = limit of detection, SDS = sodium dodecylsulphate, FMOC = 9-fluorenylmethyl

chloroformate, EME=electro membrane extraction.

capillary sample preconcentration with chemical derivatization, OPA/NAC= *ortho*-phthalaldehyde/

EOF

microchannel, or an

Electroosmotic velocit

surface vs. liquid.

y other fluid conduit.

y is dependent of the

charge in the electrical double layer, solid

vs. analytes vs. selector)

separabilit

Limitations. An EOF velocit

and char

systems is lower than the s

very pronounced in e.g., CEC s

in the hydrodynamically closed electrophoretic systems.

Chromatography The separation is

analytes between the stationar

phase accordin

mechanism differentiates mi

of the anal

chemical principles.

gration velocities

that eliminates an

ytes. Separations are based on

these analytes with the stationary phase. This

based on a distribution of the

Advanta

ges. An alternative separation mechanism to the

system. The stationary phase serves as an immobilized selector

y interferences of the selector with the

detection. CEC represents a single column chromato

implementation with EOF as a driving force.

Limitations. The chromato

graphic s

concentration of the analytes. Therefore, a chomato

pretreated sample must be further treated before the

terms of the instrumentation as well as the compactibilit

the electrophoretic and chromato

phase vs. electrolyte).

graphic s

ystems (mobile

y of

chromatographic techniques with electrophoresis is difficult in

electrophoretic separation. A coupling of the pressure driven

ystems do not provide a

graphically

graphic

electrophoretic one that enables to increase selectivity of the

y and mobile

g to the different interactions of

Nonelectrophoretic

Advanced Sample Preparation 61

y depends of a solid surface area

ge, therefore a precise control of the EOF velocit

be difficult. Hence, the reproducibility of the EOF based

ystems with suppressed EOF. It is

ystems. EOF cannot be applied

y can

separation efficiency.

implementation of the countercurrent migration effects (EOF

for an enhancement of the

y. A flat EOF profile is favourable for a hi

g

h

charged compounds in one run is possible too. EOF enables an

porous material, capillary tube, membrane,

liquid induced by an applied potential across a

on their char

ge. Therefore anal

possible, and separation of neutral, positivel

ysis of neutral compounds is

y and ne

gatively

Electroosmotic flow (EOF) is the motion of

Advantages. EOF enables movement of compounds re

gardless

DPJ Based on si

gnificant chan

velocities of the analytes between different pH

narrow range of p

acidic or basic analytes.

Limitations. Electrokinetic dispersion pla

in this technique. Only for ionizable analytes (weak

acids/bases). Analysis of neutral compounds is not possible.

ys an important role

values (BGE solution vs. sample solution with

a suppressor of analyte ionization). Lon

of sample zone is

graduall

from the BGE solution and the anal

ionized in the neutralized zone. The anal

focused at the neutralization boundar

the neutralization of the sample zone.

ytes are picked up and accumulated by

the appropriate pseudostationary phase (e.

micelles) that penetrates the micelle free

sample zone. The length of the analyte zone

after sweep is inversel

retention factor of the analyte (the ratio of the

yte amounts present in micelle and in

anal

solution), Lsweep ~ 1 / k.

y proportional to the

g.,

homogeneous electric field stren

Advantages. Electrokinetic dispersion is minimized by

gth throu

capillary. High concentration efficiency, possibly up to 5000-fold

(according to the affinity analyte vs. pseudophase), or even up

to several million-fold (when the length to the detection point is

very short and very narrow detection window is emplo

in MCE), or even more (with electrokinetic injection). Suitable

for charged as well as neutral analytes. Sample purification

aspect (migrate only analytes interacting with sweeping

pseudophase), useful for complex matrices.

Limitations. The narrow analyte zone created by sweeping tends

to broaden quickl

(separation efficiency si

capillar

pseudophase.

CFGF Counter-flow gradient focusing (CFGF)

methods can focus the analyte into a

g via simultaneous actin

y and the opposite bulk

solution flow so that the total velocity (the sum

of both velocities) is equal to zero at a unique

point (characteristic for the analyte).

g an

section in this table.

versatile wa

concentrated plu

electrophoretic velocit

y). Application is limited b

Advantages. CFGF approaches are a potentiall

y in which to simultaneousl

separate charged and neutral analytes. See also Pressure flow

Limitations. Specific electrolytes must be used in some of these

techniques. See also Pressure flow section in this table.

gnificantl

y decreases with the len

y availabilit

y of appropriate

y simple and

y concentrate and

gth of

y accordin

g to the diffusion alon

g the capillary

yed, e.

g.,

ghout the whole

SWP Anal

SWP

y titrated b

y the ion

yte will be

yte is

y during

g plug

ges in electrophoretic

Advantages. Selective concentration of the analytes havin

*K*a. Useful for the concentration of weakly

g a

Pressure flow Driving pressure or counter-pressure based

flows are provided simpl

they are controlled electronicall

important tool also in CFGF.

y b

y the pumps and

Advanta

enablin

ges. One of the basic tools in the advanced s

g a controlled transfer of solutes in various se

ystems

gments of

y. This is

the h

yphenated s

ystems (e.

electrophoretic). A counter-flow enables to increase virtually

the effective length of the capillary or channel and, b

increase the separability.

the reproducibility of the anal

instrumentation (pumps and auxiliaries) must be implemented

into the anal

Advanced Sample Preparation 63

Table 3.2. The most important electrophoretic and nonelectrophoretic techniques and tools applicable in electrophoresis.

FESS = field-enhanced sample stacking; NSM = normal stacking mode; LVSS = large volume sample stacking; FESI = field-

ent isotachophoresis; DPJ = dynamic pH junction; SWP = sweeping; EOF = electroosmotic

enhanced sample injection; tITP = transi

flow; CEC = capillary; SPE = solid-phase extraction; LLE = liquid-liquid extraction

ytical s

connections) decrease the separation efficiency.

ystem. Dead volumes (tubin

g and

ysis. An additional

Limitations. Fluctuations of these non selective flows decrease

y that,

g., electrophoretic and non

enhanced sample injection; tITP = transi ent isotachophoresis; DPJ = dynamic pH junction; SWP = sweeping; EOF = electroosmotic

flow; CEC = capillary; SPE = solid-phase extraction; LLE = liquid-liquid extraction

62 Chiral Capillary Electrophoresis in Current Pharmaceutical and Biomedical Analysis

Extraction The analytes are trapped on/in a suitable

solid/liquid-phase according to the affinities.

The distribution constant of the analytes in the

sample vs. extraction phase system determines

the extraction recovery. Separations are based

on chemical principles. The concentration

factor depends on the ratio of sample volume :

preconcentration method. The extraction selectivity can be

easily modified by the type of extractor and a specific or more

universal extraction can then be reached. Limitations. The whole analytical procedure is complex

(extraction requires conditioning, loading/sorption, washing,

labelling, if necessary, elution/desorption). In the single

capillary systems, the entire sample must pass through the

capillary, which can lead to fouling or clogging of the

separation capillary and significant decreasing of

reproducibility of the analyses when particularly problematic

samples (like biological ones) are used. A hyphenation of the

extraction techniques with electrophoresis is difficult in terms

of the instrumentation, however, SPE can be implemented to

Advantages. Microdialysis is a widely accepted sampling and

infusion technique frequently used to sample small molecules

from complex, often biological, matrices. Filtration can be

applied directly in electrophoresis avoiding a sample

collection. Both techniques can concentrate large molecules,

and purify large as well as small molecules. Limitations. Do not concentrate small molecules. In the

microdialysis, the minimum volume required for analysis

often determines the rate at which the dialysate can be

sampled. The whole analytical procedure is complex (dialysis

requires preconcentration of the analyte from dialysate). Single

column filtration systems are relatively simple but less flexible

and versatile. On the other hand, a hyphenation of the

membrane techniques with electrophoresis is more difficult in

terms of the instrumentation.

the electrophoresis easier than LLE. Membrane techniques Separations are based on physical principles,

i.e., size exclusion. In the microdialysis or

filtration, small molecules are able to diffuse

across the membrane while large molecules,

such as proteins and cell fragments, are

excluded. This is the principle of the sample

cleanup. Moreover, large molecules are

preconcentrated on the membrane in this way.

extractor volume. Advantages. An important sample purification (clean-up) and

### **3.2.1 Single column (in-capillary) electrophoretic techniques**

### **<IP_ADDRESS> Field-enhanced sample stacking**

The field-enhanced sample stacking (FESS) is easy to perform in a zone electrophoretic mode (Figure 2.2a) by simply optimizing the sample solution and the separation solution, mainly in their concentrations or electrical conductivities, to constitute different electrical field strengths between the two solutions. To perform preconcentration, the discontinuous zones having different electrical conductivities (*G*) must be constructed along the capillary axis. The analyte from a low-conductivity sample solution zone is concentrated at the boundary of a high-conductivity separation solution zone. The concentration effect is related to the conductivity ratio of these two zones according to Equation 3.1 [Simpson et al., 2008]:

$$\mathcal{C}\_{\text{stacked}} = \mathcal{C}\_{\text{t}\text{\textquotedblleft}\text{\textquotedblright}\textquotedblright}\mathcal{Y} \tag{3.1}$$

done for other stacking techniques with the zone electrophoresis separation step (see the techniques described below). Depending on the charge of chiral selector and polarity of electric field the normal or reversed stacking EKC models can be created that offer different separation selectivities. For example, depending on the charge of micelles and the polarity of the electric field, the normal (Figure 3.3a) or reversed stacking MEKC

Advanced Sample Preparation 65

(ii) Large volume sample stacking (LVSS) requires removing the sample matrix. This can be done with polarity switching where the analysed anion has higher velocity in the opposite direction than the velocity of EOF, or without polarity switching where EOF has to be suppressed as the cations migrate oppositely to EOF. It is possible to separate only cations or anions in one run and more than 100-fold concentration can be achieved.

Figure 3.1. Effect of sample matrix on LVSS. Effect of NaCl on the stacking and enantioseparation of the analytes studied. Analytes 0.01 mg/mL in 20% ACN with [NaCl] in A = 0%, B = 0.1%, C = 0.2% and D = 0.4% w/v. Injection length: 20% capillary volume.

models (Figure 3.3b) can be created.

Reprinted from ref. [Denola et al., 2007].

For schematic diagrams of the LVSS model see Figure 3.4.

where *cstacked* is the concentration of the analyte concentrated by FESS, *cinjected* is the concentration of the analyte in the sample solution injected, is the ratio of the electrophoretic velocities of the ions between the two discontinuous zones (sample zone and BGS, *1* and *2*) having different conductivities (the ratios can be written for velocities, *v1/v2*, intensities of electric field, *E1/E2*, as well as resistivities, *1/<sup>2</sup>*). From this it is obvious that the sample solution should be prepared in a low-conductivity matrix (as in other stacking modes) that is limiting in terms of application, see Figure 3.1. This technique requires suppressing EOF as EOF velocity is also proportional to the field strength and mixing of the two solutions can occur at the boundary, causing broadening of the focused zone. In practice, we often stack in the presence of EOF, albeit at lesser enrichment. In fact, at high pH, stacking occurs for anions at the rear zone boundary as opposed to the front boundary for cations at low pH. The suppression (reduction) of EOF is more important when electrokinetic injection is employed. The FESS approach is useful for charged analytes, while neutral analytes cannot be directly concentrated (with the exception of their charged complexes) [Kim J.B. & Terabe, 2003]. The FESS can be carried out in the CZE or MEKC mode where the stacking action (see below) is the same while the final separation is based on the CZE or MEKC principles [Lin C.H., 2004; Kim J.B. & Terabe, 2003]. In fact, when a chiral separation is required, the final step following stacking procedure must be principally based on the EKC (chiral MEKC, CDEKC, etc.) mechanism, see chapter 2.

Several techniques have been developed by utilizing the FESS for sample preconcentration [Lin C.H. & Kaneta, 2004; Quirino & Terabe, 2000; Chien R.L. & Burgi, 1992].

(i) Normal stacking mode is the simplest mode, it requires a rather low amount of the injected sample, the EOF must be suppressed and ca. 10-fold concentration can be easily achieved. For schematic diagrams of the normal FESS model carried out in CZE or MEKC modes see Figure 3.2 and Figure 3.3a, respectively. The schemes of FESS with CZE or MEKC separation in Figure 3.2 and Figure 3.3 can be easily modified to the chiral EKC regime implementing chiral selector into the system. Then, the final step after stacking is a chiral EKC separation (i.e., FESS-EKC). For example, the scheme of FESS with MEKC separation in Figure 3.3 can be changed from the achiral (with achiral micelles) to the chiral one (with chiral micelles). Such chiral modification can also be schematic

The field-enhanced sample stacking (FESS) is easy to perform in a zone electrophoretic mode (Figure 2.2a) by simply optimizing the sample solution and the separation solution, mainly in their concentrations or electrical conductivities, to constitute different electrical field strengths between the two solutions. To perform preconcentration, the discontinuous zones having different electrical conductivities (*G*) must be constructed along the capillary axis. The analyte from a low-conductivity sample solution zone is concentrated at the boundary of a high-conductivity separation solution zone. The concentration effect is related to the conductivity ratio of these two zones according to Equation 3.1 [Simpson et al., 2008]:

where *cstacked* is the concentration of the analyte concentrated by FESS, *cinjected* is the

*cc*

electrophoretic velocities of the ions between the two discontinuous zones (sample zone and BGS, *1* and *2*) having different conductivities (the ratios can be written for velocities, *v1/v2*,

sample solution should be prepared in a low-conductivity matrix (as in other stacking modes) that is limiting in terms of application, see Figure 3.1. This technique requires suppressing EOF as EOF velocity is also proportional to the field strength and mixing of the two solutions can occur at the boundary, causing broadening of the focused zone. In practice, we often stack in the presence of EOF, albeit at lesser enrichment. In fact, at high pH, stacking occurs for anions at the rear zone boundary as opposed to the front boundary for cations at low pH. The suppression (reduction) of EOF is more important when electrokinetic injection is employed. The FESS approach is useful for charged analytes, while neutral analytes cannot be directly concentrated (with the exception of their charged complexes) [Kim J.B. & Terabe, 2003]. The FESS can be carried out in the CZE or MEKC mode where the stacking action (see below) is the same while the final separation is based on the CZE or MEKC principles [Lin C.H., 2004; Kim J.B. & Terabe, 2003]. In fact, when a chiral separation is required, the final step following stacking procedure must be principally

Several techniques have been developed by utilizing the FESS for sample preconcentration

(i) Normal stacking mode is the simplest mode, it requires a rather low amount of the injected sample, the EOF must be suppressed and ca. 10-fold concentration can be easily achieved. For schematic diagrams of the normal FESS model carried out in CZE or MEKC modes see Figure 3.2 and Figure 3.3a, respectively. The schemes of FESS with CZE or MEKC separation in Figure 3.2 and Figure 3.3 can be easily modified to the chiral EKC regime implementing chiral selector into the system. Then, the final step after stacking is a chiral EKC separation (i.e., FESS-EKC). For example, the scheme of FESS with MEKC separation in Figure 3.3 can be changed from the achiral (with achiral micelles) to the chiral one (with chiral micelles). Such chiral modification can also be

.

> *1/*

*stacked injected c c* 3.1

is the ratio of the

*<sup>2</sup>*). From this it is obvious that the

**3.2.1 Single column (in-capillary) electrophoretic techniques**

concentration of the analyte in the sample solution injected,

based on the EKC (chiral MEKC, CDEKC, etc.) mechanism, see chapter 2.

[Lin C.H. & Kaneta, 2004; Quirino & Terabe, 2000; Chien R.L. & Burgi, 1992].

intensities of electric field, *E1/E2*, as well as resistivities,

**<IP_ADDRESS> Field-enhanced sample stacking**

done for other stacking techniques with the zone electrophoresis separation step (see the techniques described below). Depending on the charge of chiral selector and polarity of electric field the normal or reversed stacking EKC models can be created that offer different separation selectivities. For example, depending on the charge of micelles and the polarity of the electric field, the normal (Figure 3.3a) or reversed stacking MEKC models (Figure 3.3b) can be created.

(ii) Large volume sample stacking (LVSS) requires removing the sample matrix. This can be done with polarity switching where the analysed anion has higher velocity in the opposite direction than the velocity of EOF, or without polarity switching where EOF has to be suppressed as the cations migrate oppositely to EOF. It is possible to separate only cations or anions in one run and more than 100-fold concentration can be achieved. For schematic diagrams of the LVSS model see Figure 3.4.

Figure 3.1. Effect of sample matrix on LVSS. Effect of NaCl on the stacking and enantioseparation of the analytes studied. Analytes 0.01 mg/mL in 20% ACN with [NaCl] in A = 0%, B = 0.1%, C = 0.2% and D = 0.4% w/v. Injection length: 20% capillary volume. Reprinted from ref. [Denola et al., 2007].

Figure 3.2. Schematic diagrams of the normal FESS model. (A) The capillary is conditioned with a BGS (a high-conductivity buffer), the sample, prepared in a low-conductivity matrix, is then injected to a certain length, and a high positive voltage is applied; (B) focusing of the analytes occurs near the boundaries between the sample zone and the BGS because of its mobility changes; (C) stacked analytes migrate and are separated by the CZE mode. Reprinted from ref. [Lin C.H. & Kaneta, 2004].

(iii) Field-enhanced sample injection (FESI) is based on the injection of a short (usually 2- 3 mm, i.e., ca. 0.5% of the effective capillary length) water plug prior to the electrokinetical injection of the analyte. The EOF has to be reduced. Injection of a larger amount of the sample than in (ii) is possible. The injected amount is biased by the electrophoretic mobility. More than 1000-fold concentration is possible, however, injection reproducibility is influenced by depletion of the analyte in the sample solution. Selective injection is given by the charge of the sample constituents.

Figure 3.3. Schematic diagrams of stacking MEKC models. (a) A normal stacking MEKC model. (A) The sample is dissolved in a low-conductivity buffer, BGS, consisting of SDS to form the micelles; after the background and sample solution are injected, respectively, a positive voltage is applied; (B) the SDS micelles from the inlet enter the sample zone and then permit the analytes to migrate and become stacked; (C) then the SDS-analytes are separated by the MEKC mode. (b) A reversed stacking MEKC model. (A) The sample and BGS are prepared as described in Figure 3.3aA but a negative polarity is applied; (B) the EOF moves toward the inlet, the anionic analytes move toward the outlet and stack at one side of the boundary; (C) the electrophoretic current reaches approximately 95–99% of its original value, the polarity is quickly returned to positive, reversing the EOF; (D) then the SDS-analytes are separated by the MEKC mode. Reprinted from ref. [Lin C.H. & Kaneta,

Advanced Sample Preparation 67

2004].

It should be highlighted that some of the above mentioned electrophoretic on-line preconcentration approaches can be utilized simultaneously also for on-line sample purification. For example, the use of the FESI technique can eliminate potential interfering compounds from the matrix via electrokinetic injection being selective to the sample constituents according to their charge. The selectivity of the electrokinetic injection can be easily influenced by the pH of the sample solution (adjusted by an appropriate buffer if necessary) where the solute of interest has unconditionally to be ionized while the ionization of potential interfering compounds should be suppressed. The FESI can be used for the exhaustive sample injection, e.g., almost all of the ions in a sample can be injected when the volume is small. This is a real advantage to the off-line solvent extraction, evaporation to dryness and redissolving of the analyte in a small amount of dilute buffer.

Figure 3.2. Schematic diagrams of the normal FESS model. (A) The capillary is conditioned with a BGS (a high-conductivity buffer), the sample, prepared in a low-conductivity matrix, is then injected to a certain length, and a high positive voltage is applied; (B) focusing of the analytes occurs near the boundaries between the sample zone and the BGS because of its mobility changes; (C) stacked analytes migrate and are separated by the CZE mode.

(iii) Field-enhanced sample injection (FESI) is based on the injection of a short (usually 2- 3 mm, i.e., ca. 0.5% of the effective capillary length) water plug prior to the electrokinetical injection of the analyte. The EOF has to be reduced. Injection of a larger amount of the sample than in (ii) is possible. The injected amount is biased by the electrophoretic mobility. More than 1000-fold concentration is possible, however, injection reproducibility is influenced by depletion of the analyte in the sample solution.

It should be highlighted that some of the above mentioned electrophoretic on-line preconcentration approaches can be utilized simultaneously also for on-line sample purification. For example, the use of the FESI technique can eliminate potential interfering compounds from the matrix via electrokinetic injection being selective to the sample constituents according to their charge. The selectivity of the electrokinetic injection can be easily influenced by the pH of the sample solution (adjusted by an appropriate buffer if necessary) where the solute of interest has unconditionally to be ionized while the ionization of potential interfering compounds should be suppressed. The FESI can be used for the exhaustive sample injection, e.g., almost all of the ions in a sample can be injected when the volume is small. This is a real advantage to the off-line solvent extraction, evaporation to dryness and redissolving of the analyte in a small

electrokinetic

Selective injection is given by the charge of the sample constituents.

Reprinted from ref. [Lin C.H. & Kaneta, 2004].

amount of dilute buffer.

Figure 3.3. Schematic diagrams of stacking MEKC models. (a) A normal stacking MEKC model. (A) The sample is dissolved in a low-conductivity buffer, BGS, consisting of SDS to form the micelles; after the background and sample solution are injected, respectively, a positive voltage is applied; (B) the SDS micelles from the inlet enter the sample zone and then permit the analytes to migrate and become stacked; (C) then the SDS-analytes are separated by the MEKC mode. (b) A reversed stacking MEKC model. (A) The sample and BGS are prepared as described in Figure 3.3aA but a negative polarity is applied; (B) the EOF moves toward the inlet, the anionic analytes move toward the outlet and stack at one side of the boundary; (C) the electrophoretic current reaches approximately 95–99% of its original value, the polarity is quickly returned to positive, reversing the EOF; (D) then the SDS-analytes are separated by the MEKC mode. Reprinted from ref. [Lin C.H. & Kaneta, 2004].

high-conductivity BGS. A (chiral) separation by the zone electrophoresis mode then occurs. This is a simple and attractive approach for high-conductivity samples, which is growing in popularity. Another strategy introducing a sample with a higher ionic strength than the running buffer has been proposed by Landers et al. [Palmer et al., 1999]. For example, using sodium cholate as the pseudostationary phase and simply adding sodium chloride (or other ions) to the sample matrix, a reasonable enhancement in sensitivity has been achieved for a series of corticosteroids. In the first step the micelles, and not the analytes, are stacked at the boundary between the sample and buffer zone. Subsequently, the analytes migrating with the EOF are enriched in the zone with the high micelle concentration as the micelles are negatively charged and migrate in the opposite direction (Figure 3.6). Summarizing, the stacking effect is dependent on the affinity of the analytes to the micelles that were stacked before, at the boundary between the sample (with increased conductivity, e.g., by adding salt) and the buffer zone. This approach is attractive due to its robustness towards other sample constituents. Although these last two procedures have not been used for chiral analysis so far, their potential in this field is apparent. Other strategies for samples with high ionic strengths, also used in the chiral field, are based on appropriate combinations of different stacking techniques as briefly discussed, inter alia, in

Advanced Sample Preparation 69

Figure 3.5. Schematic diagrams of a pH-mediated stacking model. (A) The capillary is conditioned with a high-conductivity BGS, the cationic analytes dissolved in a lowconductivity buffer are electrokinetic injected into the capillary, and then a plug of strong acid is also electrokinetically injected; (B) a positive separation voltage is applied; (C) the strong acid titrates the sample solution to create a neutral zone causing the ions to migrate faster and become stacked; (D) the subsequent separation occurs by the CZE mode.

Reprinted from ref. [Lin C.H. & Kaneta, 2004].

section <IP_ADDRESS>.

Figure 3.4. Schematic diagrams of the LVSS model. (A) The capillary is conditioned with a BGS (a high-conductivity buffer), the sample, prepared in a low-conductivity matrix, is then injected to a certain length, and then a high negative voltage is applied (EOF is toward the inlet); (B) the anionic analytes move toward the detection end (outlet) and stack at one side of the boundary, whereas the cations and neutral species move and exit the capillary at the injection end (inlet); (C) the electrophoretic current is carefully monitored until it reaches approximately 95–99% of its original value, and the polarity is then quickly returned to positive (EOF is reversed); (D) the following separation occurs by CZE mode. Reprinted from ref. [Lin C.H. & Kaneta, 2004].

However, the FESS methods suffer generally from poor applicability to real samples. (i) For optimal precision, hydrodynamic injection is preferred since we start with the correct number of ions in the capillary. On the other hand, sample depletion and injection reproducibility could be a problem when making more than one electrokinetic injection from a vial. In a practical sense, there is a balance between the degree of enrichment and the stability of the system. This is a significant problem when performing extreme enrichment from real samples. (ii) Poor applicability to real samples is primarily because the complicated (high-conductivity) matrices increase the conductivity of the sample and reduce the efficiency of stacking. This is typical for urine or blood samples which contain salts as matrix macroconstituents. Therefore, they are usually applied in conjunction with off-line sample pretreatment. A pH-mediated FESS method was introduced by the group of Lunte and is an indirect way of changing a high-conductivity sample into a low-conductivity sample to allow field-enhanced sample stacking [Weiss et al., 2001]. Schematic diagrams are shown in Figure 3.5. In the initial step, the sample is prepared in a high-ionic strength medium and is electrokinetically injected into the capillary. Then, a plug of strong acid is electrophoretically injected and a positive separation voltage is applied. The strong acid titrates the sample solution to create a neutral zone (a high-resistance zone). Thus, a proportionally greater field will develop across the neutral zone, causing the ions to migrate faster. As a result, the analytes are stacked at the boundary between the low-conductivity zone (prepared by on-line titration of the sample solution zone, e.g., by a strong acid plug) and the at to

Figure 3.4. Schematic diagrams of the LVSS model. (A) The capillary is conditioned with a BGS (a high-conductivity buffer), the sample, prepared in a low-conductivity matrix, is then injected to a certain length, and then a high negative voltage is applied (EOF is toward the inlet); (B) the anionic analytes move toward the detection end (outlet) and stack at one side of the boundary, whereas the cations and neutral species move and exit the capillary at the injection end (inlet); (C) the electrophoretic current is carefully monitored until it reaches approximately 95–99% of its original value, and the polarity is then quickly returned to positive (EOF is reversed); (D) the

following separation occurs by CZE mode. Reprinted from ref. [Lin C.H. & Kaneta, 2004].