Enantiomeric separation by micellar electrokinetic chromatography

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Enantiomeric separation by micellar electrokinetic chromatography Koji Otsu ka* Osaka, Japan

Shigeru Terabe Hyogo, Japan Enantiomeric separation by micellar electrokinetic chromatography (MEKC) has been developed recently by using chiral surfactants or a cyclodextrin (CD) modified system (CD-MEKC). As chiral detergents, some amino acid derivatives, bile salts, glycosides and saponins are used with or without achiral micelles and other additives. In CD-MEKC, p- or y-CDs and sodium dodecyl sulphate are normally used.

Introduction Capillary electrophoresis (CE) or high-performance capillary electrophoresis (HPCE), which was first introduced by Mikkers et ~1. [ 11, Jorgenson and Lukacs [2], and Hjerten [3], has become a popular separation technique in various analytical fields because of its high resolving power. In principle, CE can separate ionic or charged substances only, and this was a serious limitation. The development of electrokinetic chromatography (EKC) [4], however, has solved such problems: EKC is a branch of CE and based on chromatographic separation principles using a homogeneous solution that contains an ionic “carrier” and can be applied to the analysis of neutral solutes. Although several modes are available in EKC [4], micellar EKC (MEKC) [5-71, which uses micellar solutions of ionic surfactants, has become the most popular method for separating small neutral molecules and

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a number of applications of MEK:C have been published [8-lo]. Enantiomeric separation is one of the important applications of chromatography, especially in the pharmaceutical, medical and biological fields. Recently, many studies on optical resolution by chromatographic techniques have appeared, mainly using high-performance liquid ch,romatography (HPLC). As for the application of CE, some papers on this area have also been published [ 111. In CE, chiral additives such as metal complexes of amino acids and cyclodextrins (CDs) have been successfully employed for enantiomeric separation. In particular, CDs are useful additives because the amounts of CDs required are low in CE due to the small volume of electrophoretic solutions, even enabling the use of e.xpensive CD derivatives. The high efficiency of CE also extends the applicability of the CD addition technique. It is suggested that this will be one of the major and most versatile techniques for analytical enantiomeric separation in the near future. The other technique where CDs are used involves immobilization of the CD in a polyacrylamilde gel. In this article, we will briefly review the application of MEKC to enantiomeric separation. There are two major methods used in MEKC for optical resolution: one is MEKC using chiral surfactants, and the other is based on cyclodex.trin modified MEKC (CD-MEKC). In the former, metal ions are sometimes used to form chelate complexes.

Enantiomeric separation by MEKC with chiral surfactants using metal chelate complexes Cohen et al. [ 121 first reported the chiral separation of some dansylated DL-amino acids (DnsDL-AAs) by MEKC using Cu(I1) complexes. They used N,N-didecyl-L-alanine (DDAla) as a chelat-

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ing detergent and added sodium dodecyl sulphate (SDS) to form mixed micelles. The metal attached to a chiral ligand of the chelating surfactant. The amino acid derivative, Dns-DL-AA, forms two diastereomeric metal chelate complexes with the Cu-DDAla-SDS, i.e., Cu-DDAla-SDS-Dns-DAA and Cu-DDAla-SDS-Dns-L-AA. Although mobilities of the diathe electrophoretic stereomeric complexes will be identical, the formation constants of these two complexes are sufficiently different, as to yield a difference in the migration velocities, so that, separation of the Dand L-forms of Dns-AA could be achieved. This resolution technique is similar to that reported by Zare and co-workers [ 131 using Cu complexes without micelles, which was the first report on optical resolution by CE.

Enantiomeric separation chiral surfactants

by MEKC with

[6]. Although the symmetry of the peaks was improved by the addition of urea and/or methanol [17], it was necessary to add SDS to the SDVal micellar solutions to enhance the selectivity. Six PTH-DL-AAS were successfully separated from each other and each enantiomeric pair was optically resolved with SDVal-SDS-urea-methanol solutions, as shown in Fig. 1 [ 181. MEKC with bile salts

Various bile salts are well known as natural anionic surfactants. Here, it is important to note that the bile salt micelles possess unique structures. Micellar aggregates of sodium deoxycholate (SDC), sodium taurodeoxycholate (STDC) and also sodium glycodeoxycholate in aqueous solutions are all characterised by helical structures and reversed micelle conformation [ 19,201. Since bile salts are also popular as optically active compounds, optical resolution by MEKC using bile salts has been examined as in the case using SDVal.

Chiral surfactants form chiral micelles. Most analytes are adsorbed onto the surface of the micelle or interact with the polar groups of the surfactants; therefore, surfactants with chiral polar groups can be used for chiral discrimination. Although many chiral surfactants are available, only a few of them have been found to be useful for enantiomeric separations by MEKC. MEKC wifh sodium N-dodecanoyl-L-valinate

Ham, Dobashi and co-workers [ 14,151 have used sodium N-dodecanoyl-L-valinate (SDVal), which is an anionic chiral surfactant, for the enantiomeric separation of amino acid derivatives such as N-(3,5dinitrobenzoyl) O-isopropyl esters and their analogues. For these enantiomers, chiral separation was achieved by using SDVal alone or without any additives, although resolution was poor. They also examined the use of mixed micellar solutions comprising SDVal-SDS, with or without the addition of methanol. In both cases chiral separation was achieved, but resolution was still poor because of the low efficiencies, probably due to adsorption of the analyte onto the capillary wall. We have also investigated the SDVal system for the enantiomeric resolution of phenylthiohydantoin-DL-amino acids (PTH-DL-AAS). By using SDVal alone in neutral or basic conditions, low efficiency due to poor peak shapes was observed

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Fig. 1. Enantiomeric separation of six PTH-DL-AAS by MEKC with SDVal. Corresponding AAs: 1 = Ser, 2 = Aba, 3 = Nva, 4 = Val, 5 = Trp, 6 = Nle; 0 denotes acetonitrile. Micellar solution, 50 mM SDVal-30 mM SDS-O.5 M urea (pH 9.0) containing 10% (v/v) methanol; separation capillary, 65 cm x 50 pm I.D.; effective length of the capillary, 50 cm; applied voltage, 20 kV; current, 17 PA; detection wavelength, 260 nm; temperature, ambient. (Reprinted with permission from ref. 18.)

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We were the first to report results on enantiomeric separations with bile salts in MEKC [21]. By using sodium taurocholate (STC) or STDC under acidic conditions, some Dns-DL-AAs were separated from each other and each pair was optically resolved, although a long separation time was required. Under the acidic experimental conditions used at pH 3.0, the electroosmotic velocity was substantially suppressed compared to that in neutral conditions. The absolute value of the electroosmotic velocity was smaller than that of the electrophoretic velocity of the mixed micelle, and each migrated in opposite directions. Therefore, the micelle migrated in the same direction as that of the electrophoretic migration of the micelle, that is, in the opposite direction to the electroosmosis i.e. from the negative to the positive electrodes. Some optically isomeric drugs, such as diltiazem hydrochloride, trimetoquinol hydrochloride, tetrahydropapaveroline, have also been successfully resolved by MEKC using bile salts [22,23]. An example is shown in Fig. 2, in which 50 mM STDC (pH 7.0) was employed. The determination of the optical purity of these components by the MEKC system using bile salts has also been reported [24]. As expected for such compounds,

STC and STDC gave good result under neutral or basic conditions. Cole et al. [25] have also reported on the enantiomeric separation of binaphthyl analogues by MEKC with bile salts. In this work, an SDC solution containing methanol gave good results. Although not many examples have been published on enantiomeric separation with bile salts, bile salt micelles seem to be effective for chiral recognition of analytes having rela,tively flat and rigid moieties in the molecule. MEKC with digitonin

Digitonin, which is a natural product of the glycoside of digitogenin, is popular as a chiral surfactant in the determination of cholesterol. We have tried to use the digitonin MEKC system for optical resolution [ 161. Since digitonin is electrically neutral and cannot be used alone as a carrier in MEKC, SDS was added to digitonin solutions to form chiral mixed micelles having negative charges. Under neutral conditions, no enanltiomeric resolution was achieved for any PTH-DL,-AAS because of the narrow migration-time range due to the higher electroosmotic velocity comlpared with the electrophoretic velocity of the digitonin-SDS mixed micelle. Therefore, an acidic micellar solution (pH 3.0) was used to suppress the electroosmotic flow and to extend the migration-time range. Under these acidic conditions, six PTH-DL-AAS were separated from each other and each enantiomeric pair was optically resolved, as shown in Fig. 3, although a long separation time was required. Here, the direction of migration of the digitonin-SDS micelle was towards the positive electrode, opposite to that of the electroosmotic f-low. MEKC with saponins

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Fig. 2. Chiral separation of trimetoquinol hydrochloride, tetrahydropapaveroline, five diltiazem-related compounds, 2,2’-dihydroxy-1 ,l’-dinaphthyl and 2,2,2-trifluoro-l-(9-anthryl)ethanol. Micellar solution, 50 mM STDC in 20 mM phosphate-borate buffer (pH 7.0); separation capillary, 65 cm x 50 pm I.D.; effective length, 50 cm; applied voltage, 20 kV; detection wavelength, 210 nm; temperature, ambient. (Reprinted with permission from ref. 23.)

Glycyrrhizic acid (GRA) and p-escin, which are saponins and natural chiral surfactants, have been employed in MEKC for the optical resolution of Dns- or PTH-DL-AAS [26]. As for CiRA, the addition of octyl-P-D-glucoside and SDS was essential to form a stable micelle and to obtain a moderate velocity of the micelle. Some Dns-DL-AAs were successfully resolved under neutral conditions although the separation time was still long as in the case of the digitonin-SDS system.

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Fig. 3. Enantiomeric resolution of six PTH-DL-AAS by MEKC with digitonin. Corresponding AAs: 1 = Trp, 2 = Nle, 3 = Nva, 4 = Val, 5 = Aba, 6 = Ala. Micellar solution, 25 mM digitonin-50 mM SDS (pH 3.0); separation capillary, 63 cm x 50 urn I.D.; effective length, 49 cm; applied voltage, 20 kV; detection wavelength, 260 nm; temperature, ambient. (Reprinted with permission from ref. 16.)

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case, chromatographic separation can be achieved in the CD-MEKC system because the micelle migrates at a different velocity from that of the CD or the aqueous phase. In this technique, any CD derivatives can also be used in CD-MEKC, even in cases where they are charged. It is a well known fact that CD can effect chiral recognition, thus the CD-MEKC system can also be applied to optical resolution. We have accomplished the enantiomeric resolution of some DnsDL-AAs by CD-MEKC using SDS solutions containing p- or y-CD [29]. Five Dns-DL-AAs were successfully separated and optically resolved with a 60 mM ‘y-CD-100 mM SDS solution, as shown in Fig. 4. We have used not only p- and y-CD, but also 2,6-di-O-methyl-P-CD or 2,3,6-tri-O-methylP-CD in SDS micellar solutions, for the resolution of the optical isomers of some pharmaceutical compounds, such as thiopental, barbital, pentobarbital, and phenobarbital [30]. In these cases, addition of some chiral compounds, e.g., d-camphor10-sulphonate or I-menthoxyacetic acid, could enhance the enantioselectivity as shown in Fig. 5.

As for p-escin, on the other hand, SDS was added to form mixed micelles and acidic solutions were employed. Nine PTH-DL-AAS were separated from each other and each pair was optically resolved within 35 min. Many other chiral surfactants are available from natural sources, and they are expected to be useful as separation carriers in MEKC for optical resolution-even though they are non-ionic.

Enantiomeric separation by cyclodex :trinmodified MEKC (CD-MEKC) The addition of CD to micellar solutions is effective for separating hydrophobic compounds [27,28]; we have described this technique as “cyclodextrin-modified MEKC” (CD-MEKC). Since CD will not interact with the micelle, CD in the micellar solution should behave as another phase in comparison with the micelle; it migrates at an identical velocity to that of the bulk solution, as CD itself is electrically neutral. Thus the analyte may be partitioned between three phases, the micelle, the CD and the aqueous phase. In either

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Fig. 4. Enantiomeric separation of five Dns-or_-AAs by CD-MEKC. Separation solution, 60 m/W y-CD100 mM SDS (pH 8.3); separation capillary, 57 cm x 75 ym I.D.; effective length, 50 cm; applied voltage, 12 kV. (Reprinted with permission from ref. 29.)

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system, i.e., capillary zone electrophoresis (CZE) or free solution CE. Compared with conventional HPLC, MEKC can be more easily utilised to achieve optical resolution, especially in terms of the preparation of columns and solutions, and therefore the use of MEKC is expected to increase. A promising application of enantiomeric separation by MEKC or CD-MEKC is the direct assay of chiral compounds in body fluids, such as plasma or urine. The direct injection of plasma samples in MEKC has been developed by Nakagawa et al. [33,34]. This technique will be especially valuable for the assay of compounds that tend to racemize during sample pretreatment. Fig. 5. Chiral separation by CD-MEKC with addition of sodium d-camphor-l 0-sulphonate: 1 = thiopental, 2 = pentobarbital, 3 = 2,2,2-trifluoro-l-(9-anthryl)ethanol, 4 = 2,2-dihydroxy-1 ,l’-dinaphthyl, 5 = phenobarbital, 6 = barbital. Separation solution, 50 mM SDS-30 mM y-CD-20 mM sodium d-camphor-l Osulphonate in 20 mM phosphate-borate buffer (pH 9.0); detection wavelength, 220 nm. Other conditions as in Fig. 3. (Reprinted with permission from

ref. 30.) Addition of urea and/or methanol often improves peak shape, hence resolution can be obtained as in MEKC with SDVal. As for other applications, some labelled amino acid enantiomers [3 I] and RS-chlorpheniramine 1321 were optically resolved by CD-MEKC. Optimization of the capacity factor is important, especially for enantiomeric separation by MEKC, because the separation factor is usually close to unity in enantiomeric separations. In CD-MEKC the capacity factor is maintained by changing the concentration of either the micelle or the CD. For the enantiomeric separation of ionic compounds, CE with the addition of CD will be superior to CD-MEKC, if the analyte has a high electrophoretic mobility. CD-MEKC and CE with the addition of CD are indeed complementary to each other.

Conclusion Although there have been only a few applications of MEKC to enantiomeric separations, further applications should appear in the near future because MEKC can separate neutral compounds as well as charged solutes. Thus a wider range of samples can be analyzed than in the normal CE

References F.E.P. Mikkers, EM. Everaerts and Th.P.E.M. Verheggen, J. Chr~nzmtogr:, 169 (1979) 11-20. J.W. Jorgenson and K.D. Lukacs, Arzal. Clzem., 53 (1981) 1298-1302. S. Hjertkn, J. Chromatog~, 270 (1983) l-6. S. Terabe, Trends Anal. Chern., 8 (1989) 129-134. S. Terabe, K. Otsuka, K. Ichikawa, .A. Tsuchiya and T. Ando, Ad Chem., 56 (1984) 11 l-l 13. S. Terabe, K. Otsuka and T. Ando, ,417ul. Chem., 57 (1985) 834-841. S. Terabe, K. Otsuka and T. Ando, ,4r7al. C/Tern., 61 (1989) 251-260. G.M. Janini and H.J. Issaq, J. Liq. Chronzufog~, 15 (I 992) 927-960. J. Vindevogel and P. Sandra, Introduction to MicelIur Electrokir?etic Chromutogr,aphy, Hiithig, Heidelberg, 1992. 10 W.G. Kuhr and C.A. Monnig, Anul. Chern., 64 (1992) 389RA07R. 11 K. Otsuka and S. Terabe, JCISCOReport, 33 (1991) 1-5. 12 A.S. Cohen, A. Paulus and B.L. K.arger, Chromutogruphiu, 24 (1987) 15-24. 13 E. Gassmann, J.E. Kuo and R.N. Zare, Science, 230 (1985) 813-814. 14 A. Dobashi, T. Ono, S. Hara and J. Yamaguchi,Anuf. Chenz., 6 1 (1989) 1984-1986. 15 A. Dobashi, T. Ono, S. Hara and J. Yamaguchi, J. Chronzutog~, 480 (1989) 413420. 16 K. Otsuka and S. Terabe, J. Chromutogr:, 5 15 (1990) 22 1-226. 17 K. Otsuka and S. Terabe, Electrophoresis, 11 (1990) 982-983. 18 K. Otsuka, J. Kawahara, K. Tatekawa and S. Terabe, J. Chromutogr:, 559 (199 1) 209-2 14. 19 A.R. Campanelli, S.C. De Sanctis, E. Chiessi, M. D’Alagni, E. Giglio and L. Scaramuzza, J. Plzys. C/?enl., 93 (1989) 1536-1542.

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20 R.O. Cole, M.J. Sepaniak, W.L. Hinze, J. Gorse and 21 22 23 24 25 26 27

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K. Oldiges, J. ChromurogK, 557 (199 1) 113-l 23. S. Terabe, M. Shibata and Y. Miyashita, J. Chromcrrogl:, 480 (1989) 403-411. H. Nishi, T. Fukuyama, M. Matsuo and S. Terabe, J. Microcol. Sep., 1 (1989) 234-241. H. Nishi, T. Fukuyama, M. Matsuo and S. Terabe, J. Chromutogx, 5 15 (1990) 233-243. H. Nishi, T. Fukuyama, M. Matsuo and S. Terabe, Anal. Chim. Acta, 236 (1990) 28 l-286. R.O. Cole, M.J. Sepaniak and W.L. Hinze, J. High Resolut. ChromurogK, 13 ( 1990) 579-582. Y. Ishihama and S. Terabe, J. Liq. Chromutogt:, 16 (1993) 933-944. S. Terabe, Y. Miyashita, 0. Shibata, E.R. Barnhart, L.R. Alexander, D.G. Patterson, B.L. Karger, K. Hosoya and N. Tanaka, J. ChromutogK, 5 16 (1990) 23-3 1. H. Nishi and M. Matsuo, J. Liq. Chromafogx, 14 (1991) 973-986. Y. Miyashita and S. Terabe, Applicution Dutu, High Performunce Cupillury Electrophoresis, Beckman,

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Kuwana and A. Nakamoto, Awl. Chenz., 63 (1991) 2979-298 1. 32 K. Otsuka and S. Terabe, J. Liq. ChromutogK, 16 (1993) 945-953. 33 T. Nakagawa, Y. Oda, A. Shibukawa and H. Tanaka, Chem. Phurm. Bull,, 36 (1988) 1622-1625. 34 T. Nakagawa, Y. Oda, A. Shibukawa, H. Fukuda and H. Tanaka, Chem. Phurm. Bull., 37 (1989) 707-711.

Dr. Koji Otsuka is associate professor at the Department of industrial Chemistry, Osaka Prefectural College of Technology Saiwai-Cho, Neyagawa, Osaka 572, Japan. Dr. Shigeru Terabe is Professor of Chemistry at the Department of Material Science, faculty of Science, Himeji institute of Technology, Kamigori, Hyogo 678- 12, Japan.

gas chromatography

Wilfried A. Kijnig Hamburg, Germany The introduction of hydrophobic cyclodextrin derivatives as chiral selectors in enantioselective gas chromatography has resulted in the wide use of this analytical method in solving stereochemical problems. The extraordinary separation efficiency of capillary gas chromatography enables the investigation of chiral constituents in complex mixtures such as essential oils, pheromones or environmental samples. Enantioselective gas chromatography features high precision in the detection of minute enantiomeric impurities and detection limits are independent of functional groups.

Introduction Enantioselectivity in gas chromatography (GC) was first described by Gil-Av. He and his associ-

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ates were able to demonstrate the complete resolution of volatile amino acid derivatives using non-racemic amino acid and peptide derivatives as chiral stationary phases (CSPs) [ 11. Enantiomeric discrimination was explained by transient formation of diastereomeric association complexes between the chiral substrates and the CSP For diamide phases, as originally applied by Gil-Av, hydrogen bonding association was assumed to be the main force of molecular interaction. This was convincingly demonstrated by Feibush et al. [2]. Although the technique was primarily limited to the separation of amino acids, many researchers involved in stereochemical problems became interested in enantioselective GC and several research groups attempted to extend the applicability of this techniques to the analysis of other chiral compounds. The thermal stability of chiral diamide phases could be substantially improved by introducing the chiral selector into a polymer. Thus with L-valine tert.-butylamide covalently bound to a polysiloxane matrix (“Chirasil-L-val”) Frank et al. [3] succeeded for the first time to resolve all protein amino acids in a single chroma-

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