Anal. Chem. 1995,67,19-25
Capillary Zone Electrophoresis Study of Naphthylethylcarbamoylated PGyclodextrins KyunWHyun Gahm and Apryll M. Stalcup*
Department of Chemistty, University of Hawaii at Manoa, Honolulu, Hawaii, 96822
Regiospecilhlly monosubstituted 1-(1-naphthy1)ethylcarbamoylated /?-cyclodextrins (NEC-/I-CDs) were successfully employed as chiral additives in capillary zone electrophoresis (CZE) to achieve chiral separation of N-(3,5-~Iinitrobemyl)phenyIglycine (3,5-DNB-PG),phenylalanine (3,5-DNB-PA),and homophenylalanine (3,5DNB-HPA). The enantioselectivity of the various sitesubstituted NEC-/?-CDsin CZE was compared with that of native /3-CD. Complexation constants of the three 3,5DNB amino acids with /?-CDwere determined from the CZE results: ~,~-DNB-L-HPA, 473 f 9 M-l; 3,5-DNBD-HPA,460 f 10 M-l; 3,5-DNB-bPA,260 f 4 M-l; 3,5DNB-D-PA,161 f 3 M-l; and ~,~-DNB-D,L-PG, 43 f 4 M-1.
The separation of chiral compounds is currently one of the most active areas in analytical science because chiralityis a leading issue in pharmacology. A recent survey indicated that 88%of all synthetic chiral drugs were marketed as the racemic mixtures.' Because many stereoisomers show different bioactivities, chiral separations are fundamental to research on the relationship between drug chirality and its efficacy.2-6 There are several ways to achieve chiral separations, including crystallization and various forms of ~hromatography.~"The known ability of cyclodextrins (CDs) to form inclusion complexes with various guest molecules led to successful applications of CDs to chromat~graphy.~J~ CD-based chiral stationary phases (CDCSPs) have several advantages. Because CDs are readily available in three sizes (a-, ,!%, and y-CD), a variety of different size enantiomers are separable by selecting the appropriate CD. Additional flexibility of CD-CSPs can be easily obtained through the derivatization of the native CD-CSPs. Derivatization of the CDs alters their physicochemical characteristics as well as their complexation mechanisms. Among several derivatized CD-CSPs, the naphthylethyl carbamate B-CD chiral stationary phases (NEC-p-CD-CSPs) have been used both in reversed and normal phase modes because of (1) Chem. Eng. News 1990,March 19, 40. (2) M e n s , E. J. Anal. Proc. 1992.29,232-234. (3) Drayer, D. E. Clin. Pharmacol. mer. 1986,40,125-133. (4) Coutts, R T.; G. B. Baker, G. B. Chirality 1989,1, 99-120. (5) Hartenstein, J.; Wagner, B. In Cardiovascular Drugs;Bristol, J. A, Ed.; John Wiley & Sons, Inc.: New York, 1986 pp 305-415. (6) Lalonde, R L.; Tenero, D. M.; Burlew, B. S.; Herring, V. L.; Bottorff, M. B. Clin. Pharmacol. They. 1990,47,447-455. (7) Zief, M., Crane, L. J. Eds. Chromatographic Chiral Separations; Marcel Dekker: New York, 1988. (8) Krustulovic,A M. Chiral Separations by HPLC; Ellis Horwood Chichester, UK, 1989. (9) Bender, M. L.; Komiyama, M. Cyclodatrin Chemisty; Springer: Berlin, 1977. (10) Hinze, W. L. Sep. PUriJ Methods 1981,10,159-237. 0003-2700/95/0367-0019$9.00/0 0 1994 American Chemical Society
their stability and resolution capability in both modes."-13 Recently, a systematic study on the resolution of several derivatized amino acids (AAs) on different CD-CSPs in the reversed phase mode indicated that 3,5DNB-AAs were resolved better on the or (S)-NEC;O-CD column than on the native p-CD ~ 0 l u m n . lThe ~ proposed mechanism of chiral recognition of the NEC-p-CD-CSPsinvolved inclusion complexationwith p-CD, n-n interaction with the naphthyl moiety, interactions with residual chiral secondary alcohols at the CD mouth, and steric hinderance. The elution order for (3,5dinitrobenzoyl)phenylglycine(3,5DNB PG) and 3,5DNRtyrosine was reversed by changing the chirality of the pendant NEC groups on NEGp-CD-CSPs.13 In contrast, the elution order for 3,5DNB-phenylalanine (3,sDNB-PA) and -homophenylalanine (3,sDNB-HPA) was independent of the chirality of the NEC groups. Further, the elution order of 3 , s DNB-HPA from (9-NEC-p-CD-CSPwas opposite that obtained from the (S)-NEC-y-CD-CSP.15Clearly, the CD makes some contribution to enantioselectivity. Several issues about these CSPs remain unanswered: the sites of substitution, the orientation of the substituent and its role in chiral recognition under different mobile phase conditions, and the integrity of the toroidal configuration of the CD upon derivatization. Given that the reported average degree of substitution varies from 3 to 812J4J5and the substitution sites are not known, the chiral recognition interaction mechanism of these phases is not clear. Capillary zone electrophoresis (CZE), a relatively recent addition to the arsenal of analytical techniques,16J7has been demonstrated as a powerful chiral separation method. Its high resolution capability and lower sample loading relative to HPLC make it ideal for the separation of minute amounts of components in complex biological mixtures.'*J9 CZE separations have been reported for peptides,20AAs,2l proteins,22DNAB catecholamines,24-B and various organic molecules.
(a-
(11) Armstrong, D. W.; Hilton, M. L.; Coffin, L LCCC 1991,9,646-652. (12) Hilton, M. L.; Chang, S. C.; Gasper, M. P.; Pawlowska, M.; Armstrong, D. W.; Stalcup, A M. J Liq. Chromatogr. 1993,16, 127-147. (13) Stalcup, A M.; Chang, S. C.; Armstrong, D. W.J Chromatogr. 1991,540, 113-128. (14) Lee, S. H.; Berthod, A; Armstrong, D. W. ]. Chromatogr. 1992,603,8393. (15) Nakatsu, C. N.; Stalcup, A M . J Liq. Chromatogr. 1993,16, 209-223. (16) Kuhr, W. G. Anal. Chem. 1990,62,403R-414R (17) Karger, B. L. Am. Lab. 1993,Oct, 23-27. (18) Jorgenson, J. W.; Lukacs, K D. Anal. Chem. 1981,53, 1298-1302. (19) Cohen, A S.; Paulus, A; Karger, B. L. Chromafographia 1987,24,15-24. (20) Mosely, M. A; Deterding, L.J.; Tomer, K B.; Jorgenson, J. W. Anal. Chem. 1991,63, 109-114. (21) Cheng, Y.F.;Dovichi, N. J. Science 1988,242,562-564. (22) Smith, R D.; Loo, J. A; Barinaga, C. J.; Edmonds, C. G.; Udseth, H. R ]. Chromatogr. 1989,480,211-232. (23) Kasper, T.J.; Melera, M.; Gozel, P.; Brownlee, R G.]. Chromatogr. 1988, 458,303-312.
Analytical Chemistry, Vol. 67,No. 1, January 1, 1995 19
In CZE, the addition of a chiral additive to a background electrolyte (BGE) is a common method for the resolution of chiral compounds. The addition of chiral selectors to the BGE has been shown to affect the migration rates of analytes. The magnitude of this change in migration of the analytes induced by the addition of a chiral additive is indicative of the strength of the analytel selector interaction and has led to the calculation of complexation constant^.^^-^^ Native and derivatized CDs, with proven chiral resolution capabilities in HPLC, have also been used for chiral resolution in CZE.3°-34 Among many possible derivatized CDs, m e t h ~ l a t e d ~ * -and ~ ~ hydroxypropyl
