Chiral Discrimination of Aromatic Amino Acids by Capillary Electrophoresis in (+)- and (-)-(18-Crown-6)-2,3,11,12-tetracarboxylic Acid Selector Modes

Abstract

Optically active (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18C6H 4 ) ( Figure 1) has been successfully employed as a chiral selector in enantioseparations of diverse amino acids and chiral primary amines in capillary electrophoresis (CE). 1-7The host-guest complexation mechanisms for the chiral recognition of crown ethers toward chiral amines have been well established. 2,6 The accurate chiral discrimination of each separated analyte has become an important task for their optical purity control and stereoselective pharmacokinetic studies in chiral drug development. The migration order of separated enantiomers is mainly determined by co-electrophoresis with enantiomerically pure standards and matching its migration time or relative migration time (RMT) with that of the reference. However, crosschecking two RMT sets measured with two chiral selectors of different enantioselectivities could enhance confidence in the identification of CE peaks and the chiral discrimination as well. Based on this concept, simultaneous chiral discrimination of 15 racemic aromatic amino acids was achieved with chiral CE in neutral and charged cyclodextrin modes in our previous report. 8 The surest way of the accurate chiral discrimination is the reversal of enantiomer migration order (EMO).9-16 It is especially desirable when peak tailing, fronting or overlapping is observed. Among the diverse ways of EMO reversal developed, suppressing or reversing the direction of electroosmotic flow has been most widely used. 9,[12][13][14][15][16] In our recent report, simultaneous EMO reversal of nine chiral profens was achieved in the normal polarity and reversed polarity modes. 16 The most certain and easy method of EMO reversal might be, however, the use of two chiral selectors with the opposite chiral recognition ability, like (+)-18C6H 4 and (−)-18C6H 4 when employing crown ether-modified CE systems. However, no attempt to exploit the crosschecking each EMO measured in these two selector modes has been made in chiral CE of amino acids to date. The present study was undertaken to investigate EMO reversal by crown ether modified-CE system in the two selector modes with (+)-18C6H 4 and (−)-18C6H 4 for the chiral discrimination of nine aromatic amino acids. When (+)-18C6H 4 was added at 5 mM to 20 mM Triscitric acid buffer (pH 2.50), each enantiomeric pairs of nine aromatic amino acids studied were baseline-resolved with similar enantioselectivity and resolution factor except for 3-hydroxyphenylglycine (Table 1). 3-Hydroxyphenylglycine showed much stronger interaction with the selector than other analytes, thereby yielding a very large separation factor (1.684) and resolution factor (21.99). In good agreement with the previous reports, 1,2,4 each D-enantiomer migrated slower than the corresponding L-isomer, indicating that the D-isomers were bound more strongly with (+)-18C6H 4 in CE. The (R)-baclofen migrated ahead of the (S)-enantiomer. When (+)-18C6H 4 was replaced in the same buffer condition with its antipode, (−)-18C6H 4 (Figure 1), ...