Evaluation of a Vancomycin Chiral Stationary Phase in Packed Capillary Supercritical Fluid Chromatography Jurgen Donnecke, 1, * Lars A. Svensson, 2 Olle Gyllenhaal, 1 Karl-Erik Karlsson, 1 ¨ ¨ Anders Karlsson, 1 Jorgen Vessman 1 ¨ 1 Di ision of Analytical Chemistry, Astra Hassle AB, S-431 83 Molndal, Sweden ¨ ¨ 2 Department of Analytical Pharmaceutical Chemistry, Biomedical Center, Box 574, S-751 23 Uppsala, Sweden Received 18 September 1998; accepted 19 February 1999 Abstract: Vancomycin immobilized on an aldehyde functionalized silica support in packed capillary columns has been used as a chiral stationary phase in supercrit- ical fluid chromatography under subcritical conditions. The use of carbon dioxide with methanol as the mobile phase modifier is shown to offer an extended scope of chiral separations compared with reversed-phase liquid chromatography, especially for basic analytes. It could be demonstrated that the chiral recognition of van- comycin for different analytes is based on several kinds of interactions. Large differences in enantioselectivity in a homologous series of local anesthetic com- pounds indicate that the fit of a host guest interaction between an analyte and vancomycin could be very precise. Nonlinear van’t Hoff plots implied that the enantiomers of metoprolol and the modifiers evaporated from their adsorption sites at different temperatures. 1999 John Wiley & Sons, Inc. J Micro Sep 11: 521 533, 1999 Key words: temperature effect; structure analogues; -adrenergic blocking agents; amine and methanol modified CO 2 INTRODUCTION The macrocyclic antibiotic vancomycin together with thiostreptone and rifamycin B were first intro- duced by Armstrong et al. 1 as chiral stationary Ž . phases CSPs in liquid chromatography. It was Ž . shown that the vancomycin CSP Chirobiotic V could be used in reversed phase, straight-phase, or polar organic-phase conditions resolving enan- tiomers of different substance classes 1,2 . Chirobi- otic V was also applied under supercritical fluid Ž . chromatography SFC conditions and compared with different types of chiral stationary phases 3. Another approach to form a vancomycin CSP was the on-column immobilization of the selector using the amino groups in a reductive amination of an aldehyde functionalized silica support in packed cap- illaries 4 . These two types of vancomycin CSPs have different selectivities depending on how they are attached to the silica. Mainly acids were resolved *Present address: Asta Medica AG, Weismullerstraße ¨ 45, DE-60314 Frankfurt a.M., Germany Correspondence to: L. A. Svensson, Division of Analytical Chemistry, Astra Hassle AB, S-431 83 Molndan, Sweden ¨ ¨ by the on-column immobilized vancomycin as was also observed in capillary electrophoresis 5 and thin-layer chromatography 6 where the selector was used as a chiral additive. However, for basic compounds low enantioselectivity was obtained in many cases. In contrast to these results, Chirobiotic V was applied to resolve basic analytes 1 . The on-column immobilization of vancomycin involves only minor modification of the amino groups. This will merely have a slight influence on the pK values a of all protolytic groups. In Chirobiotic V vancomycin is immobilized by joining on average three linkers Ž per vancomycin probably via a 3-isocyanatopropyl . silane , thereby shielding protolytic amino or aliphatic and aromatic hydroxyl residues as urea or carbamate groups, respectively 1,2 . The larger al- teration of vancomycin in Chirobiotic V in compari- son to the native form could be the reason for its different behavior in chromatography. In the present work, the merits of the on-col- umn immobilized vancomycin in packed capillary SFC are studied. The dependence on-column effi- ciency of the combination of lower viscosity and faster diffusion of analytes in supercritical or sub- critical phases is emphasized. Ž. Ž . J. Microcolumn Separations , 11 7 521 533 1999 1999 John Wiley & Sons, Inc. 521 CCC 1040-768599070521-13