Amino acids were derivatized with sulfur-containing reagents in alkaline medium and enantioresolved by HPLC on a teicoplanin chiral stationary phase. Much better resolution was achieved using methanol-based mobile phase compared with elution with acetonitrile-based solvent mixture. The value of selectivity factor for many derivatives examined in the study can be easily several tenths of 100 or even exceed 100 in some cases. This magnitude of resolution is suitable for the preparative-scale application of isolating alpha-amino acid enantiomers in the derivatized form using a short column. The resolution is believed to be a result of the hydrogen bonding and steric hindrance by the nitrogen and sulfur atoms from the isothiocyanatyl fragment of the reagent, respectively, and is enhanced as the fragment is structurally phenylated (e.g. 2,4-difluorophenyl isothiocyanate and others examined in this study). The enhancement is even more significant if the aromatic moiety of the reagent becomes more acidic through halogenation with chlorine or fluorine for the stronger pi-pi interaction. However, this type of enhancement is offset to some extent and sometimes obscured by a chlorinated reagent that is relatively large in size as compared with the fluorinated one. Judging from the chromatographic data and the elution profile, the mechanisms leading to the resolutions are believed to be different under the elution of polar-organic mobile phases (i.e. methanol- and acetonitrile-based mobile phases).
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