Optical resolution of amino acid enantiomers by high-performance liquid chromatography

Abstract

Simultaneous analysis of common protein amino acid enentiomers was achieved by a chiral derivatization and a chiral mobile phase method. In the chiral derivatization method, 2,3,4,6-tetra-O-acetyl-β- d-glucopyranosyl isothiocyanate was chosen as the reagent for the derivatization of enantiomeric amino acids to give diastereomeric thiourea derivatives. These derivatives were efficiently separated on a octadecylsilyl silica gel column by gradient elution and were detected by their absorbance at 250 nm. Derivatives of all common protein amino acid racemates, except cysteine, were resolved within about 2 h, although a few peaks were slightly overlapped. In the chiral mobile phase method, the optically active binary copper complex with N( ptoluenesulphonyl)- d-phenylglycine was used as a chiral additive in the mobile phase for the ligand-exchange chromatographic resolution of underivatized d, l-amino acids. Simultaneous resolution of common protein amino acid enantiomers on a resversed phase was achieved by a column-switching technique, utilizing two ODS columns of different lengths, and by gradient elution with acetonitrile. The column eluate was monitored fluorometrically after reaction with o-phthalaldehyde.