COMPARATIVE STUDIES OF ENANTIOSEPARATION OF SOME AMINO ACIDS AND N-PROTECTED AMINO ACIDS IN NORMAL PHASE HPLC AND ANALYSIS OF UNUSUAL PEAK SHAPE

Abstract

Comparative studies of enantioseparation of some amino acids and N-protected amino acids were performed using a Chiralpak IA column in normal phases HPLC. Better resolution results were obtained for N-protected amino acids compared with the studied amino acids at the same conditions. The effects of type and concentration of alcohol modifier in mobile phase, concentration of acidic additive trifluoroacetic acid, and column temperature on enantioseparation were also investigated. The experimental results showed that the separation factor and resolution factor were both high when ethanol was used as alcohol modifier in mobile phase. When the mobile phase contained 35% (v/v) ethanol, the separation factor of all the enantiomers was the highest. A trace amount of trifluoroacetic acid promotes enantioseparation regardless of the nature of the chiral solutes. The retention time of investigated enantiomers decreased with the increase of column temperature. The chromatographic peak shape of enantiomers separation was almost symmetrical except for the resolution of phenylalanine and 3-(3,4-dihydroxyphenyl)alanine, in which chromatographic peak of the first eluted enantiomer was unusual lower and wider than that of the second eluted enantiomer. The mechanism of the unusual peak shape from enantioseparation of phenylalanine and 3-(3,4-dihydroxyphenyl)alanine was analyzed.