ENANTIOMERIC RESOLUTION OF UNDERIVATIZED SMALL PEPTIDES BY HPLC WITH A CHIRAL CROWN-ETHER STATIONARY PHASE

Factors influencing the stereoisomeric resolution of underivatized dipeptides and a representative tripeptide on Crownpak (CR) columns have been investigated. The elution order and relative retention suggest that a combination of chiral, steric, and hydrophobic interactions effects the extent of chiral recognition and the retention achieved during separations. Some dipeptides whose amine terminus is located three atoms from the asymmetric center (such as dipeptides of D,L-glycine) were resolved, but the elution order was the opposite of that expected for the type of Crownpak column used (CR(+)). Peptides containing hydrophobic substituents were strongly retained, but their retention times could be significantly reduced, and detectability improved, by use of gradient elution. Analysis of a commercial sample of D,L-leucine-D,L-alanine revealed the stereoisomers to be present in an unexpected quantitative ratio and demonstrated the utility of these separations for quality assurance and quantitative analyses.