Micro-HPLC and standard-size HPLC for the separation of peptide stereoisomers employing an ion-exchange principle

Standard-size (4 mm ID) and micro-HPLC columns (0.5 mm ID) packed with a quinine-based ion-exchange type chiral stationary phase are comparatively evaluated for the separation of peptide enantiomers with up to six amino acid residues. The results show that downscaling the separation system in order to gain the advantages of miniaturized HPLC is possible without sacrificing separation power. Further, five different N-terminal protections (3,5-dinitrobenzoyl, 2,4-dinitrophenyl, 3,5-dinitrobenzyloxycarbonyl, carbazole-9-carbonyl, and 9-fluorenylmethoxycarbonyl) of the analytes are investigated regarding their effect on enantioselectivity. A comparison between a hydro-organic and a polar-organic mobile phase is also reported. The enantiomers of the peptides containing one to four amino acid residues were baseline resolved, while for the penta- and hexamers only partial separations were possible. In addition, all four stereoisomers of alanylalanine could be baseline separated.