Enantiomeric and diastereomeric separation of di- and tripeptides by capillary electrophoresis

Chiral resolution of a series of di- and tripeptides has been attempted by indirect and direct methods. For the indirect method, the analytes were derivatized with a reagent of high chiral purity, (-)-1-(9-fluorenyl)ethyl chloroformate, and the diastereomers produced were subsequently separated in a micellar electrokinetic capillary chromatography (MEKC) system. For the direct method, the analytes were derivatized with 9-fluorenylmethyl chloroformate and separated by MEKC using gamma-cyclodextrin (CD) or beta-CD as chiral selectors. After optimization, 9 peptides were separated when using the indirect method. Using the direct method, 9 peptides were separated with gamma-CD as the chiral selector, and two other peptides were separated with beta-CD. Complete separation of two peptides having two chiral centers, Leu-Ala and Leu-Leu, was obtained by the indirect method and Ala-Leu was separated by the direct method. The benefits of using organic modifiers to enhance enantioselectivity and separation efficiencies in the direct and indirect method were demonstrated and discussed. High separation efficiencies were obtained for both methods, plate numbers were in the range of 10(6) plates/m. Separation times were ca. 10 min using the indirect method and ca. 17 min using the direct method. It seems that the two methods are complementary.