Separation of peptide diastereomers using CEC and a hydrophobic monolithic column



A neutral hydrophobic monolith prepared by radical in situ copolymerization of lauryl methacrylate and ethylene dimethacylate has been evaluated for the CEC separation of diastereomers of small peptides using acidic mobile phases containing ACN as organic modifier. Using an acidic mobile phase, the peptides migrated due to their own electrophoretic mobility. Hydrophobic interactions with the stationary phase contributed to the separation. Peptide mobility and resolution increased with increasing the ACN content. Retention times increased with the pH of the mobile phase. Peak resolution increased with buffer pH and concentration. Di- and tripeptides composed only of L-configured amino acids migrated faster than peptides containing D-amino acids. A mixture of isomeric Asp tripeptides that could not be completely resolved by either CZE or HPLC as well as the 24mer peptides tetracosactide and 16[D-Lys]-tetracosactide could also be separated by CEC on the hydrophobic monolith.