Separation of small peptides by electrochromatography on silica-based reversed phases and hydrophobic anion exchange phases

Peptide separations are regarded as a promising application of capillary electrochromatography (CEC) and, at the same time, a suitable model to elucidate its mixed separation mechanism when charged analytes are involved. In this paper, studies on the separation of small peptides (2-4 amino acids) on a Spherisorb octadecyl silane (ODS) phase at acidic pH and on a strong anion exchange (SAX)/C18 mixed mode phase at weakly basic pH are reported. For the ODS phase a comparison of CEC, capillary zone electrophoresis (CZE) and high-performance liquid chromatography (HPLC) under identical buffer/eluent conditions is presented. The predicted retention factors for CEC under the assumption of simple superposition of HPLC retention and CZE migration matched the measured results for the peptides that had small retention factors in HPLC. For both types of stationary phases, a variation of the acetonitrile content in the mobile phase led to a wide range of retention factors, including negative values when co-electroosmotic migration was dominant. Though both the ODS and the SAX/C18 phase offer unique advantages, the SCX/C18 phase at pH 9 provides more flexibility to alter separation selectivity for the selected peptides.