Hydrophilic interaction/cation-exchange chromatography for separation of amphipathic alpha-helical peptides

Mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEX) is a novel high-performance technique which has excellent potential for peptide separations. Separations by HILIC/CEX are carried out by subjecting peptides to linear increasing salt gradients in the presence of high levels of acetonitrile, which promotes hydrophilic interactions overlaid on ionic interactions with the cation-exchange matrix. In the present study, HILIC/CEX has been applied to the separation of synthetic amphipathic alpha-helical peptides, varying in amphipathicity and the nature of side-chain substitutions in the centre of the hydrophobic or hydrophilic face. Observation of the retention behaviour of these amphipathic alpha-helical peptide analogues during HILIC/CEX and reversed-phase chromatography (RPLC) enabled the establishment of general rules concerning the applicability of these complementary HPLC techniques to peptides displaying a secondary structural motif of common occurrence.