Strong cation-exchange high-performance liquid chromatography of peptides. Effect of non-specific hydrophobic...

Strong cation-exchange chromatography (strong CEX) is probably the most useful mode of high-performance ion-exchange chromatography (IEC) for peptide separations. Although the hydrophobic character of high-performance ion-exchange packings, often giving rise to mixed-mode contributions to solute separations, has long been recognized, a systematic approach to examining the effect and magnitude of the hydrophobicity of these packings during IEC of peptides has so far been lacking. In the present study, we report the synthesis of three series of positively charged peptide polymers which vary significantly in overall hydrophobicity and polypeptide chain length (5-50 amino acid residues): Ac-(Gly-Lys-Gly-Leu-Gly)n-amide, Ac-(Leu-Gly-Leu-Lys-Ala)n-amide and Ac-(Leu-Gly-Leu-Lys-Leu)n-amide (n = 1, 2, 4 6, 8, 10). We have examined non-specific hydrophobic interactions of these peptides with both silica-and polymer-based ion-exchange packings, demonstrating how these interactions are overcome by the addition of acetonitrile to the mobile phase. It was also shown that removal of non-specific hydrophobic interactions may be necessary just to elute peptides from the ion-exchange matrix. In addition, from the observed retention times of these three peptide polymer series and other peptides which vary substantially in charge density, net charge, polypeptide chain length and hydrophobicity, we have established a simple approach to linearization and, thus, prediction of peptide retention behaviour in CEX.