Temperature selectivity effects in reversed-phase liquid chromatography due to conformation differences between helical and non-helical peptides

In order to characterize the effect of temperature on the retention behaviour and selectivity of separation of polypeptides and proteins in reversed-phase high-performance liquid chromatography (RP-HPLC), the chromatographic properties of four series of peptides, with different peptide conformations, have been studied as a function of temperature (5–80 °C). The secondary structure of model peptides was based on either the amphipathic α-helical peptide sequence Ac-EAEKAAKEXd/lEKAAKEAEK-amide, (position X being in the centre of the hydrophobic face of the α-helix), or the random coil peptide sequence Ac-Xd/lLGAKGAGVG-amide, where position X is substituted by the 19 l- or d-amino acids and glycine. We have shown that the helical peptide analogues exhibited a greater effect of varying temperature on elution behaviour compared to the random coil peptide analogues, due to the unfolding of α-helical structure with the increase of temperature during RP-HPLC. In addition, temperature generally produced different effects on the separations of peptides with different l- or d-amino acid substitutions within the groups of helical or non-helical peptides. The results demonstrate that variations in temperature can be used to effect significant changes in selectivity among the peptide analogues despite their very high degree of sequence homology. Our results also suggest that a temperature-based approach to RP-HPLC can be used to distinguish varying amino acid substitutions at the same site of the peptide sequence. We believe that the peptide mixtures presented here provide a good model for studying temperature effects on selectivity due to conformational differences of peptides, both for the rational development of peptide separation optimization protocols and a probe to distinguish between peptide conformations.