Role of helix formation for the retention of peptides in reversed-phase high-performance liquid chromatography

Abstract

In order to get insight into the role of helix formation for retention in reversed-phase HPLC, we have studied the isocratic retention behavior of amphipathic and non-amphipathic potentially helical model peptides. Plots of the logarithmic capacity factor in absence of organic solvent (ln k 0) versus 1/ T were used to derive the enthalpy, Δ H 0, the free energy, Δ G 0, the entropy of interaction, Δ S 0, and the heat capacity change, Δ C p. Retention of all peptides was accompanied by negative Δ C p revealing that hydrophobic interactions play a large role independent of peptide sequence and secondary structure. Δ H 0 was negative for the amphipathic analogs and was attributed mainly to helix formation of these peptides upon interaction with the stationary phase. In contrast, Δ H 0 was considerably less exothermic or even endothermic for the non-amphipathic analogs. The differences in helix formation between the individual analogs were quantified on the basis of thermodynamic data of helix formation previously derived for peptides in a hydrophobic environment. Correlation of the helicity with the free energy of stationary phase interaction revealed that helix formation accounts for ∼40–70% of Δ G 0, and is hence in addition to the hydrophobic effect a major driving force of retention.