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

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 k0) versus 1/T were used to derive the enthalpy, ΔH0, the free energy, ΔG0, the entropy of interaction, ΔS0, and the heat capacity change, ΔCp. Retention of all peptides was accompanied by negative ΔCp revealing that hydrophobic interactions play a large role independent of peptide sequence and secondary structure. ΔH0 was negative for the amphipathic analogs and was attributed mainly to helix formation of these peptides upon interaction with the stationary phase. In contrast, ΔH0 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 ΔG0, and is hence in addition to the hydrophobic effect a major driving force of retention.