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 r eversed-phase HPLC, we have studied the isocratic retention behavior of amp hipathic and non-amphipathic potentially helical model peptides. Plots of t he logarithmic capacity factor in absence of organic solvent (ln k(0)) vers us 1/T were used to derive the enthalpy, DeltaH(0), the free energy, DeltaG (0), the entropy of interaction, DeltaS(0), and the heat capacity change, D eltaC(p). Retention of all peptides was accompanied by negative DeltaC(p) r evealing that hydrophobic interactions play a large role independent of pep tide sequence and secondary structure. DeltaH(0) was negative for the amphi pathic analogs and was attributed mainly to helix formation of these peptid es upon interaction with the stationary phase. In contrast, DeltaH(0) was c onsiderably less exothermic or even endothermic for the non-amphipathic ana logs. The differences in helix formation between the individual analogs wer e quantified on the basis of thermodynamic data of helix formation previous ly derived for peptides in a hydrophobic environment. Correlation of the he licity with the free energy of stationary phase interaction revealed that h elix formation accounts for similar to 40-70% of DeltaG(0), and is hence in addition to the hydrophobic effect a major driving force of retention. (C) 2001 Elsevier Science B.V. All rights reserved.