The present study sets out to extend the utility of reversed-phase liquid chromatography (RP-HPLC) by demonstrating its ability to monitor dimerization and unfolding of de novo designed synthetic amphipathic alpha-helical peptides on stationary phases of varying hydrophobicity. Thus, we have compared the effect of temperature (5-80 degrees C) on the RP-HPLC (C8 or cyano columns) elution behaviour of mixtures of peptides encompassing amphipathic alpha-helical structure, amphipathic alpha-helical structure with L- or D-substitutions or non-amphipathic alpha-helical structure. By comparing the retention behaviour of the helical peptides to a peptide of negligible secondary structure (a random coil), we rationalize that "temperature profiling" by RP-HPLC can monitor association of peptide molecules, either through oligomerization or aggregation, or monitor unfolding of alpha-helical peptides with increasing temperature. We believe that the conformation-dependent response of peptides to RP-HPLC under changing temperature has implications both for general analysis and purification of peptides but also for the de novo design of peptides and proteins.