To better study collagen-mediated cellular and enzymatic activities, a generally applicable solid-phase methodology has been developed by which aligned triple-helical peptides (designated THPs or "minicollagens") ranging from 79 to 124 residues can be assembled. Reversed-phase HPLC is typically the purification method of choice following chemical synthesis of small proteins of this size, as well as one of the analytical techniques used to verify product purity. We have thus compared the effects of different stationary phases (C18, C4, or diphenyl), organic modifiers (acetonitrile or isopropanol), support pore sizes (120 angstroms, 300 angstroms, or nonporous), and counterions for the reversed-phase HPLC analysis of THPs. Large pore C18 or C4 reversed-phase HPLC gave broad peaks, resulting in poor resolution of the desired THP from synthetic impurities. Broad peaks were presumably due to conformational instability of THPs to reversed-phase conditions and subsequent slow cis-trans isomerization of the peptide bonds. Peak sharpness was improved greatly by use large-pore diphenyl reversed-phase HPCL. We found that THPs can be best resolved from synthetic impurities by diphenyl or non-porous C18 reversed-phase HPLC using water-acetonitrile gradients. These results most likely reflect conditions which maintain the native conformation of collagen-like triple-helices.