Use of sodium perchlorate at low pH for peptide separations by reversed-phase liquid chromatography. Influence of perchlorate ion on apparent hydrophilicity of positively charged amino acid side-chains.

The reversed-phase liquid chromatography (RPLC) behavior of synthetic model peptides containing positively charged amino acid residues was studied in the presence or absence of 100 mM sodium perchlorate in order to determine the effect on apparent side-chain hydrophilicity of a charged residue at low pH. The peptides used in this study were either non-helical peptides or amphipathic alpha-helical peptides, where the effect of the negatively charged perchlorate ion on a charged residue in either the hydrophobic face or hydrophilic face of the helix was monitored. We have shown that the addition of 100 mM perchlorate to RPLC separations of positively charged peptides performed in a 20 mM aqueous phosphoric acid-acetonitrile system resulted in an increase in retention time of a peptide relative to the same peptide in the absence of perchlorate. This effect occurred independent of conformation, i.e., whether comparing the effect of positively charged residue substitutions in the hydrophobic or hydrophilic face of an amphipathic alpha-helix or in a peptide with negligible secondary structure. From these results, suggesting that positively charged side-chain hydrophilicity is decreased by ion-pairing with the perchlorate ion, we have shown practical examples where mixtures of non-helical and amphipathic alpha-helical peptides showed enhanced resolution in the presence of perchlorate at pH 2, compared to in its absence. In addition, it was shown that an aqueous phosphoric acid-perchlorate-acetonitrile mobile phase may show markedly different selectivity for peptide separations at low pH compared to the more traditional aqueous trifluoroacetic acid-acetonitrile system.