Ion‐interaction CZE: The presence of high concentrations of ion‐pairing reagents demonstrates the complex mechanisms involved in peptide separations

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Abstract
We have furthered our understanding of the separative mechanism of a novel CE approach, termed ion‐interaction CZE (II‐CZE), developed in our laboratory for the resolution of mixtures of cationic peptides. Thus, II‐CZE and RP‐HPLC were applied to the separation of peptides differing by a single amino acid substitution in 10‐ and 12‐residue synthetic model peptide sequences. Substitutions differed by a wide range of properties or side‐chain type (e.g., alkyl side‐chains, polar side‐chains, etc.) at the substitution site. When carried out in high concentrations (400 mM) of pentafluoropropionic acid (PFPA), II‐CZE separated peptides in order of increasing hydrophobicity when the substituted side‐chains were of a similar type; when II‐CZE was applied to the mixtures of peptides with substitutions of side‐chains that differed in the type of functional group, there was no longer a correlation of electrophoretic mobility in II‐CZE with relative peptide hydrophobicity, suggesting that a third factor is involved in the separative mechanism beyond charge and hydrophobicity. Interestingly, the hydrophobic PFPA− anion is best for separating peptides that differ in hydrophobicity with hydrophobic side‐chains but high concentrations of the hydrophilic H2PO4− anion are best when separating peptides that differ in polar side‐chains relative to hydrophobic side‐chains. We speculate that differential hydration/dehydration properties of various side‐chains in the peptide and the hydration/dehydration properties of the hydrophilic/hydrophobic anions as well as the electrostatic attractions between the peptide and the anions in solution all play a critical role in these solution‐based effects.