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

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 H 2 PO 4 − 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.

Counterion condensation in short cationic peptides: Limiting mobilities beyond the Onsager–Fuoss theory

“…Nonelectrostatic interactions, absent in DH theory, may also play a large role in determining the degree of ion‐pairing. This is illustrated by the fact that pentafluoropropanoate has been used to separate peptides with substitutions that did not affect the total charge . A semiquantitative treatment in terms of ion‐pairing between the analyte and the BGE ions does, however, capture the general features of the experimental trends.…”

Mixed‐mode hydrophilic interaction/cation‐exchange chromatography: Separation of complex mixtures of peptides of varying charge and hydrophobicity

“…Briefly, the II-CZE approach consists of combining the powerful CZE mechanism (separations based on differences in solute massto-charge ratio) located in the BGE with that of a hydrophobically based mechanism also located in the BGE consisting of high concentrations of perfluorinated acids (TFA; pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (HFBA)). The mechanism for the separation of peptides by II-CZE has been discussed previously [28]. In summary, the differences in electrophoretic mobility observed between peptides with subtle differences in hydrophilicity/hydrophobicity at the substitution site are due to interactions of peptides with the bulk BGE.…”

Recent developments in CE and CEC of peptides

“…The principle of II-CZE has been successfully applied also for the separation of peptides differing by a single amino acid substitution in deca-and dodecapeptides with model amino acid sequences [98]. Substitutions differed by a wide range of properties, e.g., alkyl side chains, polar side chains, and charged side chains.…”