High performance liquid chromatography separation of structurally related enkephalins on quaternary ammonium-embedded stationary phase in isocratic mode

Separation of twelve enkephalins was investigated on a quaternary ammonium-embedded stationary phase (Stability BS-C23). Variation of buffer pH of the mobile phase highlighted the complex relationship between repulsive/attractive electrostatic interactions and the reversed-phase partitioning mechanism. The effect of three different anions employed as additives (phosphate, chloride and perchlorate) was examined at various concentrations and two pH values (acidic and neutral). At pH 2.5, an increase in the anion eluent concentration resulted in a higher retention factors of positively charged enkephalins. This effect was more pronounced when perchlorate ions were added to the mobile phase rather than phosphate and chloride ions, due to chaotropic and ion-pairing effects. In contrast, at pH 7.5, retention factors of negatively charged enkephalins decreased when these salts were added, due to an anion-exchange mechanism. Perchlorate caused a sharper decrease than chloride and phosphate anions did. The results presented here provide insight into the possible adjustment of retention and separation of peptides on a mixed-mode stationary phase (BS-C23) by a careful control of the buffer pH, the nature and concentration of anions, added to the buffer, and organic modifier content.