MIXED-MODE RETENTION OF PEPTIDES ON PHOSPHATE-MODIFIED POLYBUTADIENE-COATED ZIRCONIA

Zirconia HPLC packing materials were found to be potentially advantage ous for large-scale protein separations due to their excellent pH stab ility and mechanical stability. However, Lewis acid sites on zirconia' s surface cause irreversible adsorption of proteins due to their inter actions with hard Lewis bases such as the carboxyl groups in proteins. Although the Lewis acid sites can be effectively blocked by adsorbing phosphate ions onto zirconia's surface, proteins and peptides cannot be eluted using a typical reversed-phase mobile phase. In this work, w e found that the separation of peptides on a phosphate-modified polybu tadiene-coated zirconia (PBD-ZrO2) can be brought about by using a mob ile phase containing both an organic modifier and a high concentration of sodium perchlorate. The salt is needed to cancel the Coulombic int eractions between the negatively charged stationary phase and the posi tively charged proteins. To understand the retention mechanism of prot eins and peptides on phosphate-modified PBD-ZrO2, this work was aimed at the study of the surface characteristics of the phosphate-modified PBD-ZrO2. We found that the phosphate-modified PBD-ZrO2 phase has both reversed-phase and cation-exchange characteristics under the acidic m obile-phase conditions used for protein and peptide separations. The P BD coating provides hydrophobic moieties, and the phosphate ions adsor bed on zirconia's surface provide cation-exchange sites. Reversed-phas e separation of a peptide standard mixture and cation-exchange separat ion of a cationic peptide standard mixture on the same phosphate-modif ied PBD-ZrO2 column shows excellent column resolution in both modes. A lthough mixed-mode stationary phases provide unique selectivity, the s econdary equilibrium on phosphate-modified PBD-ZrO2 can cause peak bro adening. Applications of the phosphate-modified PBD-ZrO2 to peptide se parations are demonstrated here.