Mixed-mode retention of peptides on phosphate-modified polybutadiene-coated zirconia

Zirconia HPLC packing materials were found to be potentially advantageous for large-scale protein separations due to their excellent pH stability and mechanical stability. However, Lewis acid sites on zirconia's surface cause irreversible adsorption of proteins due to their interactions 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, we found that the separation of peptides on a phosphate-modified polybutadiene-coated zirconia (PBD-ZrO2) can be brought about by using a mobile phase containing both an organic modifier and a high concentration of sodium perchlorate. The salt is needed to cancel the Coulombic interactions between the negatively charged stationary phase and the positively charged proteins. To understand the retention mechanism of proteins 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 mobile-phase conditions used for protein and peptide separations. The PBD coating provides hydrophobic moieties, and the phosphate ions adsorbed on zirconia's surface provide cation-exchange sites. Reversed-phase separation of a peptide standard mixture and cation-exchange separation of a cationic peptide standard mixture on the same phosphate-modified PBD-ZrO2 column shows excellent column resolution in both modes. Although mixed-mode stationary phases provide unique selectivity, the secondary equilibrium on phosphate-modified PBD-ZrO2 can cause peak broadening. Applications of the phosphate-modified PBD-ZrO2 to peptide separations are demonstrated here.