Reversible Immobilization of Chelating Affinity Surfactants on Reversed Phase Adsorbents for Protein and Peptide Separations under Metal Affinity Chromatography

Alkyl-bound silica was modified using chelating surfactants and the resulting adsorbent was used in immobilized metal affinity chromatography of proteins and peptides. Brij-76, a non-ionic amphiphilic surfactant with an alkyl moiety and an ethylene oxide chain, was reversible adsorbed to alkyl silica (C18). The hydroxyl group at the end of the ethylene oxide chain was chemically modified previously with an iminodiacetate functionality as chelating agent of transitional metal ions. Cu(II) was studied as immobilized ion for the adsorption of peptides and proteins. Three chromatographic supports were prepared having different Cu(II) capacities. For a low Cu(II) capacity case, the generated adsorbent behaved as a controlled access media preventing the adsorption of large molecular weight proteins, such as BSA, while small peptides, such as Angiotensin III, or amino acids could be retained. For a medium and high Cu(II) capacity, the synthesized adsorbent no longer behaved as a controlling access media and all molecules in this study, either large or small, were retained by the immobilized ion. Nonetheless, most of the BSA was strongly retained by the system and a pH change did not remove any of the adsorbed BSA while the small molecules were removed by the same pH change.