Coupling of ligands to primary hydroxyl-containing silica for high-performance affinity chromatography. Optimization of conditions.

Silicas of different particle and pore sizes were derivatized with three different silanes. The functionalized silica contained either epoxide, methacrylate or amino groups. These groups were further modified to yield primary hydroxyl functions. Activation of the resultant primary hydroxyl groups for the purpose of chemically coupling proteins was studied with a variety of reagents and optimized for p-nitro-phenyl chloroformate. The effect of pH on the efficiency of coupling proteins (BSA and trypsin) to p-nitrophenyl carbonate-silica was studied in detail. Slightly acidic conditions (pH 6) gave the highest yields. In a dynamic recycling process, bovine pancreatic trypsin inhibitor was immobilized to activated primary hydroxyl-silica packed into a stainless-steel column. The high-performance affinity chromatography purification of trypsin on this column is demonstrated.