Preparation and characterization of temperature-responsive affinity chromatographic stationary phase modified with heparin

The silica beads modified by poly (N-isopropylacrylamide-co-N-acryloxysuccinimide) and heparin, which was then used as temperature-responsive affinity chromatographic stationary phase were prepared via radical polymerization. Grafting ratio of polymers grafted on surfaces was 9.45% calculated by thermogravimetric analysis (TGA) and the heparin content on surfaces was 3.6 mg/g determined by Elson-Morgan method. The packing column with the modified silica beads as the HPLC stationary phase had temperature responsive features, which was proved by separating benzene and hydrocortisone. The results also indicated that the strong affinity interaction occurs between thrombin and the modified column, with recovery of 81.4%. The releasing time of thrombin was shortened by controlling the column temperature, which provided a fast and efficient method for protein separation.