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.

Key words: high-performance liquid chromatography, poly(N-isopropylacrylamide), heparin, thrombin