The preparation of silica matrices of different pore diameter, the synthesis of different pore diameter silica-based polymer-bonded phases and their chromatographic characteristics in the separation of proteins are described. The results obtained show that macroporous packings have a relatively small surface area and low capacity, and are therefore, suitable for the quick separation and analysis of biopolymer. The separation mechanism changes as the pore diameter increases and the mobile phase is altered. The effect of acetonitrile concentration on the retention of proteins on a 10 nm pore diameter silica-based polymer-bonded phase is discussed, together with the effect of salt concentration and pH value of the eluent on the retention of proteins on 300 nm pore diameter silica-based polymer-bonded phases.