Spherical silica gel with a diameter of 5 μm and pore size of 30 nm was activated by KH-560 and coupled with three kinds of ligands (3-aminopyridine

2-mercapto benzimidazole and 2-mercapto-1-methylimidazole) to prepare the high performance hydrophobic charge-induction chromatography (HPHCIC) column. The chromatographic properties of three columns were compared and evaluated with three model proteins (Lysozyme

BSA and IgG). The column with 2-mercapto-1-methylimidazole as ligand displayed a proper chromatography and separation behavior which controlled by pH. This work demonstrated that the novel synthetic materials can be utilized as the media of high performance hydrophobic charge induction chromatography.