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Abstract: A new immobilized metal affinity chromatography (IMAC) matrix was prepared by coordinating Cu2+ with cross-linked chitosan coated on non-porous silica gel (Cu-CTS-SiO2). Macroporous structure could be formed on the coated layer by imprinting polyethylene glycol (PEG) in chitosan film. The surface morphology changes on Cu-CTS-SiO2 bead prepared in different condition were confirmed by scanning electron microscopy (SEM). Effects of chitosan and PEG content in coating solution, the molecular mass of PEG on the surface macropore formation and adsorption capacity of bovine serum albumin (BSA) were investigated. Results indicated that coating solution with 2% chitosan and 10% PEG 20000 was optimal. Batch experiments were also conducted for elucidating the optimal pH, the adsorption isotherm and adsorption kinetics of BSA. Adsorption isotherm of trypsin on the same adsorbent was also performed. Results showed that the support itself had low non-specific interaction with both BSA and trypsin. The maximum adsorption capacity for BSA and trypsin on the prepared IMAC adsorbent could reach 192 mg and 5000 IU, respectively calculated by every gram of chitosan. The binding and eluting condition for BSA were tested on column filled with the adsorbent. Crude BSA sample could be purified on the IMAC column