Abstract：The multifunctional stationary phase with the properties of weak cation exchange (WCX), hydrophobic and hydrogen bonding interaction was synthesized by bonded the glutathione (GSH) to the silica surface. Five standard proteins myoglobin (Myo), ribonuclease (RNase), cytochrome (Cyt-C), α-Chymotrypsin (α-Chy) and lysozyme (Lys) were selected to evaluate the column efficiency. The experiments show that the stationary phase has the good separation performance for proteins both in hydrophobic interaction chromatography (HIC) and ion exchange chromatography (IEC). More importantly, the proteins separation in both HIC and IEC mode can be accomplished only by a single column and a kind of mobile phase. In order to compare the resolution of multifunctional column with other column, two popular commercial columns of TSKgel Ether-5PW in HIC mode and TSKgel CM-5PW in IEC mode were tested under optimum condition. It was found that the multifunctional column has a good resolution both in IEC and HIC mode compared with commercial TSKgel columns. The eluted order of five proteins were the same on multifunctional and TSKgel CM-5PW column in IEC mode, but there is a big different between multifunctional column and TSKgel Ether-5PW column in HIC mode, the eluted order of Myo and RNase as well as α-Chy and Lys are changed completely. This indicates that the selectivity of the multifunctional column is significantly better than that of the TSKgel columns. The chromatographic retention behaviors and mechanisms for proteins in glutathione bonded column were studied. The results demonstrate that there are two retention mechanisms for a basic protein, at a low salt concentration, the retention time of proteins is decreased with the increasing of salt concentration and the proteins were separated by electrostatic force, while in high salt concentration the retention time of proteins is increased with the increasing of salt concentration and proteins were separated by hydrogen bonding interaction force. This multi-interaction forces retention mode can effectively improve the selectivity of HPLC column. The research provides a new solution for high performance separation of proteins, peptides and amino acids.