Abstract: Using phenyl-β-D-glucopyranoside(PGL) and 4-methoxyphenyl-β-D-glucopyranoside(MGL) as the competitive molecules owing to its structural analogues of gastrodin(GAS), the separation performance of GAS by usurushiol methacrylate bonded SiO₂ column was investigated, and its thermodynamics behavior was also analyzed. The result showed that the optimum separation conditions of urushiol methacrylate bonded SiO₂ chromatographic column were as follows: 0.05% phosphoric acid water solution(3∶97, volume ratio), flow rate 0.4 mL/min, column temperature 25 ℃, detection wavelength 220 nm and injection volume 20 L. Under this chromatographic condition, the resolution of urushiol methacrylate bonded SiO₂ chromatographic column to PGL-GAS and MGL-GAS was 7.8 and 14.0, respectively. The thermodynamic studies revealed that the ΔH, ΔΔH and ΔΔG values of both PGL-GAS and MGL-GAS were negative, and the ΔΔS values were greater than zero, during the process of separation. It indicated that the separation of GAS and its structural analogues on urushiol methacrylate bonded SiO₂ column was a thermodynamic spontaneous process, which was driven both by the entropy and enthalpy. The retention and resolution of analytes decrease with the increasing column temperature. After the crude extract was purified by usurushiol methacrylate bonded SiO₂ column, the impurities were significatly reduced, and the purity of gastrodin increased from 17.06% to 93.45%. The urushiol methacrylate bonded SiO₂ column exhibited good separation performance and stability during the separation process.