A novel stationary phase for high performance liquid chromatography was prepared using urushiol methacrylate as the chromatographic ligand. The mixed urushiol methacrylate was prepared using urushiol and methacryloyl chloride via a substitution reaction and then coated onto the surface of spherical silica by physical adsorption. The spherical silica was chemically modified with 3-methacryloyloxypropyl trimethoxysilane. Then, the urushiol methacrylate-bonded silica stationary phase (USP) was synthesized via the surface radical polymerization of urushiol methacrylate and the pendant vinyl groups onto the surface of the spherical silica. The stationary phase was characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis, scanning electron microscopy, and elemental analysis. The results revealed that the urushiol methacrylate was successfully immobilized on the spherical silica surface after the surface polymerization reaction, and that it had excellent monodispersity. The stationary phases were packed in a stainless-steel hollow column by the slurry packing method, with methanol as the slurry solvent and absolute ethanol as the propelling solvent. The chromatographic performance of the stationary phases were investigated for the separation of Gastrodia elata extract. Acetonitrile-0.05% phosphoric acid solution (3:97, v/v) was employed as the mobile phase at a flow rate of 0.4 mL/min, with the detection wavelength of 220 nm. The separation performance for Fructus evodia extract was also studied, using acetonitrile-water (50:50, v/v) as the mobile phase at a flow rate of 0.5 mL/min, and the detection wavelength was 290 nm. This column showed good separation performance for both these extracts. Out of the five peaks observed for the Gastrodia elata extract, one was attributed to gastrodin, but the other four peaks need to be further verified. Two peaks assignable to evodiamine and rutaecarpine were observed for the Fructus evodia extract. Compared with C18 column, the USP column allowed for more effective separation of the components from the Gastrodia elata extract, with baseline separation; on the other hand, the chromatographic conditions for the separation of the components of the Fructus evodia extract were more environmentally friendly and safer. Because of the low flow rates adopted for the separation of the Gastrodia elata and Fructus evodia extracts, the amount of mobile phase used could be reduced. This study provides not only a new method for the separation and purification of gastrodin and evodiamine in real samples, but also a new strategy for the preparation of chromatographic stationary phases. It expanded the application of raw lacquer in chromatographic separation materials.
Keywords: bonded silica stationary phase; evodiamine; gastrodin; high performance liquid chromatography (HPLC); urushiol.