Method Development for Purification of ?-oryzanol from Hydrolyzed Rice Bran Acid Oil by Semi-preparative Chromatography

The objective of this study was to develop a method for isolation and purification of gamma-oryzanol from hydrolyzed rice bran acid oil (RBAO) using semi-preparative chromatography by first applying silica coated-thin layer chromatography (TLC) to determine the suitable mobile phase. Subsequently, column chromatography was carried out to determine the effects of purification conditions such as the amount of and particle sizes of the sample silica gel, and elution modes, on the percentage of gamma-oryzanol yield and recovery. The results from the TLC suggested that 75:25 (v/v) hexane to ethyl acetate mixture was a suitable mobile phase. The semi-chromatographic results indicated that the column containing 10 g of 25-40 mu m silica gel with isocratic elution gave the highest yield (84%) of purified gamma-oryzanol (> 95% purity). Further application of a step-gradient elution with 85:15 (v/v), followed by 75:25 (v/v) hexane to ethyl acetate mixture increased chromatographic resolution (Rs), resulting in enhanced separation efficiency, which in turn led to a higher yield of purified gamma-oryzanol of 90%.