Study On The Purification Of Rebaudioside A By MPLC

Natural sweetener stevia glycosides have high economic value, among which rebaudioside A (RA) is the most valuable of all. Most domestic manufacturers obtained RA of high purity through recrystallization method, resulting a mixture of steviosides in the mother liquid, wherein the content of RA is about10-30%. However, it is unable to be separated through a conventional process, resulting in the mother liquor to be accumulated or sold as low-value-added products. Thus, it’s highly meaningful to investigate the process of purifying RA from the crystilized mother liquor.Firstly, carboxyl group was successfully introduced to the monodisperse PS-DVB:the first step, lOg dried PS-DVB was dissolved by dichloroethane and reacted with65%concentrated nitric acid and98%concentrated sulfuric acid solution with a molar ratio of2:5for5hours at50℃. Then, the resin with amino group was futher prepared, using tindichloride as a reducing agent, reacting at75℃for12hours. Finally, the reaction of bonding the carboxylic acid:amino resin and chloroacetic acid with a mass ratio of1:2reacted at60℃for12hours.Secondly, two modified and the previous resin were equipped into a standard HPLC column, the resolution distinction for ST and RA was investigated, besides, the effects of the mobile phase, flow rate, column temperature were studied to optimize the the HPLC condition. Finally, ST and RA from crystalized mother liquor reached baseline separation.At last, a medium pressure preparative liquid chromatography (MPLC) was used to isolate RA. Purity, yield rate and recovery rate were observed to study the proportion of mobile phase, sample concentration, sample volume as well as the mobile phase flow rate. The optimal operating conditions were:the proportion of mobile phase (acetonitrile/water) was80/20(v/v), sample concentration280mg/ml, sample volume0.4ml, and the flow rate4ml/min. Under the optimal operating conditions, the obtained RA purity was90.22%, the recovery rate achieved79%. This method provided a favorable reference for the RA industrialization research.