Study On The Preparation Of CHC By Preparative High Performance Liquid Chromatography

CHC(Chenghai Chlorin)is the main hydrolysate of pheophorbide a.CHC itself and its derivatives are widely used in fluorescence detection and photodynamic therapy of cancer.However,the composition of the CHC product obtained by the hydrolysis of pheophytin a is more complicated and difficult to separate.Therefore,the separation and purification of crude CHC products is of special significance.Conventional separation methods have low separation efficiency,and preparative high performance liquid chromatography(Prep-HPLC)separation and purification products have high efficiency and good repeatability.This research topic uses natural algae as raw materials,designs raw material organic solvent extraction,chemical continuous fractional degradation,high performance liquid chromatography stepwise analysis and preparative high performance liquid chromatography preparation process routes,extraction and preparation of high purity CHC,The following aspects have been studied in detail:(1)The analytical conditions of the ammonium formate buffer system suitable for CHC were optimized by high performance liquid chromatography technology,and the Z7-C18 ME chromatographic column made by Dalian Institute of Chemical Physics was used.The phase is ammonium formate methanol.The HPLC analysis spectrum combined with the molecular ion peak signal measured by the mass spectrometer preliminarily determines that the content of the main component CHC is 71%,and the remaining impurities include oxidative degradation products such as purpurin-18.(2)Obtain the preparative chromatographic conditions by formulas according to the HPLC conditions.Under reversed phase conditions,a Z7-C18 ME column with a diameter of 10 mm was used for semi-preparation.In semi-preparation of CHC,the detection wavelength was reduced from 400 nm to 254 nm,and the resolution of each peak on the spectrum was changed,making CHC collection easier,And the purity of the obtained CHC reaches more than 98%.Using the innovative Tongheng LC6000 preparation system,a separation and purification method that can be continuously prepared has been developed.The crude CHC product was separated and purified on a 50 mm diameter preparative chromatographic column,which improved the purity of the CHC product.High performance liquid chromatography analysis showed that the purity was over 98%.