Analysis And Preparation Of CHP In Spirulina Platensis

Pheophorbide a is a natural degradation product of chlorophyll and an important starting material for the synthesis of chlorinated compounds.However,the extracted pheophorbide a product has low purity and complex composition,and is isolated and purified to obtain high purity.The pheophorbide a sample is difficult,so the analysis and preparation of pheophorbide a is of great significance.Atmospheric pressure silica gel column chromatography has lower efficiency and lower separation efficiency,while high performance liquid chromatography(HPLC)column pressure and column efficiency,especially on the basis of analytical techniques,developed high performance liquid chromatography The technology enables samples with difficult separation to be rapidly scaled and continuously prepared to obtain high purity products.In this research,the crude product of CHP(Chenghai Pheophorbide)was extracted and prepared from Spirulina platensis in Chenghai Lake,Yunnan Province,and the following aspects were studied in detail:The analytical conditions of the acetonitrile-water-buffered salt system suitable for CHP were optimized by high performance liquid chromatography.The Z7-C18 ME column was prepared by Dalian Institute of Chemical Physics,and the mobile phase was acetonitrile: water: ammonium acetate buffer salt(100mM)=17:2:1(volume ratio).HPLC analysis of the spectrum and the molecular ion peak information measured by mass spectrometry to determine the main component CHP content of 57%,the remaining impurities including CHP isomer,pyropheophorbide a and CHP-O-heteromorphous crystal Oxidative degradation products.The conditions of normal phase chromatography and reversed phase chromatography of crude CHP products under methanol-ammonium acetate buffer salt system were optimized.The final analysis conditions were as follows: using Z7-C18 MEcolumn,the mobile phase was methanol: ammonium acetate buffer salt(100mM)=9:1(volume ratio),semi-prepared using a 10 mm diameter Z7-C18 ME column under reversed phase conditions,and the purity of CHP obtained was over 95%.Using the optimized reversed-phase chromatographic conditions to expand the preparation scale,and using the innovative Tongheng LC6000 preparation system,a separation and purification method that can be continuously prepared was developed.The CHP crude product was separated and purified on a 50 mm diameter preparative column to improve the purity of the CHP product.The HPLC analysis showed a purity of over 95%.The average recovery rate is 94.3%.It was found that the CHP and CHP epimer were converted into an equilibrium state in solution state,and the ratio between the two was stable at about CH:CHP epimer =17:3.The stability of CHP in different organic solvents was tested.The results showed that CHP was poorly stable in DMSO and tetrahydrofuran solvents.CHP was rapidly converted to CHP epimer and reached equilibrium.However,the time for CHP to reach equilibrium in methanol and dichloromethane is slower.