Study on Separation and Analysis of Complicated Systems by Liquid Chromatography Methods
|School||Central South University|
|Keywords||high performance liquid chromatography, open column chromatography, preparative high performance liquid chromatography,liquid chromatography with mass spectrometry Eucommia ulmioides geniposidic acid, geniposide, pinoresinol diglucoside, syringaresinol diglucoside, and chlorogenic acid 5, 10, 15, 20-tetraphenyporphine cobalt and 5,10,15,20-tetraphenylporphine separation, determination and characterization|
Two complicated systems,namely extract from Chinese traditional herb and porphine catalyst,were studied by the high performance liquid chromatography,open column chromatography,preparative high performance liquid chromatography,or liquid chromatography with mass spectrometry.The main content consists of two parts:（1）considering the bioactive components from Eucommia ulmoides as object,extraction, separation,determination and characterization of iridoids and lignans from Eucommia ulmoides were studied.Five bioactive compounds were attained and identified as geniposidic acid,geniposide,pinoresinol diglucoside,syringaresinol diglucoside,and chlorogenic acid by means of HPLC,UV,IR,1HNMR and MS.（2）Purification,characterization and determination of 5,10,15,20-tetraphenylporphine cobalt（CoTPP）and its middle product 5,10,15,20-tetraphenylporphine（TPP）were studied systematically by means of column chromatography,HPLC,UV-VIS,IR and HPLC-MS.It provided the method for large-scale produce of porphine catalyst and the industrial application of cyclohexane biomimetic catalytic oxidation to produce cyclohexanone.Details are as following:1.Extraction bioactive components from Eucommia ulmoidesExtraction methods using solvent extraction（SE）,enzyme-assisted aqueous extraction（EE）,semi-bionic extraction（SBE）and supercritical CO2 fluid extraction（SFE）were evaluated for the yields of geniposidic acid,geniposide,pinoresinol diglucoside,syringaresinol diglucoside, aucubin and chlorogenic acid from Eucommia ulmoides.Semi-bionic extraction was chosen for its high yields.2.Preparation bioactive components from Eucommia ulmoides by silica gel column chromatographyGeniposidic acid,geniposide,pinoresinol diglucoside,and syringaresinol diglucoside were prepared by silica gel column chromatography.The purities of geniposidic acid,geniposide,pinoresinol diglucoside,and syringaresinol diglucoside were 98.69%,96.54%, 92.18%,and 93.77%,respectively. 3.Preparation bioactive components from Eucommia ulmoides by preparative high performance liquid chromatographyGeniposidic acid,geniposide,and chlorogenic acid were prepared by preparative high performance liquid chromatography.The purities of geniposidic acid,geniposide,and chlorogenic acid were 95.61%,93.28% and 96.57%,respectively.The adsorption mechanism of geniposidic acid on C18column was studied by frontal analysis.4.Determination and characterization of bioactive components from Eucommia ulmoidesTwo methods base on high performance liquid chromatography coupled with photodiode array detector（HPLC-DAD）were established to determination of lignans,iridoids and chlorogenic acid,respectively.（1） Pinoresinol diglucoside and syringaresinol diglucoside were separated and analyzed on a Kromasil RP-C18column by HPLC;（2）Geniposidic acid,geniposide,and chlorogenic acid were separated and determined by HPLC.Five bioactive components from Eucommia ulmoides were identified as syringaresinol diglucoside,pinoresinol diglucoside,geniposidic acid, geniposide,and chlorogenic acid by chemical and spectra analysis.The animal experimental result indicated that there were significantly statistic differences in maximum effectiveness,maximum effective percentage and duration between two different groups and that intravenous administration of the extract of Eucommia ulmioides dose-dependently reduced blood pressure in the SHRs.5.Study fingerprint ofEucommia ulmioides by HPLCHPLC fingerprint Spectrum ofEucommia ulmioides was established. Chromatography conditions:column,Kromasil RP-C18column;mobile phase,methanol-0.1%acetic acid（20:80,v/v）;detection wavelength,240 nm;flow rate,0.8 mL/min;and injection volume,20μL.Geniposidic acid was used as the reference compound.6.Purification of CoTPP and TPPTPP was separated and purified through extraction and aluminum oxide column chromatography.The purity of purified product was 99.85%.The total recovery was 88.98%.CoTPP was separated through extraction（using efficient solvent A）and was purified by crystallization. The purity of purified CoTPP was from 29.57%,38.79%,32.34%to 76.41%,81.37%,and 74.29%,respectively.The recovery was 85.13%.7.Quantification of TPP by RP-HPLCTPP was determined by the reversed phase high performance liquid chromatography.The mobile phase consisted of methanol-water（85:15, v/v）and the quantitative analysis was conducted with calibration curve method.The result indicated that the calibration curve was linear in the range of 0.5-2.5μg/mL of TPP with r=0.9993,the minimum detection limits to 0.2μg/mL and the recoveries were between 95.2%and 106.1%. The structure of TPP was identified by means of UV-VIS,IR,LC-ESI-MS.8.Quantification of CoTPP by RP-HPLCCoTPP was determined by HPLC.The mobile phase consisted of methanol-acetonitrile-chloroform（7:3:1,v/v）and the quantitative analysis was conducted with calibration curve method.The result indicated the calibration curve was linear in the range of 1.0-5.0μg/mL of CoTPP with r=0.9997,the minimum examination limits to 0.5μg/mL and the recoveries were between 95.05%and 102.4%.The structure was identified by means of UV-VIS,IR.9.Industrial application of analytical methodSimultaneous determination of CoTPP and TPP by HPLC.The mobile phase consisted of methanol- acetonitrile- chloroform（7:3:1,v/v） and the quantitative method was rapid,simple and precise.This method provided excellent results in industrial application to produce cyclohexanone.