Application Research For Separation And Determination Of The Active Components In Euphorbiae Semen By HPCE

Objective:The first chapter aims to establish a simple,fast and efficient capillary electrophoresis for the separation and determination of Euphorbia factors L1,L2 and L3with sodium dodecyl sulfate as electrolyte additives.This established method will provide a guidance for the analysis of three important diterpenoid in Euphorbiae semen and its formulations.In the second chapter,try to use cyclodextrin derivatives as electrolyte additive to establish a simple,rapid and effective capillary electrophoresis method for the simultaneous separation and determination of six phenolic acids in Euphorbiae semen.This established method could be applied for the separation and quantification phenolic acid compounds in Euphorbiae semen.The last chapter aims to determine the pK_avalues of six phenolic acids by pressure-assisted capillary electrophoresis.It will provide a reference for the determination of pK_avalues of phenolic acids by capillary electrophoresis.Methods:1.The experiment was carried out using a P/ACE MDQ system equipped with a diode array detector and an uncoated fused-silica capillary(40.2 cm×75μm id)with an effective length of 30 cm.Samples were injected into the capillary at 0.3 psi for 3 s.Several important experimental parameters such as the type of solvents and content,the type of buffer and concentration,the type of surfactant and concentration,detection wavelength,separation voltage and temperature were investigated.The satisfactory separation results of Euphorbia factors L1,L2 and L3 were achieved at the optimized condition when 60%(v/v)methanol was solvent,5.0 mmol/L ammonium acetate(p H=6.86)containing 30 mmol/L sodium dodecyl sulfate was electrolyte solution,25 k V of electric field across the capillary was applied at 25℃,and the detection wavelength was at 280 nm.2.The experiment was carried out using a P/ACE MDQ system equipped with a diode array detector and an uncoated fused-silica capillary(40.2 cm×75μm id)with an effective length of 30 cm.The influence of several important experimental parameters were investigated,such as the content of solvents,the type of buffer and concentration,the type of cyclodextrin and concentration,detection wavelength,separation voltage and separation temperature.The optimum condition of simultaneous separation and determination of the six phenolic acids in Euphorbiae semen were as follows:the detection wavelength was at 260 nm,35 mmol/L sodium tetraborate buffer,3.0 mmol/L Heptapkis(2,6-di-o-Methyl)-β-cyclodextrin,5%(v/v)methanol,the separation voltage and temperature were 12.5 k V and 25℃,respectively.3.The experiment was performed by using a P/ACE MDQ system equipped with a diode array detector.The separation was carried out in an uncoated fused-silica capillary(40.2 cm×75μm id)with an effective length of 30 cm.The influence of inlet pressure on the measurement of pK_avalue was investigated.The satisfactory measurement condition of pK_avalue was obtained when inlet pressure was 0.1 psi.Results:Under the optimum capillary conditions,Euphorbia factors L1,L2,and L3 could reach baseline separation within 15 min.The linear relationships of these three diterpenoids were good with correlation coefficients from 0.9945 to 0.9995.The intraday precision ranged from 1.3%to 2.1%and the interday precision ranged from 2.7%to3.1%.The average recoveries of real samples were in the range of 98.5%-103.8%.Under the optimum capillary conditions,these six phenolic acids in Euphorbiae semen realized baseline separated within 15 min.Good linear relationships were achieved with correlation coefficients from 0.9925 to 0.9990.The intraday precision ranged from 2.1%to 2.6%and the interday precision ranged from 1.0%to 2.1%.The average recoveries of real samples were in the range of 98.8%-104.9%.The measured pK_avalues for the six phenolic acids were Chlorogenic acid(3.96),Ferulic acid(4.57),p-Coumaric acid(4.65),Vanillic acid(4.56),Caffeic acid(4.63)and Gallic acid(4.40),respectively.Conclusion:In this study,a hydrophobic interaction electrokinetic chromatography method was established for the separation and determination of Euphorbia factors L1,L2,and L3 in Euphorbiae semen and its formulations.This developed method is novel,simple,and rapid,which is a promising provide guidance for simultaneous separation and determination of more hydrophobic terpenoids.And a simple and rapid capillary zone electrophoresis with cyclodextrin derivative as modifier was established to simultaneous separate and determine of these six phenolic acids in Euphorbiae semen.The proposed method provides an alternative method for the quality control of phenolic acids in Euphorbiae semen and comprehensive development of Euphorbiae semen.In addition,the pK_avalues of these six phenolic acids in Euphorbiae semen were successfully determined by pressure-assisted capillary electrophoresis.Which provided a reference for the determination of pK_avalues of phenolic acids by capillary electrophoresis.