Application Of Capillary Electrophoresis In Analysis Of Traditional Chinese Medicines

Capillary electrophoresis has been developed as an important separation technique in the last decades.As a powerful analytical technique,CE has many advantages such as rapidly,simple sample pretreatment,low solvent consumption,high resolving power,small amount of samples and reagents required and varieties of separation modes.It has been widely used in different areas of biochemistry,environment and chemistry.However,the application of CE was limited by poor detection sensitivity due to short optical path length across the capillary.Therefore,a series of on-line enhancement techniques and pretreatment methods have been developed.This thesis focused on the optimizing conditions to improve sensitivity of CE,the enhancement techniques including MSPD and online enrichment combined technology were studied.This dissertation is mainly concerned with the following aspects:(1)A simple and effective method was developed for determining lignans in Schisandrae Chinensis Fructus by using a micro-matrix solid phase dispersion(MSPD)technique coupled with microemulsion electrokinetic chromatography(MEEKC).Molecular sieve,TS-1,was applied as a solid supporting material in micro MSPD extraction for the first time.Parameters that affect extraction efficiency,such as type of dispersant,mass ratio of the sample to the dispersant,grinding time,elution solvent and volume were optimized.The optimal extraction conditions involve dispersing 25 mg of powdered Schisandrae samples with 50 mg of TS-1 by a mortar and pestle.A grinding time of 150 s was adopted.The blend was then transferred to a solid-phase extraction cartridge and the target analytes were eluted with 500 μL of methanol.Moreover,several parameters affecting MEEKC separation were studied,including the type of oil,SDS concentration,type and concentration of cosurfactant,and concentration of organic modifier.A satisfactory linearity(R>0.9998)was obtained,and the calculated limits of quantitation were less than 2.77 μg/mL.Finally,the micro MSPD-MEEKC method was successfully applied to the analysis of lignans in complex Schisandrae fructus samples.(2)A simple,efficient and environmental friendly method was proposed for determining five sesquiterpenoids of Curcuma wenyujin by micro matrix solid phase dispersion(MSPD)extraction coupled with MEEKC separation.TS-1 was applied as a solid support for extraction of sesquiterpenoids for the first time.Some parameters affecting extraction and separation efficiency were investigated.The optimal condition involved dispersing sample(200 mg)with 200 mg of TS-1 for 150 s,and then 1000 μL of methanol were used to elute five target analytes.Finally,they were well separated using a running buffer containing 1.3%SDS,5%1-butanol,0.5%ethyl acetate and 10%acetonitrile in 10 mM borate buffer at pH 9.0.Consequently,the developed method was fully validated and successfully applied to determine the five sesquiterpenoids including curdione,curcumenol,germacrone,furanodiene and f-elemene in Curcuma wenyujin origin’s Chinese herbal medicines.Furthermore,hierarchical cluster analysis was performed based on the contents of target compounds for distinguishing steamed and non-steamed drugs.The present study provided a promising method for fast investigation and discrimination of chemical difference in steam&non-steamed Chinese medicines from Curcuma wenyujin origin.(3)A on-line stacking preconcentration method by FASS in CZE was developed and validated for the simultaneous determination of three organic anions(Protocatechuic acid,Danshensu and Cinnamic acid).The main parameters that affect the separation and sensitivity were investigated and optimized.The best separation was achieved in 10 mmol/L NaH2PO4 with 40%(v/v)methanol and the samples were diluted in pure water.Compared with normal CZE injection,42-51 folds improvement in concentration sensitivity was achieved for three organic anions.The calibration curve was linear over a range of 0.25-10.0 μg/mL,with correlation coefficients of 0.9991-0.9993.The limits of detection(LODs)for the three organic anions were less than 0.010 μg/mL.The method is simple,fast,and sensitive and it is considered feasible for fast screening of organic anions in traditional Chinese medicines.(4)Three-step stacking by field-enhanced sample injection(FESI),sweeping,and micelle to solvent stacking(MSS)in co-EOF capillary zone electrophoresis(CZE)is presented for anionic analytes(Danshensu,Protocatechuic acid and Vanillic acid)).Ionic liquid was used for EOF reversal,and chemical stability of the capillary was investigated.Injection of ionic liquid micelles produced a zone of micelles solution.Sweeping of the FESI zone was by electrokinetic injection of analytes.MSS was by short injection of 60%methanol that released the swept analytes from ionic liquid micelles.The sensitivity enhancement factors were 1677-2424.The LODs were between 2.2-4.6 ng/mL,repeatability(intra-column and inter-column)was RSDs≤4.46%,and linearity was R≥0.992.It can become a referenced method in Traditional Chinese Medicines and biological samples.