Novel CE Methods For The Determination Of Some Active Components In Chinese Herbal Medicines

Capillary electrophoresis(CE)is a new-style separation technique which was established and quickly developed in the early 1980s.Nowadays,CE has been widely used in the field of chemistry,life sciences,pharmaceutics,and environmental sciences,due to the advantages of higher separation power,short analysis time,small sample and reagent consumption and simplicity.In this dissertation,we have carried out major innovative work about the study and application of reversed voltage in CE. Different CE mode with reversed voltage was used to separate and determine active components of Chinese herbal medicine.On the basis of the previous literatures,in this dissertation,some original studies are carried out as followings:1.A nonaqueous capillary electrophoresis(NACE)method was developed for the separation and determination of chlorogenic acid and caffeic acid in Hawthorn fruit.2.The large volume sample stacking(LVSS)method was combined with NACE method to improve the sensitivity of chlorogenic acid and caffeic acid in Hawthorn fruit.3.A reverse-flow micellar electrokinetic chromatographic(RF-MEKC) method was developed for the separation of quercetin,rutin and chlorogenic acid,and this method was applied for analyzing these components in Houttuyniae and Hawthorn fruit.The dissertation consists of two parts and four chapters.Part one is about recent advance in theories and application of CE,part two is about the author's research:Partâ… Review of the development of capillary electrophoresis,application of CE in Chinese herbal medicines and application of reversed voltage in CE.Chapter 1:The principle and characteristics of CE were simply described.The application of CE in Chinese herbal medicines and the application of reversed voltage in CE were reviewed. Partâ…¡Study and application of CE with reverse voltage for the separation and determination of activity components of Chinese herbal medicinesChapter 2:A nonaqueous capillary electrophoresis(NACE)method was developed for the separation and determination of chlorogenic acid and caffeic acid from Hawthorn fruit extracted with methanol.The separation conditions of the two organic acids were optimized with respect to the concentrations of Tris,acetic acid and applied voltage.Baseline separation was obtained for the two analytes within 10 min,using a running buffer containing 35 mM Tris,0.7%acetic acid in methanol and -25 kV voltage.The linear calibration range was 20-200μg/ml for chlorogenic acid and caffeic acid.The relative standard deviations(n=5)of the migration time and peak area of chlorogenic acid and caffeic acid were 2.82%and 2.29%(intra-day),and 4.02%and 3.14%(inter-day),respectively.The proposed method had been used to determine the two acids in Hawthorn fruit.The recovery results for chlorogenic acid were 98.7%,98.2%and 97.5%(added 25,50,100μg/mL standard solution of chlorogenic acid respectively);for caffeic acid were 95.6%,102.5%and 99.4% (added 25,50,100μg/mL standard solution of caffeic acid respectively).Chapter 3:The large volume sample stacking(LVSS)method was combined with NACE method to improve the sensitivity of chlorogenic acid and caffeic acid from Hawthorn fruit extracted with methanol.The separation conditions of the three analytes were optimized with respect to the concentrations of Tris,acetic acid,sample injection time and applied voltage.Baseline separation was obtained for using a running buffer containing 35 mM Tris,0.7%acetic acid in methanol,50 s injection time and -25 kV voltage.For chlorogenic acid and caffeic acid,the linear calibration range was both 0.625-20μg/mL,the detection limits(S/N=3)were 0.08 and 0.05μg/mL,respectively.The recovery results for chlorogenic acid were 98.7%-102.6%, for caffeic acid were 96.1%-103.3%.Chapter 4:A reverse-flow micellar electrokinetic chromatographic(RF-MEKC) method was developed for the separation and determination of quercetin,rutin and chlorogenic acid in Houttuyniae and Hawthorn fruit.The separation conditions of the three analytes were optimized with respect to the concentrations of SDS,NaH₂PO₄, pH and applied voltage.Baseline separation was obtained for the three analytes within 15 min,using a running buffer containing 70 mM SDS,30 mM NaH₂PO₄,pH 2.1 and -15 kV voltage.The linear calibration range was 25-400μg/mL for quercetin and rutin;12.5-200μg/mL for chlorogenic acid.The recoveries were 98.5-103.7%, 95.8-97.6%and 99.1-103.8%for quercetin,rutin and chlorogenic acid,respectively. The detection limits of quercetin,rutin and chlorogenic acid(S/N=3)were 2.52,3.1 and 3.64μg/mL,respectively.