Analysis Of Isoflavones In Radix Puerariae By Capillary Electrophoresis With Ionic Liquids As Additives

Ionic liquids(ILs), or named as room temperature molten salt, are composed of various anions and organic cations which possess fascinating properties, such as low volatility, excellent solubility and great conductivity. Hence, they have been received more and more interests in numerous fields. At present, the IL has been widely used in the analysis of natural products by capillary electrophoresis(C E). In this study, eight isoflavones as the main active components in Radix Ruerariae(RP) were simultaneously determined by CE. RP is the dry root of P. lobata(Willd.) Ohwi or P. theomsonii Benth, which has various biological activities, were used as medicine or functional food. Isoflavones are the main bioactive compounds in RP, but only puerarin is used as quality control marker in C hinese Pharmacopoeia(2010 edition). Therefore, a fast, simple and accurate analytical method for the simultaneous determination of multiple isoflavones in RP is significant for its quality control. In the present study, by using ILs as additives, surfactants or oil phases, CE methods included capillary zone electrophoresis(CZE), micellar electrokinetic chromatography(MEKC) and microemulsion electrokinetic chromatography(MEEKC) were developed for the analysis of eight isoflavones in RP samples. The thesis consists of five main parts(chapters).The first part is literature review. The applications of ILs in CE analysis of natural products were summarized, including C ZE, MEKC, MEEKC and non-aqueous capillary electrophoresis(NAC E). In addition, a brief introduction of RP was also presented.Second part is to develop a simple and repeatab le C ZE method for the simultaneous determination of eight isoflavones in three RP samples(P. thomsonii from Guangxi, P. lobata from Guangxi and P. lobata from Hubei). By comparing the effect on the separation of eight isoflavones, four ILs composed of different cations and anions as additives were compared. The results indicated that BMIm BF4 in borate buffer system was good for the separation. Finally, the fast analysis(about 9 min) of eight isoflavones was achieved by careful optimization of related parameters. The results showed that IL revealed good selectivity in the separation of isoflavones, and the selectivity was resulted from the electrostatic attraction between the cations and the dissociated groups of analytes.MEKC for the analysis of investigated isoflavones were studied in the third part. Different micelles systems including(1) monomeric surfactant including an anionic surfactant sodium dodecyl sulfate(SDS), a cationic surfactant hexadecyl trimethyl ammonium bromide(C TAB), a neutral surfactant polyoxyethylene sorbaitan monolaurate(Tween-20) and an ionic liquid type surfactant 1-dodecyl-3-methylimidazolium tetrafluoroborate(C12MIMBF4), and(2) monomeric surfactants with BMIm BF4 as additive, and(3) mixed micelles of SDS+Tween-20 and C12MIMBF4+Tween-20 was systematically compared. Finally, both SDS with BMIm BF4 as additive and mixed micelles SDS+Tween-20 had highest separation efficiency for the eight investigated compounds. Furthermore, the SDS with BMIm BF4 as additive is more stable(within 20 hours) than mixed micelles SDS+Tween-20, which may resulted from the stabilized effect of BMIm BF4. The developed method was fully validated(limit of detection, limit of quantification, intra-day precision, inter-day precision, and recovery) and was successfully applied to determine the eight analytes in three Radix Puerariae samples. The present study indicated that SDS with ILs as additive are suitable for the analysis of isoflavones by MEKC.Chapter four is analysis of eight isoflavones by MEEKC. The oil phase is one of the key components of microemulsion in microemulsion electrokinetic chromatography(MEEKC) analysis. In the present study, three different oil phases includ ing 1-butyl-1-methylpyrrolidinium bis(trifluoromethanesulfonyl) imide(BMPy[N Tf2]), 1-butyl-3-methylimidazolium hexafluorophosphate(BMIm PF6) and n-octane were applied and compared for the MEEKC analysis(SDS as surfactant and n-butanol as co-surfactant) of eight isoflavones from pueraria. The investigated isoflavones could be well separated by all of those three microemulsion systems after careful opt imization. And the MEEKC with n-butanol as co-surfactant and n-octane as oil phase was the best choice(symmetry peaks, high resolutions of peaks with short analysis time) for the analysis of investigated isoflavones. And then the developed method was fully validated(limit of detection, limit of quantification, intra-day precision, inter-day precision, and recovery) and was successfully applied to determine the eight analytes in three Radix Puerariae samples. In addition, although the MEEKC with classic oil phase(n-octane) showed better results for the analysis of isoflavones in the present study, the MEEKC with ionic liquids(BMPy[NTf2] and BMIm PF6) also showed great separation potential for analytes, which may be further applied in the analysis of other natural products.In sum, the results in the present study indicated that P. lobata contains much abundance of isoflavones than P. thomsonii, and puerarin is the main isoflavone in RP. Furthermore, IL was applied in all the C E experiments, and different ro les such as additives, surfactants or oil phases were played by ILs in different CE modes. The applications of ILs can improve the separation efficiency of CE analysis of natural products. Therefore, more applications of ILs will be helpful for the natural products analyses.