Separation And Analysis Of Polyphenols In Chuju By Two-dimentional Liquid Chromatography

Chuju is one of the four most famous chrysanthemums of China, which has many functional and bioactive activities, such as antineoplastic, antiphlogosis, antioxidant, antimyocardial ischemia, antiviral. Plant polyphenols is the most important effective constituent of Chuju. Separation and analysis of polyphenols in Chuju makes for research on bioactive activities of Chuju, thus realizing efficient industrial application. Separation and analyzation of polyphenols of Chuju are commonly applied high-performance liquid chromatography (HPLC), although the good resolution of all the possible components present on this type of complex profiles is quite difficult to achieve due to their huge chemical complexity and diversity. A method with high resolving power need to be utilized.Two innovative analytical approach with high peek capacity and orthogonality was applied to obtain the profiling of phenolic compounds in Chuju, rapid separation and identification by comprehensive2DLC while qualification and quantification by heart-cutting2DLC. The main work of this thesis is summarized in3aspects as follows:Firstly, hydrophilic interaction (HILIC) and reversed phase liquid chromatography (RPLC) were chosen to build a2D HILIC×RPLC separation system with the application of sample loops to transfer and connect. The narrow diameter Lichrospher diol-5column (150x1.0mm,5μmd.p., HiChrom, Reading, UK) and rapid analyzing Cortecs C18+column (50×4.6mm,2.7μm d.p., Waters, MA, US.) were chosen as the1st and the2nd chromatography, respectively.150μL sample loops were applied in the center of this system after optimizing. The peek capacity, components distribution and orthogonality were evaluated. The theoretical and effective peek capacities were up to1117and649;25polyphenol peeks were laid on the2chromatography differently and complementarily; the surface coverage proposed by Gilar (SCG) was0.56that indicating good orthogonality of this2D-LC system.Secondly, the extracting methods of Chuju were optimized and chosen, the result showed ultrasonic-assisted extraction was the most effective while the Chuju sample and solvent were1:30. The optimized sampling volume was2μL because the solvent was80%ethanol solution.23peeks in the2D-LC separation space were found by the optimized comprehensive2D HILIC xRPLC system, and13of them were allowed tentative identification according to comparing with retention times and maximum absorption wavelengths between the sample and standards. There are4phenolic compounds first found in Chuju. Moreover, isorhamnetin-3-O-glucoside is first found in Dendranthema morifolium.Thirdly, multiple heart-cutting2DLC with peek capacity up to2536of polyphenols was built by choosing conventional reversed Zorbax80A Extend-C18column (150×4.6mm,5μm d.p., Agilent, CA, US) as the2nd RPLC while cutting the6-60min fractions from the1st HILIC every two minutes.133peeks were achieved by separation of Chuju extract using this system.18polyphenols were identified according to comparing with retention times and maximum absorption wavelengths between the samples and standards while another18polyphenols were identified by comparing between the samples and what reported in literature. The36polyphenols were quantitative analyzed using standard curves. The results showed that Chuju was abundant in phenolic compounds. The contents of3respective components, chlorogenic acid, isochlorogenic acid A, luteolin-7-O-glucoside were0.21%,0.81%,0.14%, respectively.The comprehensive and heart-cutting2DLC systems which achieved good separation of polyphenols in Chuju constructed in this work built new analysis methods for polyphenols in Chuju and provided new directions for research on complex plants.