Dissertation > Biological Sciences > Biochemistry > Lipids

High Speed Countercurrent Chromatography for C. Elegans Lipidomics Study

Author WangYan
Tutor WuZhiYong
School Northeastern University
Course Analytical Chemistry
Keywords High Speed Countercurrent Chromatography (HSCCC) Lipidomics Thin LayerChromatography (TLC) Liquid Chromatography-Tendern Mass Chromatography(LC-MS/MS)
Type Master's thesis
Year 2011
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Lipidomics was firstly proposed by Han and Gross in2003, which is a new research area in life science following genomics and proteomics. Generally speaking, lipidomics involves the analysis and identification of all lipids and molecules inacting with them in organisms, tissues or cells, the study of the structure and function of lipids, and the exploration of the relationship between lipid metabolism and the physiological, pathological process in cell, organ and organisms. Lipidomics become a hot area since it was formally proposed.High speed counter current chromatography (HSCCC) is a liquid-liquid partition technology developed in the last20years. Comparing to HPLC, HSCCC does not use solid support. Thus, HSCCC has following outstanding advantages:1. no irreversible adsorption;2. high sample recovery rate;3. wide selection range of sovent system;4. convenient and easy to change sovent system. In addition, HSCCC system is cheap and robust. HSCCC has now been widely applied in the fields such as biochemistry, biological engineering, medicine, natural product chemistry, organic synthesis, and so on.Firstly, the develpment of Lipidomics and the related analysis technologies were summarized, and the separation principle, work rules and application of HSCCC were systematically introduced. Application of HSCCC for prefractionation of row biological sample was proposed for reducing the complexity.C. elegans was selected as an experimental model for Lipidomics study. Cultivation of the nematodes was described in detail. The two lipid extraction methods, chloroform-methanol extraction and methyl tertiary butyl ether (MTBE) extraction, were evaluated using thin layer chromatography (TLC) and liquid chromatogram-mass chromatogram (LC-MS).In Chapter3, the lipids extracted from plasma and the nematodes were separated by thin layer chromatography (TLC). The separation influencing factors such as solvent saturation degree, humidity as well as the spreading styles were discussed. In addtion, Image J, an image processing software, was applied in TLC image processing. The method repeatability and the linear relationships of lipids content with the closed area of sgray scales from the separated spots were studied.In Chapter4, high speed countercurrent chromatography was applied in the seperation of lipids extracted from C.elegans. Several HSCCC sovent systems were tested with TLC, the solvents includes hexane-ethanol-water (6:5:1, V/V), hexane-ethyl acetate-methanol-water (5:5:7:3and1:1:1:1, V/V), petroleum ether-dichloromethane-methanol-propyl alcohol-water (1:5:6:1:4and1:3:5:6:1:4, V/V), normal butanol-methyl tertiary butyl ether-acetonitrile-water (3:1:1:5, V/V), and so on. Both solvent system and HSCCC seperation conditions were optimized. The HSCCC seperation result of C. elegans lipids sample was characterized by TLC and LC/MS.In this work, high speed counter current chromatography was for the first time used in lipidomics. The complexity of sample was reduced afte HSCCC separating, and the recovery of lipids sample was good because of no irreversible adsorptio.

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