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Multicomponent Analysis by Micro-Liquid Chromatography-Capillary Electrophoresis

微量液相色谱-毛细管电泳多组分分析

基本信息

批准号：
15350049
负责人：
TERABE Shigeru
金额：
$ 9.6万
依托单位：
University of Hyogo (2004-2005)Himeji Institute of Technology (2003)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15350049/
关键词：
Micro Liquid Chromatography Capillary Electrophoresis LC-CE Two Dimensional Separation Metabolome Analysis Cell Extracts from B.subtilis Cell Extracts From E.coli Flavin Coenzymes Monolithic Silica Column マイクロチップ電気泳動生物発光検出オンライン固相抽出 /

项目摘要

Liquid chromatography-capillary electrophoresis two dimensional separation system was developed for the analysis of multicomponent mixtures in one run. Micro liquid chromatography was used as the first step. The conventional capillary electrophoresis system was employed as the second step. Reversed phase monolithic silica based columns of 200μm inside diameter, 25cm or 50cm in length were used with the gradient elution technique for micro LC separation. A fused silica capillary of 50μm inside diameter, 50cm in length to the detector was used for CE separation. As standard samples mixtures of 54 or 118 compounds which were expected to be included in metabolites in the cell were employed. Cell extracts of Bacillus subtilis or Escherichia coli were also used to show the separation capability of the developed system. The eluent from the LC column was fractionated every minute (2μl) to 24 fractions, solvent in each fraction was evaporated and the residue was resolved for the second CE analysis. Since early eluent contained polar compounds, dynamic pH junction technique was applied to the CE analysis as an online sample preconcentration technique. Later eluting analytes were hydrophobic and hence sweeping was employed. The standard 54 compounds are successfully separated by this technique. Sixty three compounds among 118 which contained many non-UV absorbing compounds were successfully identified. The technique was applied to the analysis of cell extracts of B.subtilis or E.coli and many components were separated and more than 20 compounds were identified.An analytical method of flavin coenzymes was also developed with a micro LC system in combination with solid phase extraction with reversed phase monolithic silica column. Flavin coenzymes in E. coli were quanitated in the level of sub-μg/ml. Microchip electrophoresis with chemiluminescent detection was also developed for high sensitivity analysis of ATP.

建立了液相色谱-毛细管电泳二维分离系统，用于多组分混合物的一次分析。微液相色谱法作为第一步。第二步采用常规毛细管电泳系统。采用内径200μm、长25 cm或50 cm的反相硅胶整体柱，梯度洗脱，进行微量液相色谱分离。使用内径50μm、距检测器长50 cm的熔融石英毛细管进行CE分离。作为标准样品，使用了预期包含在细胞代谢物中的54或118种化合物的混合物。枯草芽孢杆菌或大肠杆菌的细胞提取物也被用来显示所开发的系统的分离能力。将来自LC柱的洗脱液每分钟（2μl）分馏为24个馏分，蒸发每个馏分中的溶剂，并将残留物分离用于第二次CE分析。由于早期的洗脱液中含有极性化合物，动态pH连接技术被应用于CE分析作为一种在线样品预浓缩技术。后来洗脱的分析物是疏水性的，因此采用了扫描。用该技术成功地分离了54种标准化合物。在118种含有大量非紫外吸收化合物的化合物中，成功鉴定了63种化合物。将该技术应用于枯草B和大肠杆菌细胞提取物的分析，分离出多种组分，鉴定出20多种化合物，并建立了反相硅胶整体柱固相萃取-微量液相色谱法分析黄素辅酶的方法。E.大肠埃希菌的定量水平为亚μg/ml。微芯片电泳荧光检测技术也被开发用于ATP的高灵敏度分析。