Multicomponent Analysis by Micro-Liquid Chromatography-Capillary Electrophoresis

建立了液相色谱-毛细管电泳双向分离系统，用于一次分析多组分混合物。微液相色谱法是第一步。第二步采用常规毛细管电泳系统。采用内径200μm、长度25cm或50cm的反相单片硅基色谱柱，采用梯度洗脱技术进行微LC分离。采用内径50μm、长度50cm的熔融石英毛细管进行CE分离。作为标准样品的54或118种化合物的混合物，预计将包括在细胞的代谢物。还用枯草芽孢杆菌或大肠杆菌的细胞提取物来证明所建立的系统的分离能力。LC柱洗脱液每隔1分钟（2μl）分24个馏分，每个馏分蒸发溶剂，分离残留物进行二次CE分析。由于早期的洗脱液中含有极性化合物，动态pH结技术作为一种在线样品预富集技术被应用于CE分析。后来的洗脱物是疏水性的，因此采用了清扫。该方法成功地分离了54种标准化合物。118个化合物中有63个化合物不吸收紫外线。将该技术应用于枯草芽孢杆菌和大肠杆菌的细胞提取物的分析，分离出许多成分，鉴定出20多种化合物。建立了反相硅胶整体柱固相萃取相结合的微量液相色谱系统分析黄素辅酶的方法。以亚μg/ml水平定量测定大肠杆菌中黄素辅酶的含量。微芯片电泳与化学发光检测也被开发用于ATP的高灵敏度分析。

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.