Multicomponent Analysis by Micro-Liquid Chromatography-Capillary Electrophoresis

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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.