A Novel Method For The Sequential On-line Analysis Of Enzyme Reactions Based On Capillary Electrophoresis And Its Application

We have developed an easy-to-operate and effectiye method for performing the sequential on-line analysis of enzyme reactions based on capillary electrophoresis. The system was constructed by passing two capillaries through a sample vial at a distance of5μ m. Direct on-line sample injection and sequential CE analysis were achieved by periodically switching the high-voltage power supply off and on without any physical disturbance of the capillary inlet, The sample was injected via concentration diffusion with in-column derivatization of the amino acids occurring at the interface of the capillaries. The effects of the separation voltage and the injection time on the sequential analysis of a standard mixture of the amino acids alanine (Ala) and glutamate (Glu) were investigated, and the calibration curves of the peak area versus the sample concentration were obtained. High reproducibility of the sequential injections was demonstrated with RSD values (n=20) of1.01%,1.25%and0,80%for peak height, peak area and migration time, respectively. Using the presented method, we have studied a GPT-catalyzed enzyme reaction with Ala as one of the substrates and Glu as one of the products. A sequential on-line CE enzyme assay was carried out by simultaneously monitoring the Ala consumption and the Glu formation every30s from the beginning to the end of the reaction. The Michaelis constants for the reaction were obtained and were found to be in good agreement with the results of traditional off-line enzyme assays. Therefore, our method has great potential for usage in sequential on-line CE analysis of chemical reactions with in-column chemical derivatization of the analytes for UV or LIF detection.