Visualization of electrophoretically mediated in-capillary reactions using a complementary metal oxide semiconductor-based absorbance detector

Abstract

A method for real-time visualisation of reactions performed in-capillary by the technique of electrophoretically mediated microanalysis (EMMA) is described, using a two dimensional imaging detection system. The UV absorbance detector is based on a complementary metal oxide semiconductor (CMOS) active pixel sensor. Imaging of analyte peaks absorbing at 200 nm and migrating over length of 14 mm in the capillary dimension allowed measurement of velocities and lengths of reactant and product zones. By contrast with use of single point detection, velocities of species generated by reaction anywhere within the capillary are readily measured with CMOS imaging: this is of particular benefit for EMMA experiments where reaction occurs during zone overlap. For the oxidation of glutathione by hydrogen peroxide, reaction times were varied over the range 0.5–20 s by changing voltages for electrokinetic injection and zone migration, and reactant and product peak areas were obtained for kinetic analysis of the reaction. The use of EMMA conditions with CMOS imaging allows the whole process of reaction, separation and quantification to be carried out in nanolitre volumes on-capillary in a single run on a time scale of less than 5 min.