Capillary Electrophoretic Methods for Protein and Microorganism Separations

Capillary electrophoresis (CE) is an analytical technique for the separation of molecules in a capillary under an applied electric field. CE generates high resolution separations in relatively short analysis times, with low reagent, and sample volume requirements. Two of the most common modes of CE are capillary zone electrophoresis (CZE) and capillary isoelectric focusing (cIEF). CZE separates analytes based on their size-to-charge ratios. It is the most common mode of CE due to its ability to separate molecules with high resolution and relative simplicity. cIEF is an orthogonal technique that separates analytes based on their isoelectric point (pI), which is the pH at which the average net charge of the molecule is zero. CZE and cIEF offer distinct advantages for high resolution separations of molecules in numerous fields including pharmaceutical, proteomic, genomic, and microbial analysis. CE instrumentation typically employs optical detection methods. However, optical detectors limit the information that you can obtain about the analytes. In this work, I improve instrumentation and methods for CE separations with two alternative detection schemes. (1) I developed an in-line cIEF interface to remove the concentration of ampholytes required for cIEF prior to MS detection. With the interface, an over 90% increase in area under the curve protein signal was observed. (2) I developed methods that enabled separation and collection of intact microorganisms for subsequent offline detection and analyses of cultured and uncultured species. These data represent significant improvement vs. existing methods. These efforts highlight the utility of CE for a diverse range of analytes with enhanced sensitivity and characterization.