One of the major barriers to the wider implementation of CE technology, either in capillary or microchip form, is poor concentration sensitivity when conventional UV absorbance detection is employed. This project involves the investigation of three separate approaches to improve the sensitivity and utility of capillary electrophoresis (CE) methodologies for the biochemical and clinical laboratories. The proposed work involves precise manipulation of local conditions within capillary tubes to affect molecular-level processes that occur during CE separation processes. In particular, we will investigate the use of rapid polarity switching (RPS) and also several stacking methodologies to enhance the rate(s) of in-column reaction(s) and to improve concentration sensitivity of CE-based analyses. Most of the proposed work involves in-line chemical reactions of small molecules using electrophoretically mediated micro- analysis (EMMA). In prior and on-going work, we have shown that RPS has proven to be a useful methodology to quickly mix the reagents of one small molecule reaction, the Jaffe reaction between creatinine and picrate, and we will extend the use of this approach to other small molecule systems. Complementary work with transient isotachophoretic stacking (tITP) of the in-line generated product of the Jaffe reaction via a concentrated plug of hydroxide appears to be quite effective, and will continue, but simpler tITP systems will also be explored to gain fundamental understanding of the stacking dynamics. The third phase of this work will explore the use of both micellar electrokinetic (MEKC) modes of on-line stacking and separation for in-capillary reactions forming uncharged products. Through a new collaboration, we will use nuclear magnetic resonance (NMR) spectroscopy to investigate and understand the interactions between micelles and analytes during stacking and separation processes. In all, this project will elucidate the general capability of various dynamic in-line techniques to enhance the sensitivity attainable with small molecule EMMA, and may lead to new approaches for rapid, low-volume bioassays in the clinical laboratory. NARRITIVE This project involves the investigation of three separate approaches to improve capillary electrophoresis (CE) methodology for the biochemical and clinical laboratories. Partly because of the characteristically poor concentration sensitivity, CE-based methods, in either capillaries or on microchips, have not been fully developed and this compelling mode of analysis has therefore been underutilized. The proposed work is aimed at increasing the sensitivity of CE- based modes of analysis either by manipulating, through one of several means, the local concentration of ions within discrete reagent zones in a capillary, or by increasing the rate of in-column reactions that are part of the assay method. This work may lead to development of new approaches for rapid, low-volume and sensitive bioassays in the clinical laboratory. ? ? ?
