Improving sensitivity for capillary electrophoresis for the analysis of physiological samples

The focus of this research is the development of methods to improve sensitivity and detection limits of CE for the analysis of physiological samples. pH-mediated stacking is an online preconcentration technique used to titrate high-ionic strength biological samples to low-ionic strength and induce field-amplified sample stacking. An investigation of the mechanism pH-mediated stacking of biologically important anions was first conducted in order to identify separation parameters critical to achieve the high peak efficiency and resolution needed for lowering detection limits. Peak efficiency was highly dependent on background electrolyte pH and analyte mobility, and was shown to increase with increasing background electrolyte and sample ionic strength. Information learned in this study was used to develop a new enhanced pH-mediated stacking technique in which a discontinuous buffer system is used to manipulate pH and achieve multi-million plate peak efficiencies for anions. The application of pH-mediated stacking in the determination of biomarkers of oxidative DNA damage in microdialysis samples of the rat cerebral cortex was also demonstrated. The detection limits and sensitivity necessary for measuring biomarker concentration in microdialysates by CEEC could not be achieved without the use of pH-mediated stacking. Using this method, the concentration of 8-oxoguanine in vivo was determined to be 3.2 nM. In order to improve selectivity and gain more qualitative information with CEEC, an integrated dual-series electrode system was also designed. Both series oxidation and series oxidation-reduction modes were demonstrated, and the system was successful in distinguishing between analytes based on half-wave potentials. The response for the four phenolic acids was linear over the concentration range tested (10--100 muM), and the detection limit was ∼1 muM for phenolic acids.