Email: sarnett@ku.edu
Sensitivity Enhancement in Capillary Electrophoresis for the Analysis of Physiological Samples
The focus of my research is primarily the development of capillary electrophoresis (CE) techniques for the analysis of biological samples. One of my areas of research is the investigation of a technique termed pH-mediated stacking of anions, or “base stacking.” CE has become a powerful analytical technique for the analysis of physiological samples such as plasma and microdialysate. However, sample destacking can occur during the analysis of these high-ionic strength samples, resulting in poor separation efficiency and reduced sensitivity. This technique has previously been developed in our group to analyze microdialysate samples and achieve on-line preconcentration of analytes. Base stacking has been successfully used to analyze compounds in microdialysate samples, the mechanism has not been fully investigated. To elucidate this mechanism, six anions in Ringer's solution were analyzed by CE-UV with base stacking. Peak efficiency was shown to increase with decreasing sample ionic strength and increasing background electrolyte (BGE) ionic strength. The minimum length of base injection required to stack each analyte was shown to increase as a function of analyte mobility. Resolution was shown to degrade with increasing injection times, however when resolution was not an issue, sample injection time could be increased to 6 minutes. A linear relationship between peak area and sample injection time was demonstrated and sub-micromolar detection limits were achieved with CE-UV. Substantial differences in efficiency and the optimal injection time ratio of sample to base were observed with different BGEs. Peak efficiencies of more than 6 million plates can be achieved by manipulating pH of the BGE, which is approaching theoretical CE peak efficiency. A discontinuous BGE system was subsequently developed for improved peak efficiency and resolution for multi-million plate separations. Future work will include further development of the pH-mediated stacking technique with multi-million plate counts for application to a wider range of both cationic and anionic analytes. Information gained in these studies will allow the broader development of CE separations for physiological samples.
I have also been involved in the development of a rapid and sensitive method to quantitate 8-oxoguanine (8oxoG) and 8-hydroxy-2’-deoxyguanosine (8OHdG), biomarkers for oxidative DNA damage, in microdialysate samples using CE with electrochemical detection (EC). This method can be used to monitor biomarker concentrations in animal models of stroke or myocardial infarction with higher temporal resolution than previous analytical techniques due to the small sample volume required by CE. On-column anodic detection is performed with a carbon fiber working electrode, and a laser-etched decoupler was used to isolate the working electrode from the separation current. Standards in Ringer’s solution and rat microdialysate samples were concentrated on-column, with no sample pretreatment, using base stacking. The detection limit for 8oxoG and 8OHdG using this method was two orders of magnitude lower than previously published for CE-EC with a 3-fold improvement in temporal resolution over existing LC techniques. Basal 8oxoG concentration was determined to be 3.2 nM in the rat cerebral cortex extracellular fluid. (No 8OHdG was detected.) The method is currently being applied to the in vivo analysis of 8oxoG and 8OHdG concentration during induced ischemia-reperfusion to study mechanisms of oxidative DNA damage during an animal model for stroke. The CE-EC method for 8-OHdG will also be applied to rat heart microdialysate collected during an animal model for myocardial infarction.
I was also involved in the design of a carbon microdisk electrode for use in an amperometric immunoselective detection system for CE at Dublin City University. I designed screen-printed electrodes for use in an environmental assay for pesticides in the triazine class. Once assay conditions are optimized for a capillary-based separation, the method could be adapted to a disposable microchip system for on-site environmental analysis of triazines. Currently, the design and manufacture of microelectrodes has been optimized with a new carbon ink formulation. Future work will include the optimization of surface immunochemistry for detection of atrazine.
