Collections > Electronic Theses and Dissertations > Development and Application of Microfluidic Capillary Electrophoresis-Electrospray Ionization Devices for the Analysis of Biological Samples
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The development and optimization of tools and techniques that characterize biological systems on the molecular level are necessary, and can provide significant benefits to society. Capillary electrophoresis (CE) has many advantages when analyzing biological samples, including low sample volume requirements, fast analysis times, and high separation efficiencies. Performing the CE separation on a microfluidic device can yield improvements due to integration of multiple functional components, automation, and efficient heat dissipation. The work describes the development of microchip capillary electrophoresis-electrospray ionization (CE-ESI) devices for various applications focused on the analysis of biological samples. First, microchip CE-ESI was coupled with commercial liquid chromatography (LC) to perform a multi-dimensional separation. A tryptic protein digest mix was utilized to probe the separation performance of the LC-CE-ESI system. In 50 minutes, a peak capacity of 1500 was observed. The technique was also used to characterize the glycopeptides of a monoclonal antibody. Increasing the mass spectrometer acquisition speed resulting in improved sampling in both the CE and LC dimensions. Microchip CE-ESI was also investigated for use in hydrogen deuterium exchange mass spectrometry (HX MS). A state of the art commercial LC system was compared to microchip CE-ESI for both separation and deuterium uptake/recovery performance. The microchip CE-ESI method proved to be much faster than the LC counterpart with improved separation performance. For the CE-MS method, a peak capacity of 62 was observed in a 1 minute separation, compared to a 4 minute LC-MS separation with a peak capacity of 31. Overall, the deuterium uptake and recovery between the two methods were similar. Sequence coverage and peptide redundancy scores for the CE-MS method were lower compared to the LC-MS, due to the narrow duration of the CE-MS peaks, lower sensitivity of the mass spectrometer used, and a much smaller injection volume. Future improvements to the mass spectrometer as well as integrating sample processing with the CE-ESI microchip will likely improve these metrics. Next, microchip CE-ESI at low temperatures was investigated. A Peltier device was used to cool the microchip; temperatures between 30 °C and 0 °C were investigated. At lower temperatures, a linear increase in theoretical plates was observed, as was an increase in analyte migration time. Furthermore, lowering the temperature of the separation decreased deuterium back exchange, indicating the potential of low temperature microchip CE-ESI for HX MS experiments. Finally, integrated sample processing was investigated by coupling a solid phase extraction (SPE) bed on a CE-ESI microchip. Injection broadening incurred during the transfer of analyte from the SPE bed to the CE separation channel resulted in a decrease in the separation performance. Utilizing transient isotachophoresis (tITP) focusing following the transfer improved the separation performance of the SPE-CE-ESI method and maintained the pre-concentration values. For a four peptide mix, enrichment values of between 70 and 2500 were observed. The method was applied to a phosphorylase B tryptic digest. A peak capacity of 147 was observed for a 5 minute separation. Over two orders of magnitude of pre-concentration was observed with a simultaneous increase in peak capacity. However, the number of observed peptides in the electropherogram decreased from 150 to 97. Overall, the work described in this thesis illustrates the potential of microchip CE-ESI for the analysis of biological samples, demonstrating high separation efficiencies, fast analysis times, and integration with other functional elements.