UBC Theses and Dissertations
Applications of capillary electrophoresis - mass spectrometry interfaced by a flow-thourgh microvial electrospray ionization sprayer Zhao, Shuai Sherry
Capillary electrophoresis – electrospray ionization – mass spectrometry (CE-ESI-MS) combines the superior separation capability of CE and detection and characterization ability of MS. Different CE separation modes can be coupled to ESI-MS, employing an interface with a flow-through microvial. In the first part of the thesis, recent development of CE and CE-MS applications in the analysis of complex samples are reviewed. Capillary isoelectric focusing (cIEF) is an important tool for the separation and characterization of amphoteric molecules based on isoelectric points. Minute structural changes on a large protein can result in changes in isoelectric point, and the changes can be detected by slab gel isoelectric focusing or capillary isoelectric focusing. A systematic study on the interactions among carrier ampholytes, sample media and capillary inner coatings was carried out to provide guidelines for choosing feasible combinations that can achieve isoelectric focusing and successful chemical mobilizations. Within the 0.1%-1% (w/v) carrier ampholytes concentration range, small forward EOFs will ensure a higher chance of good focusing and successful electrophoretic mobilization, while a negative EOF will hinder these processes. Feasible combinations of experimental conditions are summarized. Using the optimized conditions, we reported the direct observation of the shape of focused ampholyte bands in the cIEF process by online cIEF- ESI-MS. The ampholyte bands directly detected by MS have the potential to enable a more accurate pI determination for unknown amphoteric molecules. Immunoglobulin G from rabbit serum is used to demonstrate this possibility. In Chapter 6, a CE-MS method was developed to monitor the concentration variations of major nutrients and/or metabolites in human embryonic stem cell CA1S culture medium over a culturing cycle. Concentration changes for nutrients and/or metabolites in the culturing media provided information on the cell growth behavior without destructing living cells. In the last part of the thesis, an atmospheric ion lens was applied to the flow-through microvial CE-ESI-MS interface to improve the electrospray ionization and sampling efficiency. A mixture of amino acids was tested to show the increased signal-to-noise ratios. The atmospheric ion lens also gives more flexibility when choosing the EOF and chemical modifier flow rates.
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Attribution-NonCommercial-NoDerivs 2.5 Canada