Miniaturized Fractionation and Purification of Analytes of Interest in Complex Mixtures: Protein Enrichment and Purification with Capillary Electrophoresis and Identification of Pesticide Chemicals in Bio-oils
Doctor of Philosophy
Dr. Ken Yeung
The ability to isolate and detect an analyte of interest is vital for many different facets of life today, from disease detection to environmental safety. The goal of the method development discussed within this thesis is to isolate and identify unique analytes from complex matrices and to do so in a scaled down approach. Removal of an analyte from a diverse background of molecules is often required for successful detection and determination of analyte properties. As well, many samples are only available at low volumes and at trace levels so miniaturized techniques that only require small sample volumes are designed and discussed herein. Two unique classes of analytes are examined with one common theme of isolation for successful detection.
Proteins and peptides are enriched and purified from permanently charged and buffering ions, which are common biological contaminants, using discontinuous buffers with capillary electrophoresis. This method only uses sub-microliter quantities of sample and exploits the isoelectric point of the biological macromolecules in buffers of different pH. Successful removal of sodium chloride, TRIS, phosphate, and MES is demonstrated. Subsequent enhanced mass spectral detection and separation of a protein mixture exhibited the improved analyte detection by employing discontinuous buffers. Magnetic beads are used to isolate phosphorylated peptides at micro to nanoliter volumes by covalent derivatization of the post-translational modification and selective enrichment, further demonstrating the online CE sample preparation capabilities.
The second challenging sample examined is bio-oil, a complex mixture of thousands of chemicals. The novel pesticide properties of this sample are explored as the chemical complexity of pyrolysis bio-oil from tobacco leaves is reduced through isolation by solid phase extraction, liquid-liquid extraction, and heating. Mass spectrometry is used to identify the compounds after separation by gas chromatography. Three problematic microorganisms in Canada, S. scabies, C. michiganensis, and P. ultimum, are negatively affected by the bio-oil. It is determined that the most abundant phenolic species are not responsible for the observed pesticide activity from the bio-oil. A few minor components are identified that could contribute to the observed antimicrobial activity, but no abundant, active chemicals could be identified with the current technology.
Booker, Christina J., "Miniaturized Fractionation and Purification of Analytes of Interest in Complex Mixtures: Protein Enrichment and Purification with Capillary Electrophoresis and Identification of Pesticide Chemicals in Bio-oils" (2012). Electronic Thesis and Dissertation Repository. Paper 373.