Development of novel separation techniques for biological samples in capillary electrophoresis

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Chang, Huan-Tsung
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Edward S. Yeung
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The Department of Chemistry seeks to provide students with a foundation in the fundamentals and application of chemical theories and processes of the lab. Thus prepared they me pursue careers as teachers, industry supervisors, or research chemists in a variety of domains (governmental, academic, etc).

The Department of Chemistry was founded in 1880.

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This dissertation includes three different topics, general introduction of capillary electrophoresis (CE), gradient in CE and CE in biological separations, and capillary gel electrophoresis (CGE) for DNA separation;Factors such as temperature, viscosity, pH and the surface of capillary wall affecting the separation performance have been well demonstrated in Chapters 2 to 4. A pH gradient between 3.0 and 5.2 is useful to improve the resolution among eight different organic acids. Flow gradient due to the change in the concentration of surfactant which is able to coat to the capillary wall to change the flow rate and its direction is also shown as a good way to improve the resolution for organic compounds. Temperature gradient caused by joule heat has been shown by voltage programming to enhance the resolution and shorten the separation time for several phenolic compounds. We also showed that self-regulating dynamic control of electroosmotic flow in CE by simply running separation in different concentration of surfactant has less matrix effect on the separation performance. One of the most important demonstrations in this dissertation is that we proposed on-column reaction which gives several advantages including the use of small amount of sample, low risk of contamination, time saving and kinetic feature. We used this idea with laser induced fluorescence (LIF) as a detection mode to detect an on-column digestion of sub-ng of protein. This technique also has been applied to single cell analysis in our group;An entangled polymer, poly(ethylene oxide), was used to separate 11 fragments of Hae III digest of [phi]X174RF DNA. The effects of voltage, gel concentration and molecular weight of polymer are well studied. Also, the effects of the nature and concentration of labeling dyes, and the capillary length on the resolution of DNA fragments has been studied with different lasers. DNA sequence of PGEM/U with four different terminal dyes has been shown. Mixed gel matrices provides single-base resolution and better separation performance for DNA fragments up to 500 base pairs have been shown;Lastly, CE with LIF has been shown as a powerful technique to detect attomol levels of amine compounds, which are neurotransmitters. This method also has been used to determine the norepinephrine and epinephrine at femtomole level in single adrenomedullary cells.

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