Identifier

etd-11122012-170655

Degree

Doctor of Philosophy (PhD)

Department

Chemistry

Document Type

Dissertation

Abstract

The goal of this dissertation is to investigate electroosmotic flow (EOF) and electric field dynamics during capillary electrophoresis (CE) experiments using methods based on periodic photobleaching of fluorophores added to the separation buffer at nanomolar concentrations. The methods provide time resolved EOF and local electric field information during an experiment, which can be applied to fundamental studies to provide better understanding of CE techniques. The potential of the EOF monitoring method to improve CE migration reproducibility was investigated in Chapter 2. The EOF monitoring method and four other methods from the literature were applied to the same electrophoretic separations, and their performance for improving reproducibility was compared. The EOF monitoring method significantly improved migration reproducibility, in general; however, much simpler neutral marker method performed nearly well. Biological sample adsorption is a common cause of EOF variability and poor reproducibility for CE. In Chapter 3, the effects of biological samples on EOF dynamics were investigated. Model compounds representing major components of a biological cell and complex biological samples were introduced into the CE system while EOF was monitored continuously. Due to sample adsorption, EOF rates decreased and vacancy peak widths, used for EOF monitoring, increased. It was found that protein molecules had the greatest impact on EOF. Discontinuous solutions in a capillary (zones of different pH, ionic strength or composition) result in generation of different EOF and local electric fields down the length of the capillary. The EOF monitoring method was expanded by adding a charged marker (fluorescein), and this improved method was employed to investigate EOF dynamics and local electric field changes during CE with discontinuous solutions, which were generated by introducing a low ionic strength buffer zone into the capillary. Faster EOF rates in the capillary and faster fluorescein electrophoretic velocities within the sample plug were observed due to high local electric field. Unexpected fluorescein concentration changes were observed during the experiments. These observations led to use of computer simulations in an attempt to understand and reproduce the electrophoresis results. The simulation results, which were obtained using Simul 5.0 indicated the experimental results are consistent with the CE theory.

Date

2012

Document Availability at the Time of Submission

Release the entire work immediately for access worldwide.

Committee Chair

Gilman, S. Douglass

DOI

10.31390/gradschool_dissertations.1721

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