Date of Award

Summer 2022

Document Type

Open Access Thesis

Department

Biomedical Engineering

First Advisor

Guiren Wang

Abstract

The utility of field amplified sample stacking and pulsed field electrophoresis toward improving electrophoretic outcomes was studied with regards to the electrophoretic separation of three organic dyes rhodamine B, 2’,7’-dichlorofluorescein, and fluorescein salt in microchip electrophoresis conditions. In this study, there were four experimental groups: nonstacking nonpulsed, nonstacking pulsed, sample stacking nonpulsed, and sample stacking pulsed.

Electrophoretic outcomes were evaluated by examining the electrophoretic separations under an epifluoresence detection method, plotting the signal intensities vs. time, and comparing separation resolutions and signal-to-noise ratios between experimental groups. From this it was shown that sample stacking nonpulsed conditions yielded the best outcome when evaluating the signal intensity vs. time plot. At a distance of 0.750 mm from the microchannel channel cross-section, the separation resolution between RB and DCF was 0.944 in the sample stacking nonpulsed group which was an improvement compared to the separation resolution of 0.885 between dyes RB and DCF in the nonstacking nonpulsed group. However, the separation resolution between DCF and FS in the sample stacking nonpulsed group was worse than that of the same dyes in nonsample stacking nonpulsed group at 0.3933 compared to 0.885. The sample stacking nonpulsed group exhibited the fastest electrophoretic migration of the organic dyes with FS reaching at 0.750 mm in 0.653 seconds and nonsample stacking pulsed was the slowest group with regards to electrophoretic migration with FS reaching 0.750 mm in 0.959 seconds.

The signal intensities of the fluorescent dyes in the sample stacking groups were significantly lower than those of the nonsample stacking groups and so the potential roles of electrokinetic instability and electrokinetic mixing in preventing signal amplification were investigated. Electrokinetic instability was shown to occur at electric field strengths equal to and greater than 20 kV/m in our MCE setup, suggesting that electrokinetic instability and/or electrokinetic mixing may have been a factor in preventing signal amplification in the FASS groups.

These hindrances to achieving optimal electrophoretic outcomes could be overcome by reducing the switching frequency in pulsed field DC electrophoresis and by reducing the gating and injection voltage in sample stacking conditions. This way, mixing could be reduced in nonsample stacking pulsed and sample stacking pulsed groups and EKI could be reduced in sample stacking and sample stacking pulsed groups and separation outcomes with higher separation resolutions and signal-to-noise ratios could be achieved.

Rights

© 2022, Travis Geoffrey Stewart

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