Doctoral Dissertations

Date of Award

12-1991

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Chemistry

Major Professor

Michael J. Sepaniak

Abstract

Capillary electrokinetic separation methods have become increasing popular in the past decade due to their high efficiency, short analysis times, and ability to analyze minute samples. These advantages are a direct result of performing separations in small diameter capillaries, typically less than 100 μm in internal diameter. These capillaries are able to dissipate heat produced by the passage of current through the electrophoretic medium more effectively than other electrophoretic separation formats, allowing the use of very high electric field strengths. Unfortunately the size of the capillaries employed results in band volumes of less than 10 nL, which often contain less than 1 picomole of analyte, making detection of eluting bands a formidable task. Furthermore, on-column detection is usually required to prevent band broadening. Laser-based fluorometric detection is one of the most successful detection schemes that has been applied to capillary electrokinetic separation methods. The high power of laser beams result in high sensitively in spite of the short pathlengths created by on-column detection. Furthermore, laser beams are highly collimated, allowing them to be easily focused into the flow channel of the capillary. The major limitation of this method is the lack of available fluorophores. To increase the applicability of this method, fluorescent labels are often attached to the analyte prior to detection. However, this can require either time consuming sample preparation or the use of a post column reactor. A more desirable situation would involve the labeling reaction occurring during the separation process. Presented herein are two labeling methods that allow the reaction to be performed concurrent to separation. The detection of proteins and surfactants is performed using fluorescent hydrophobic probes. Similarly, the detection of metal ions is accomplished using fluorescent chelating agents. The detection of fluorescently labeled species can usually be performed at a single wavelength; however, a mixture of native fluorophores may require monitoring several different wavelengths to maximize sensitivity. This is most conveniently accomplished using a multichannel detector such as a diode array. Multichannel detection permits the optimization of emission wavelength and bandpass to enhance sensitivity and detector selectivity. Described herein is the characterization of a fluorometric photodiode array detection scheme for capillary electrokinetic separation methods. Analytical figures of merit and a discussion of the ability of multichannel detection to improve analytical selectivity will be presented.

Download
Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Share

COinS