Doctoral Dissertations

Date of Award

8-1999

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Chemistry

Major Professor

Michael J. Sepaniak

Committee Members

David C. Joy

Abstract

During the past few decades, the development of chromatography and electrophoresis has been an essential factor for the significant advancements achieved in biotechnology. Today, efforts continue to improve upon the accuracy, speed, and precision of these methods. DNA analysis by capillary electrophoresis (CE) is a good example of how the best attributes of different methods can be brought together to develop analytical methodology that offers significant improvements over existing technology. Despite the many attributes of CE, method validation continues to be problematic. In order to reproducibly achieve high efficiency and good resolution of DNA fragments, deactivating the surface of the separation column is essential. There exist many variations to the original method first suggested in 1985 by Stellan Hjerten. In this work, scanning electron microscopy (SEM) was utilized to examine various columns coated with non cross-linked polyacrylamide. At very low concentrations of acrylamide (~2.5%), no noticeable polymer layer is present. However, as the concentration of acrylamide exceeds 2.5%, a noticeable thickness and non-uniformity is observed. The use of coated columns can then be employed for the size-, selective capillary electrophoresis (SSCE) separations of DNA fragments. Since the development of this method in the early 1990’s, several papers have discussed the theoretical aspects of utilizing aqueous solutions of soluble polymers for the separation of DNA fragments. However, the instrumentation required to directly evaluate fundamental processes such as variance in SSCE has been limited by the lack of novel instrumentation necessary to perform these experiments. In this work, experimental measurements of variance under static and dynamic conditions are reported. The determination of static diffusion coefficients and their contribution to total band variance is reported. The fact that diffusion accounts for less than half of the total variance observed led to the conclusion that other processes occurring during DNA fragments separations (i.e., DNA - polymer entanglement/disentanglement interactions) contribute significantly to band variance. Upon optimizing conditions for DNA analyses by SSCE, a novel class of cyanine intercalation dyes reported to offer superior fluorescence sensitivity relative to ethidium bromide was also evaluated in this work. Despite an improvement in sensitivity of DNA/dye complexes when employing the cyanine intercalation dyes, the labeling mechanisms and kinetics proved to be problematic in achieving appreciably lower detection limits by CE.

In another area of research, the potential of utilizing modestly selective stationary phases on microsensors was evaluated. Phases commonly employed in gas chromatography (GC) and liquid chromatography (LC) were bonded onto prepared silicon substrates. The relative affinity and selectivity of these phases for semivolatile organic compounds was determined by exposing these “sensors” to solutions followed by analysis by gas chromatography/mass spectrometry (GC/MS). It was found that improving wettability of the substrate prior to phase deposition was essential to achieve uniform films. Although the relative affinity and selectivity of these films are modest, these phases may be suitable for part of a higher-order, generalized approach to sensing.

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