The use of nonaqueous solvents in capillary electrophoresis (CE) was investigated. Nonaqueous solvents used included methanol, ethanol, acetonitrile, dimethylformamide, and molten salts. Both indirect, and direct UV detection as well as electrochemical detection were examined. The behavior of electroosmotic flow and Joule heating was examined as well as selectivity patterns for a variety of inorganic and organic ionic species. Analytes examined included inorganic anions, alkali and alkaline earth metal ions, alkanesulfonates, alkyl sulfates, linear alkylbenzensulfonates, derivatized and free amino acids, and fatty acids. Selectivity was dependent not only on the nature of the solvent, but also on the nature and concentration of the electrolyte. With many inorganic ions completely reversed separation order relative to aqueous systems was observed. Although the addition of metal ions caused changes in resolution, complete separation of alkanesulfonates (C₂-C₁₆) and alkyl sulfates (C₈-C₁₈) was possible by a change from protic (methanol) to aprotic conditions (addition of acetonitrile). The partial separation of positional isomers of linear alkyl benzensulfonate homologues (C₁₀-C₁₅) was achieved in methanol/acetonitrile mixtures. The complete separation of alkali and alkaline earth metal ions, which has not been reported to date, and separation of potassium and ammonium were observed in a methanol/imidazole electrolyte. Significantly different migration order was achieved for dansylated amino acids compared to aqueous systems. Changes in selectivity of free amino acids were possible in basic and acidic electrolytes. Complete separation of a wide range of fatty acids (C₁-C₂₀) was possible in less than 20 min. The unique selectivity patterns in nonaqueous solvents was related to changes in solvation and ion-interaction effects. The ability to easily adjust selectivity via choice of electrolyte and to inject aqueous samples directly into the nonaqueous electrolytes suggests that such systems may offer significant advantages for many analytical problems. The results illustrate that when selectivity, efficiency, quantitation, and detection limits are taken into consideration, nonaqueous CE approaches should, in many situations, offer a unique alternative to aqueous based CE analyses. Electroosmotic flow was appreciable, and was cathodal in basic solutions. Reversal of electroosmotic flow was observed in the presence of an excess of a strong acid (HCl or HClO₄) in the separation electrolyte. The nature of electrolyte anion (chloride, perchlorate, and acetate) and solvent (methanol/acetonitrile mixtures) had significant effects on the electroosmotic flow in acidic conditions. Evidence was observed for ion adsorption (protons and anions) on to silica surfaces and ion-interaction in the electrolyte. Adsorption and ion-interaction could be used to control both the direction and magnitude of electroosmotic flow. Reproducibility of electroosmotic flow was good under appropriate experimental conditions (%RSD = 1.1).

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1994. / Vita. Abstract. Includes bibliographical references (leaves 222-229). Also available via the Internet.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

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The synthesis of the sodium salt of heptakis (2, 3-di-O-acetyl-6-O-sulfo)-β-cyclodextrin was modified to increase the isomeric purity to 98.5%. This salt was used to obtain the organic-solvent-soluble, single-isomer, charged tetrabutylammonium salt of heptakis (2, 3-di-O-acetyl-6-O-sulfo)-β-cyclodextrin. Its isomeric purity was higher than 99%, as determined by CE, and its structure was confirmed by NMR and ESI-MS analysis. The hydrophobic single-isomer cyclodextrin was utilized to separate the enantiomers of weak base analytes in aprotic media by capillary electrophoresis. The effective mobilities and separation selectivities follow trends observed with negatively charged cyclodextrins in amphiprotic solvents. The properties of the dissolved cyclodextrin are altered by its counter ion, thereby affecting the separations of enantiomers. The aprotic media allow the modification of the separation selectivity, since the binding strength of the enantiomers to the cyclodextrin is intermediate between that reported in aqueous and methanolic buffers.

Chromosomal mediated antimicrobial resistance in Neisseria gonorrhoeae can develop as a result of three main processes including the alteration of target enzymes, changes in transmembrane transport channels and active efflux pump function. Single nucleotide polymorphisms (SNPs) of target genes such as DNA gyrase (gyrA) and topoisomerase (parC), together with mutations in the promoter regions of the efflux pumps norM and mtr can confer resistance to the macrolides, penicillins and fluoroquinolones. These SNPs were analyzed using the SNaPshot method to allow for rapid detection of resistant isolates. Oligonucleotides were developed in the 5’ to the 3’ direction, ending one nucleotide adjacent to the specific SNP of interest. Single base extension reactions were performed and were detected using capillary electrophoresis. The SNaPshot procedure from Applied Biosystems employed in this study adds a single fluorescently-labelled nucleotide complementary to this SNP at the 3’ end by a primer extension polymerase reaction. Then using capillary electrophoresis, the labelled nucleotide is detected, enabling differentiation between A, C, T, or G. SNP results obtained were verified using DNA sequencing and both single and multiplexed reactions were carried out to increase the efficiency of the procedure. Spiked urine samples were also observed to determine if SNPs could be detected clinically. Single reactions enabled the characterization of all confirmed and relevant SNPs. With multiplex primer extension, multiple peaks were observed, each corresponding to one of the SNPs in the gene. This technique was explored for its applicability to detect SNPs of gyrA and parC mutations. Observable SNP detection limits were seen in spiked urine samples at 108 cells/mL in as early as 4 hours. DNA sequencing results confirmed the SNPs identity in each case. Thus, capillary electrophoresis using the SNaPshot protocol is another way to rapidly identify clinically resistant strains of Neisseria gonorrhoeae. This technique has also been shown to reduce analysis time compared to DNA sequencing and produces the same results. / February 2005

The synthesis of the sodium salt of heptakis (2, 3-di-O-acetyl-6-O-sulfo)-β-cyclodextrin was modified to increase the isomeric purity to 98.5%. This salt was used to obtain the organic-solvent-soluble, single-isomer, charged tetrabutylammonium salt of heptakis (2, 3-di-O-acetyl-6-O-sulfo)-β-cyclodextrin. Its isomeric purity was higher than 99%, as determined by CE, and its structure was confirmed by NMR and ESI-MS analysis. The hydrophobic single-isomer cyclodextrin was utilized to separate the enantiomers of weak base analytes in aprotic media by capillary electrophoresis. The effective mobilities and separation selectivities follow trends observed with negatively charged cyclodextrins in amphiprotic solvents. The properties of the dissolved cyclodextrin are altered by its counter ion, thereby affecting the separations of enantiomers. The aprotic media allow the modification of the separation selectivity, since the binding strength of the enantiomers to the cyclodextrin is intermediate between that reported in aqueous and methanolic buffers.