No description available.

No description available.

No description available.

No description available.

Velocity-difference induced focusing (V-DIF) with a dynamic pH junction in capillary electrophoresis (CE) using a sample with a pH different from that of the background electrolyte (BGE) was developed in our group, but the mechanism was not well understood. In this work, the mechanism of this focusing technique was first studied using an appropriate dye to monitor the pH of the BGE and the sample during the focusing process. A mechanism was proposed based on the experimental results. This technique was then applied to serotonin to improve the detection limit when CE was used with a UV absorption detector. It was also applied to focus amino acids, peptides, and proteins to improve the concentration sensitivity. It is found that the pKa rather than the pI of the analytes is the key criterion for selecting the pH for the sample and for the BGE to obtain the optimum focusing for these molecules. Since UV detection only provides migration time information, more structure information is obtained by using a photodiode array (PDA) and mass spectrometer (MS) for peak identification. Comparisons were made between the PDA detection and MS detection for aromatic amino acids with V-DIF using a dynamic pH junction. This V-DIF technique was then applied to non-aromatic amino acids with MS detection. It was used at low pH with positive ESI-MS detection and at high pH with negative ESI-MS ionization. The results of the two methods were compared and discussed. Finally, the preparative operation of continuous flow counterbalanced CE (FCCE) was studied. The effects of larger sample volumes and multiple capillary systems on improving the purification yield were investigated.

This thesis includes a theoretical and experimental analysis of electrophoresis of colloidal particles in non-Newtonian polymeric fluids with shear-rate dependent viscosities. A model is derived that predicts field dependent electrophoretic mobility in shear-thinning Carreau uids. The latter effect is experimentally investigated for submicron particles in solutions of linear polyacrylamide using capillary electrophoresis. The mobilitiies of the particles studied in these solutions did not depend on field strength, yet the mobilities were consistently an order of magnitude greater than in water and glycerol solutions with similar bulk viscosities. The increase in particle mobility could be attributed to several mechanisms, however it is consistent with the depletion of polymer apparent viscosity of the fluid as it migrates by electrophoresis is the same as experienced by the particle in Brownian diffusion. A comparison of particle mobility in both glycerol and polyacrylamide solutions to diffusion coefficients of the particles measured by dynamic light scattering supports this conclusion.

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.

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.

Velocity-difference induced focusing (V-DIF) with a dynamic pH junction in capillary electrophoresis (CE) using a sample with a pH different from that of the background electrolyte (BGE) was developed in our group, but the mechanism was not well understood. In this work, the mechanism of this focusing technique was first studied using an appropriate dye to monitor the pH of the BGE and the sample during the focusing process. A mechanism was proposed based on the experimental results. This technique was then applied to serotonin to improve the detection limit when CE was used with a UV absorption detector. It was also applied to focus amino acids, peptides, and proteins to improve the concentration sensitivity. It is found that the pKa rather than the pI of the analytes is the key criterion for selecting the pH for the sample and for the BGE to obtain the optimum focusing for these molecules. Since UV detection only provides migration time information, more structure information is obtained by using a photodiode array (PDA) and mass spectrometer (MS) for peak identification. Comparisons were made between the PDA detection and MS detection for aromatic amino acids with V-DIF using a dynamic pH junction. This V-DIF technique was then applied to non-aromatic amino acids with MS detection. It was used at low pH with positive ESI-MS detection and at high pH with negative ESI-MS ionization. The results of the two methods were compared and discussed. Finally, the preparative operation of continuous flow counterbalanced CE (FCCE) was studied. The effects of larger sample volumes and multiple capillary systems on improving the purification yield were investigated.

Thesis (PhDAppliedPhysics)--University of South Australia, 2000.