Studies in capillary electrophoresis

This study employs either free solution capillary electrophoresis or micellar electrokinetic chromatography (MEKC) in three selected application areas; analysis of small inorganic anions in water, analysis of humic acids in water and soil, and the separation of tuberculosis and related microorganisms. Three independent detection strategies; on capillary detection using absorbance (both direct and indirect) and fluorescence (direct) allowed data to be acquired for each application.;A critical evaluation of two common capillary electrophoresis injection techniques displayed dramatic differences in sensitivity and linearity. Compared to hydrodynamic injection (gravity), electrokinetic injection allowed increased sensitivity, but quantitation suffers from poor linearity and bias of sample injection amount. With electrokinetic injection, an increase in sample ionic strength or addition of background ions can significantly reduce the peak area which created quantitation problems. Improvement in the linearity and quantitation of electrokinetic injection was possible via the addition of sodium octane sulfonate, as an ionic strength normalizer, to the sample and calibration standards. Linearity of anion standards with electrokinetic injection and sodium octane addition was between 0.45-90 ppm. Without the sodium octane sulfonate, linearity was not possible between any three calibration points.;The environmentally significant humic and fulvic acids were analyzed by free solution capillary electrophoresis. Relative differences between absorbance detection, at 214 nm, and fluorescence detection, at 313 excitation wavelength and emission above 400 nm, allowed insights into peaks that are selectively fluorescent at the fluorescent wavelength pair. to determine the relative absorbance and fluorescence signals of the humic acids, the same capillary, buffer and electrophoresis conditions were chosen for two separate capillary electrophoresis detectors. With different fulvic acid samples, capillary electrophoresis and fluorescence detection uncovered the selective detection of new peaks that are not detected by absorbance at 214 nm. Using fluorescence detection combined with capillary electrophoresis, a minimum of eight separate peaks are resolvable for fulvic acids and fingerprinting from different fulvic acid origins was possible.;Mycobacterium tuberculosis and other related organisms were analyzed by micellar capillary electrophoresis to give a separation of derivatized fatty acids from the cell walls of the mycobacteria. The fatty acids from the cell walls were extracted and then the acid group was derivatized with the fluorescent label, 4 bromo methyl 6,7 dimethoxy coumarin. Unlike the low carbon chain length fatty acids, it was found that the high molecular weight fatty acids in the cell wall are both weakly fluorescent in aqueous conditions and they do not partition into the aqueous phase once in the sodium dodecyl sulfate micelle. High resolution separations of different mycobacteria cell wall fatty acids show differences in the number and position of the peaks for the lower molecular weight, micelle partitioning, fatty acids. It was necessary to investigate more polar surfactants, i.e. sodium laureth-13 carboxylate, to allow partitioning of the hydrophobic mycolic acids into the aqueous phase. It was found that capillary electrophoresis can be used with the sodium laureth-13 carboxylate surfactant to separate mycolic acids and different species of mycobacteria, i.e. tuberculosis.