Development and application of microanalysis NMR methods

The goals of this dissertation were to develop anionic capillary isotachophoresis (cITP) coupled with microcoil NMR detection. This technology was used for the analysis of heparin oligosaccharides and to provide insights into the behavior of ions in the cITP stacking process.; Heparin oligosaccharides are highly sulfated linear polysaccharides that display a wide range of biological activities through their interaction with proteins. Structure elucidation of heparin is complicated by its microheterogeneity arising from variations in sulfation patterns and stereochemistry. Although progress has been made in the structure determination of heparin oligosaccharides, the structures of only those sequences that are most abundant or have specific biological activity have been determined. Hence, sensitive and specific analytical techniques are needed for progress in this area.; Although, NMR spectroscopy is a powerful tool for molecular structure determination, it trails other analytical techniques in sensitivity and typically requires milligram quantities for analysis. Obtaining such quantities of pure oligosaccharides, especially less abundant rare sequences, is a tedious and laborious process often involving several separation steps. Microcoil NMR technology is a relatively inexpensive method of enhancing NMR mass sensitivity and allows structure elucidation using only microgram amounts of material. However, one limitation of microcoil probes is their relatively poor concentration sensitivity. In this research, this problem is addressed by coupling microcoil NMR detection with cITP, a sample separation and preconcentration method. On-line cITP-NMR is a potentially powerful technique for the separation and identification of heparin enzymatic digests.; Results are presented showing the application of microcoil NMR for the structure elucidation of microgram amounts of heparin-derived oligosaccharides. The methods developed in this research are expected to reduce NMR sample requirements of unknown oligosaccharides by as much as 50-100 fold. These methods have the potential to accelerate the discovery of novel oligosaccharides and significantly advance efforts to relate the structure of specific saccharide sequences to their biological function.; On-line cITP-NMR was also used in this dissertation to provide unique insights into the stacking behavior of ions by providing both qualitative and quantitative information about the behavior of buffer and sample components during the cITP experiment.