Microcoil NMR techniques for improved sensitivity and throughput
Abstract
Improved NMR probes in terms of sensitivity and throughput have led to enhanced developments in hyphenated techniques and improved the utility of NMR for the analysis of natural products, small molecule metabolites and proteins. In this thesis the development of several microcoil NMR probes is presented. First a unique dual solenoidal microcoil NMR difference probe is described. The difference probe has the ability to acquire a single difference spectrum from samples contained in two separate capillaries. This method was applied to the characterization of potential binding ligands for the protein human serum albumin (HSA). Through a pulse sequence technique known as Saturation Transfer Difference, two potential ligands were evaluated, whereby the complete difference spectrum not only identifies the bound ligand, but also performs a second difference in eliminating the broad background resonances of the protein itself by virtue of the unique probe circuit. Second, the design and construction of a larger volume flow-through micrcocoil NMR probe for the routine analysis of 20 μL samples is described. The aim of this work is to provide a NMR active volume that is a closer match for eluate volumes exhibited by popular separation methods, while still taking advantage of the increase in mass sensitivity exhibited by solenoidal microcoils. The detection cell for this probe was created using a novel laser-heated HF etching technique. Mass sensitivity for this probe is five times better than that of a standard 5 mm probe. This probe is designed to be used with hyphenated LC and preconcentration techniques for identifying components from complex mixtures such as biofluids. Additional studies focus on improving the sensitivity of this larger volume probe. Overall, this work represents further progress in the application of small-volume NMR in bioanalysis and opens up directions for further optimization of hyphenated NMR methods through probe development.
Degree
Ph.D.
Advisors
Raftery, Purdue University.
Subject Area
Analytical chemistry|Biophysics
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