Separation and Identification of Peptides by Supercritical Fluid Chromatography Coupled with Mass Spectrometry
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The presence of certain proteins in physiological fluids could be used as an early diagnostic tool for disease; however, because of the large concentration range of proteins and the number of distinct chemical species the detection and quantification of these proteins is problematic. This research focuses on the ability to separate proteins using supercritical fluid chromatography (SFC), a form of chromatography that uses supercritical carbon dioxide (scCO2) as the mobile phase. This project was divided into two parts. The first is the synthetic aspect that involves reacting an amino acid, in this case tyrosine, with dimethyl-tert-butyl-chlorosilane which substitutes onto the hydroxyl group to increase its solubility in scCO2. The synthesis, purification, and characterization of this novel molecule have been successfully completed. The second part of the project is the optimization of the chromatograph itself, necessitating a complete rebuild of an extant SFC. Much of the internal controls were bypassed or replaced; at this stage, the SFC is capable of injecting and detecting large organic compounds and amino acids. Research efforts are now focused on separating the silylated tyrosine from nonderivatized amino acids. Once achieved, the synthesis will be scaled up to include other amino acids and ultimately small peptides, which should separate more readily and provide identification of target proteins.