This research is to develop a new technology, Ultra High Pressure Liquid Chromatography (UHPLC), for the rapid separation and analysis of complex mixtures of biological origin. UHPLC uses pressures from 10,000 to 100,000 psi in order to pump mobile phase throug capillary columns packed with exceedingly fine particles (below 1 to 1.5 micron) of packing material. This technique is currently capable of separating hundreds of compounds in under 30 minutes. We will develop the high pressure hardware and acquire the knowledge needed to operate chromatography columns at such pressures. Stationary phase support materials based on non-porous as well as porous silica will be synthesized or acquired, and characterized with regar to performance. Stationary phases for reversed phase will be studied in detail but normal phase, ion exchange and chiral phases will be studied as well. Theory will be developed to describe the effects of high pressure on the performance of these columns in isocratic separations. This theory will includ the effects of pressure on diffusivity. Measurements of the diffusion coefficients of model compounds will be made as a function of pressure. Experimental and theoretical investigations of the impact of column diameter and column material on heat generation and transfer will be made. We will also develop and test theory of chromatographic band spreading under gradient elution conditions. The goal of this theory is to predict optimal particle diameter, column length, and flow and gradient rate, in order to maximize peak capacity with limits on available pressure and time. Finally, UHPLC-Mass Spectrometry will be used to demonstrate the power of this system in peptide analyses.
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