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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Metallobiochemistry Study Section (BMT)
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Edmonds, Charles G
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University of North Carolina Chapel Hill
Schools of Arts and Sciences
Chapel Hill
United States
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Patel, Kamlesh D; Jerkovich, Anton D; Link, Jason C et al. (2004) In-depth characterization of slurry packed capillary columns with 1.0-microm nonporous particles using reversed-phase isocratic ultrahigh-pressure liquid chromatography. Anal Chem 76:5777-86
Lan, K; Jorgenson, J W (2001) A hybrid of exponential and gaussian functions as a simple model of asymmetric chromatographic peaks. J Chromatogr A 915:1-13
Lan, K; Jorgenson, J W (2001) Spatial and temporal progressions of spatial statistical moments in linear chromatography. J Chromatogr A 905:47-57
Lan, K; Jorgenson, J W (2000) Theoretical investigation of the spatial progression of temporal statistical moments in linear chromatography. Anal Chem 72:1555-63
MacNair, J E; Patel, K D; Jorgenson, J W (1999) Ultrahigh-pressure reversed-phase capillary liquid chromatography: isocratic and gradient elution using columns packed with 1.0-micron particles. Anal Chem 71:700-8
Lan, K; Jorgenson, J W (1999) Automated measurement of peak widths for the determination of peak capacity in complex chromatograms. Anal Chem 71:709-14
Lan, K; Jorgenson, J W (1998) Pressure-induced retention variations in reversed-phase alternate-pumping recycle chromatography. Anal Chem 70:2773-82
Hsieh, S; Dreisewerd, K; van der Schors, R C et al. (1998) Separation and identification of peptides in single neurons by microcolumn liquid chromatography-matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and postsource decay analysis. Anal Chem 70:1847-52
Opiteck, G J; Ramirez, S M; Jorgenson, J W et al. (1998) Comprehensive two-dimensional high-performance liquid chromatography for the isolation of overexpressed proteins and proteome mapping. Anal Biochem 258:349-61
MacNair, J E; Opiteck, G J; Jorgenson, J W et al. (1997) Rapid separation and characterization of protein and peptide mixtures using 1.5 microns diameter non-porous silica in packed capillary liquid chromatography/mass spectrometry. Rapid Commun Mass Spectrom 11:1279-85

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