Development and Evaluation of New Stationary Phases for Liquid and Supercritical Fluid Chromatography and Their Use in Bio-Analytical Separations
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A series of studies have been undertaken that encompass different aspects of chromatography; these include fundamental studies on chromatographic retention, synthesis and characterization of chromatographic materials, and application of chromatography to analyze a complex biological sample. First, we report on the evaluation of chromatographic retention of a relatively new stationary phase containing a polar group embedded in a hydrophobic backbone using supercritical fluid chromatography (SFC). The amide-based column was compared with columns packed with bare silica, C18, and a terminal-amide phase using the the linear solvation energy relationship model. The results show that the C18-amide stationary phase offered a combination of hydrophobic interactions and electron donating abilities. Under the chromatographic conditions used, the C18-amide column was able to provide baseline resolution of a five-component test mixture, while the other columns tested did not. Furthermore, the initial studies of using nanodiamonds as sationary phase under SFC conditions showed potential.In a second study, superficially porous silica particles were modified to contain an amino-phenyl layer at the surface. After surface coverage optimization, the amino-modified layer was crosslinked via “Friedel-Craft knitting” and packed into a chromatographic column. The stationary phase showed reversed phase properties, although a retention map indicated some HILIC-like characterstics at high concentration of acetonitrile in the mobile phase. The “Friedel-Craft knitting” resulted in an improved hydrolytic stability of the stationary phase. The new phase lost about 40-50% of retention after exposure to harsh acidic conditions for more than 20 hours (2,400 column volumnes), while a non-knitted phase loss about 75% of retentivity in less than 5 hours (600 column volumnes). It was also demonstrated that the amino groups at the surface of the silica can be converted to an intermediate diazonium salt, which can then be used to introduce further modification of the silica particles to provide chromatographic selectivity. Finally, chromatographic approaches were used to continue studies aimed at the elucidation of the composition of the human ascites. By use of liquid chromatography-mass spectrometry, turicine was confirmed as one of the components present in the ascites biofluid. High resolution mass spectrometry allowed to establish unique fragmentation patterns of unrecognized species that were not identified in existing mass spectrometric databases.