Utilization of Long Columns Packed with Sub-2 μm Particles Operated at High Pressures and Elevated Temperatures for High-Efficiency One-Dimensional Liquid Chromatographic Separations Public Deposited

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  • March 22, 2019
Creator
  • Franklin, Edward Gordon
    • Affiliation: College of Arts and Sciences, Department of Chemistry
Abstract
  • As Ultrahigh Pressure Liquid Chromatography (UHPLC) techniques have become increasingly popular across a number of scientific disciplines, the strongest emphasis has remained on its ability to yield moderately high separation efficiencies on much faster timescales than were previously achievable. This has been accomplished through the use of commercial instrumentation capable of generating maximum pressures of 18,000 psi and 15 – 25 cm columns packed with 1.8 – 2.0 μm particles, providing theoretical plate counts upwards of 60,000. While this represents a major step forward when compared to typical HPLC approaches, many applications involving highly complex samples, such as those encountered in “-omics” settings, stand to benefit from even higher efficiency separations. The work presented in this dissertation explores the combined benefits of using ultrahigh pressures (up to 50,000 psi), small particles (sub-2 μm) and elevated temperatures (up to 85°C). Efforts were made to improve microcapillary column packing procedures by investigating the individual and synergistic effects of experimental packing parameters on ultimate column performance. Findings were used to pursue two of the most enduring objectives in the separation sciences: faster speeds and higher efficiencies. By packing 20 – 30 cm columns with fully porous 1.0 μm particles, plate counts in excess of 100,000 were achieved with column dead times of approximately 1 minute. Alternatively, 1.9 μm particles were used to pack columns several meters in length to achieve very high separation efficiencies. A 360 cm x 50 μm ID column produced over 106 plates with a column dead time of just over 100 minutes at 25°C. Likewise, ~ 200 cm columns were used in the analyses of proteomic samples under gradient elution conditions to generate peak capacities approaching 1000 in just over three hours at 65°. These results suggest a real applicability of high-efficiency one-dimensional approaches to proteomics and similar fields of study.
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  • In Copyright
Advisor
  • Jorgenson, James
Degree
  • Doctor of Philosophy
Graduation year
  • 2012
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