| The possibility of fast separation of intact proteins with sub-2μm nonporous silica (NPS)-based chromatographic packings was investigated. The chemically modified packings were packed into chromatographic cake (or named caky column) which has diameter to be much greater than its thickness or column length for fast separation of intact proteins in several minutes, even several seconds. It is convinced that column efficiency would increase as the particle diameter decreases. When such small particles were packed into a traditional column, the high efficiency and high peak capacity were achieved, but abnormal huge backpressure was produced, too, bringing big problem to conventional liquid chromatograph. If flow rate of mobile phase is lower than a certain value, the high column efficiency cannot be obtained. Because of the very large crossing-section area and very short column length, chromatographic cake only produces suitable back pressure, even though very small particles employed and chromatographic running under very high flow rate. Packings for reversed phase and hydrophobic interaction liquid chromatography were synthesized and tested, fast separation of intact proteins was achieved on conventional HPLC. The whole thesis consists of five parts.1. Introduction. The recent development of fast separation of intact proteins in liquid chromatography was reviewed, including ultrahigh pressure liquid chromatography, chromatographic cake, monolithic column and high temperature fast chromatography, the characteristics of each method were outlined. It contains 50 references,42% of them sourcing from recent three years.2. Synthesis and characterization of sub-micrometer and micrometer silica particles. The methods for producing sub-micrometer and micrometer non-porous silica (NPS) particles were discussed in detail. Sub-2μm NPS particles were successfully produced in our Lab, then the NPS particles were chemically modified for reversed phase liquid chromatography (RPLC) and hydrophobic interaction chromatography (HIC), respectively. Using optical microscope, scanning electron microscope and laser particle size analyzer, the silica particles and packing materials were characterized. Comparing sub-2μm NPS particles producing in our Lab with commercial ones, it was found that both diameter and surface morphology are similar, but quite different from porous particles, the sub-2μm NPS particles have a smooth surface.3. Fast separation of intact proteins by chromatographic cakes with sub-2μm NPS-based RP-C18 stationary phase. Seven intact proteins were well separated within 48s. Comparing with packing materials based on commercial NPS particles, similar performance but much faster separation was observed by our NPS columns.4. Fast HIC of intact proteins:2.5μm commercial butyl nonporous resin (NPR).2.5μm butyl-NPR was packed into different sizes of chromatographic cakes, then applied for the rapid separation of intact proteins on conventional HPLC within 48s. Under high flow rate(10ml/min), the mixture of seven intact proteins was separated completely with an acceptable back pressure(<20MPa). Meanwhile, rapid cycling of the separation process was conducted, it was convinced that the chromatographic cake was capable of both rapid separation and rapid equilibration. Through the plot of conditional plate height or resolution versus flow rate, it was concluded that column efficiency increased as flow rate increased.5. Fast protein separation by sub-2μm NPS HIC-PEG600.630nm,650nm and 1μm NPS microspheres were synthesized, further modified with PEG600. The NPS-based HIC packings were packed into different sizes of chromatographic cakes, then applied for the fast separation of intact proteins on conventional HPLC within minutes, even seconds. At last, the column capacity, permeability and efficiency were tested. Through the plot of conditional plate height or resolution versus flow rate, it was concluded that column efficiency increased as flow rate increased. |
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