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Ionic Liquid Modified Silica-a Novel Multi-interaction Stationary Phase For Protein Separation And Purification

Posted on:2016-10-22Degree:MasterType:Thesis
Country:ChinaCandidate:Y X WangFull Text:PDF
GTID:2191330461958854Subject:Analytical Chemistry
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Ionic liquids(ILs) are a kind of novel liquids which are solely composed of organic cations and organic or inorganic anions.Currently the research and application of ILs have become an important aspect in liquid chromatography. This thesis selects ILs as ligands by its characters of cation-based conjugate ring structuers. We synthesized a series of stationary phase modified with N-methylimidazolium,4-methylthiazole, pyridine and pyrazine. The effect of influential factors of the dual-functional stationary phase on the separation of protein was discussed in detail. We carried on a further research on the mixed retention mechanism of proteins in mixed-mode chromatography using stoichiometric displacement theory (SDT).The dissertation includes the following four chapters:1. Literature ReviewThis chapter introduced the advantages of the mixed-mode chromatography over traditional mode chromatography and the different types of ligands of mixed-mode stationary phase. Then the preparation, properties and application of ionic liquids-based stationary phase were reviewed as well.2. The preparation and chromatographic performance of mix-mode chromatography stationary phase modified with N-methylimidazolium ligandA novel kind of mix-mode chromatography(MMC) stationary phase based on silica gel was prepared using N-methylimidazolium as the ligand, which provided both hydrophobic association and electrostatic interaction. The results demonstrated that when adding trifluoro acetic acid as the ion-pairing reagent in the mobile phase in RPLC mode, most of the tested protein standards except myoglobin could not be retained on this stationary phase at all. When using the mobile phase at neutral pH, all of the basic proteins tested could still not be retained on this stationary phase by electrostatic repulsion. On the contrary, all of the acidic proteins were adsorbed too tightly to be eluted. When operated in RPLC/IEC mode, basic proteins still could not be retained, however eight kinds of acidic proteins can be separated completely with this stationary phase. It showed special selectivity and better resolution to acidic proteins compared with commonly used commercial C18 column. The influence of acetonitrile concentration, salt concentration and pH value was experimentally investigated. The resultsshowed that with the acetonitrile concentration increasing, the retention of proteins reduced by hydrophobic interaction, and with the sodium perchlorate concentration increasing, the retention of proteins reduced as well due to the electrostatic interaction. With pH value changing, both the hydrophobic interaction and electrostatic interaction proved to be factors influencing the chromatography behaviors. The result indicated that with the increase of pH value, the retention time of proteins showed lower trends. In addition, the above stationary phase was also employed to separate proteins from egg white. The basic protein lysozyme could not be retained and eluted out directly with solvent, and two kinds of acidic proteins, ovalbumin and ovotransferrin, could be separated completely. It confirms further this stationary phase has good selectivity and resolution to the acidic proteins.3. Studies of mixed retention mechanism of the stationary phase modified with N-methylimidazolium ionic liquidWe carry on a further research on the mixed retention mechanism of proteins in mixed-mode chromatography using stoichiometric displacement theory (SDT). Two modes were separately set up for the retention behavior of proteins. In the RPLC mode, the retention followed the SDT which showed that the stationary phase had the reversed-phase chromatographic performance. In the EC mode, the retention only satisfied the first set of linear equation. The results agreed with this idea-the electrostatic interaction being a kind of selective force. It indicated that it was reliable to use SDT to explain the mixed retention mechanism of proteins in this mixed-mode chromatography stationary phase, and all the parameters carried explicit physical meanings.4. Studies of chromatographic performance of mix-mode chromatography stationary phase 4-methyIthiazole, pyridine and pyrazineA series of bonded stationary phase for proteins separations had been prepared by a similar bonding method.We choosed three other cation-based ILs including 4-methylthiazole, pyridine and pyrazine to be the ligand. They are a type of organic aromatic compounds with a cations in common, but these ILs have their own repective characteristics yet, which determines the separation process have some consistent features and also have clear difference at the same time. Pyrazine and pyridine modified stationary phase could separate proteins under conventional reversed-phase method.4-methylthiazole modified stationary phase was different, which had a similar chromatographic behavior to N-methylimidazolium modified stationary phase. We found that this kind of stationary phase could not retained proteins by conventional reversed-phase mode. But all these three stationary phase had a good separation efficiency under RPLC/IEC mode. The factors affecting retention were involved in this thesis, including acetonitrile concentration, sodium perchlorate concentration and pH value. The result indicated that all these stationary phase could show multi-interaction mechanism.The chromatographic behaviors were regular in a certain degree. Another important point here was that both acidic and basic proteins could be retained on the other three ILs-modified stationary phases, unlike N-methylimidazolium modified as ligand, they can not show the unique selectivity to the acidic proteins.
Keywords/Search Tags:Ionic liquid, Mixed-mode chromatography, Protein separation, Reversed-phase liquid chromatography, Ion-exchange chromatography
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