| With the development of biotechnology and life science, both recombinant protein drug production and proteomics research depend largely on fast and efficient protein separation technology. Therefore, developing new separation materials, better separation modes, and more sensitive detection methods would be beneficial to meet the higher requirements of modern separation science.Mixed-mode chromatography (MMC) is a chromatographic method in which multiple modes of interaction occur between the stationary phase and the solutes in the feed. Although the ways proteins interact with the chromatography sorbents are generally considered in terms of single modes, such as ionic or hydrophobic interactions, protein chromatography often involves multiple modes of interaction, some of which are unintended "secondary" interactions with the sorbent bead or the spacer-linker structure carrying the nominal ligand. However, it was discovered that MMC could become a new technique for improving separation power by using suitable approaches. The advantages of mixing modes deserve much wider recognition. Mixed-mode columns provide a unique tool to easily adjust the separation selectivity of acids, bases, zwitterions, and neutral molecules, via variation of the pH, ionic strength, or organic modifier. Beyond enhanced selectivity, MMC can also reduce the number of column steps needed for protein purification, and indeed has been shown to solve protein purification problems that are otherwise intractable.Although many types of MMC have been developed, such as RPLC/IEC, RPLC/HILIC and HILIC/IEC, MMC is still dominated by one retention mechanism and assisted by another. Some types of IEC/HIC mixed-mode columns are also commercialized, but all of them are dominated by ion-exchange interaction, assisted by hydrophobic interaction. The development of a MMC column with improved efficiency rather than a general mixed-mode column in a broad sense is required. The former shows superiorities in column capacity and selectivity over two separate commercial columns and could thus replace two normally commercial columns. This thesis was divided into five parts as following:1. Review:This chapter gives a comprehensive review on some aspects of MMC, including the definition, development, preparation, application, tendency, mixed-mode retention mechanism and click chemistry.2. "Thiol-ene click chemistry" preparation of WCX/HIC stationary phases:A novel dual-function mixed-mode stationary phase based on PGMA/EDMA microspheres was synthesized by thiol-ene click chemistry and characterized by infrared spectroscopy and elemental analysis. The new system displays both HIC character in a high salt concentration mobile phase and WCX chromatography character in a low salt concentration mobile phase. It can be used to separate proteins in both IEC mode and HIC mode. The resolution and selectivity of the stationary phase were evaluated in both HIC mode and IEC mode using protein standards. In comparison with the conventional WCX and HIC columns, the results were satisfactory and acceptable. Protein mass and bioactivity recoveries of more than 96% can be achieved in both HIC mode and IEC mode using this column. The results indicate that the novel dual-function mixed-mode column in many cases can replace the use of two individual WCX and HIC columns. In addition, the effects on protein separation of different ligand structures in the dual-function stationary phase and the pH of the mobile phase used were also investigated in detail. The results show that electrostatic interaction of the ligand with proteins must match the hydrophobicity of the ligand, which is an important factor to prepare the dual-function stationary phase. On the basis of this dual-function mixed-mode chromatography column, a new two dimensional liquid chromatography technology with a single column system was also developed in this study, and was used to renature and purify recombinant human interferon-y from inclusion bodies.3. Preparation and application of SCX/HIC dual-function stationary phase:In this chapter, a novel SCX/HIC dual-function stationary phase was prepared, which is based on porous PGMA/EDMA microsphere containing a hydrophobic linker and a sulfonic group. The stationary phase can display HIC character in a high salt mobile phase and IEC character in a low salt mobile phase. The resolution and selectivity of the dual-fuction stationary phase were evaluated under both HIC and IEC modes with standard proteins. The results indicated that the SCX/HIC dual-function stationary phase can give high selectivity and resolution under both HIC and IEC modes and can be comparable to the conventional IEC and HIC columns. Compared with traditional column, the latter can only be performed under one chromatographic mode, but the former can replace two individual SCX and HIC columns for protein separation. Based on this SCX/HIC dual-function stationary phase,2DLC-1C was set up and can completely separate eight standard proteins in 130min. In addition, the above 2DLC-1C was also employed to separate four kinds of active proteins completely from egg white.4. "Thiol-ene click chemistry" preparation of WAX stationary phases:A novel WAX stationary phase based on PGMA/EDMA microspheres was synthesized by thiol-ene click chemistry. The stationary phase displays WAX chromatography character in a low salt concentration mobile phase. The stationary phase can not give a good separation in HIC mode, so it can not be called "dual-funtion" stationary phase. By optimizing the condition of separating proteins in egg white with the WAX stationary phase, ovotransferrin and ovalbumin can be purified by one step.5. Cu (I) click chemistry preparation of AEX stationary phase:"Cu (I) click chemistry" was used as an effective strategy for coupling 3-Diethylamino-l-propyne onto the azide-microsphere to obtain a novel weak anion exchange (WAX) stationary phase, which showed better resolution, better separation ability for protein separation compared with commercial WAX packings. The stationary phase can not give a good separation in HIC mode, so it could not be called "dual-funtion" stationary phase. The results in HIC and IEC mode indicated that the hydrophobicity of the ligand is not suitable to match the magnitude of the electrostatic interaction. After the quaternization of tertiary amine group with benzyl chloride, benzyl group was introduced onto the surface of the WAX stationary phase. The separation results became better in IEC mode, but there was no significant improvement in HIC mode. |
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