Preparation Of Tetrazole-functionalized Stationary Phase And Their Chromatographic Properties

Lliquid chromatography(LC)has been widely applied in the separation and purification of biopolymer.Ion-exchange chromatography(IEC)and immobilized metal-ion affinity chromatography(IMAC)are two important kinds among LC methods.In this dissertation,tow kinds of tetrazole-functionalized stationary phase were prepared successfully,and were employed in separation and purification of biopolymer by IEC and IMAC.The obtained results are summerized as in the following:1.A novel tetrazole-functionalized silica stationary phase was prepared successfully for use in weak cation exchange chromatography.It was prepared conveniently by modifying silica gel initially withγ-glycidoxypropyltrimethoxysilane,then with 3-hydroxypropionitrile and finally with ammonium-catalyzed(3+2)azide-nitrile cycloaddition.The prepared stationary phase was characterized and evaluated for its separation performance, protein retention behavior,loading capacity and chemical stability.The results show that five proteins could achieve a good separation on this column,and their retention behavior was consistent with IEC.Notably,the column with tetrazole-functionalized stationary phase showed good loading property and long-term stability,and no significant irreversible adsorption.Finally,the column was used to carry out a single-step purification of lysozyme from egg white.The purity of the purified lysozyme was 89.6%,and specific activity was 66.823IU/mg.2.Another novel tetrazole-functionalized silica stationary phase was developed for use in IMAC of proteins.It was prepared by modifying silica gel initially withγ-aminopropyltriethoxysilane,then with 3-chloroypropionitrile and finally with ammonium-catalyzed(3+2)azide-nitrile cycloaddition.After the chelation of metal ion(e.g.Cu²⁺,Ni²⁺and Co²⁺),the chromatographic characteristic of the column was evaluated in terms of separation ability with different metal ions at different pH in the mobile phase.The result indicated that four proteins could achieve better separation,and protein retention behavior was consistent with IMAC.