Preparation of Immobilized Metal Affinity Chromatographic Packings Based on Monodisperse Hydrophilic Non‐porous Beads and Their Application |
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Authors: | Chun‐Miao BO Bo‐Lin GONG Wen‐Zhi HU |
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Institution: | 1. Key Laboratory of Energy & Chemical Engineering, Ningxia University, Yinchuan, Ningxia 750021, China;2. Tel.: 0086‐0951‐2062300;3. Fax: 0086‐0951‐2062860;4. Laboratory of Bio‐functional Chemistry, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060‐0810, Japan |
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Abstract: | Three hydrophilic immobilized metal affinity chromatographic packings for HPLC have been synthesized by chemical modification of 3.0 µm monodisperse non‐porous poly(glycidyl methacrylate‐co‐ethylenedimethacrylate) (PGMA/EDMA) beads. The retention behavior of proteins on the metal ion chelated columns loaded with copper(II), nickel(II) and zin(II) ion was studied. The effect of pH on the protein retention was investigated on both the naked and metal ion chelated columns in the range from 4.0 to 9.0. Four proteins were quickly separated in 3.0 min with linear gradient elution at a flow rate of 3.0 mL/min by using the synthesized Ni2+‐IDA (iminodiacetic acid) packings. The separation time was shorter than other immobilized metal affinity chromatography reported in the literature. Purification of lysozyme from egg white and trypsin on the commercially available trypsin was performed on the naked‐IDA and Cu2+‐IDA columns, respectively. The purities of the purified trypsin and lysozyme were more than 92% and 95%, respectively. |
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Keywords: | monodisperse non‐porous bead immobilized metal affinity chromatography protein purification |
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