Preparation of Immobilized Metal Affinity Chromatographic Packings by Immobilization of Carboxymethylated Asparate (CM-Asp) Based on Monodisperse Hydrophilic Non-porous Beads and Their Application

The hydrophilic immobilized metal affinity chromatographic packing was prepared by immobilization of carboxymethylated asparate (CM‐Asp) as chelating ligand and Ni²⁺ as center ion on the base of monodispersed, 3.0 µm non‐porous monodisperse poly(glycidylmethacrylate‐co‐ethylenedimethacrylate) (P_GMA/EDMA) particles. The retention behavior of proteins and the effect of pH on the retention in the range from 4.0 to 9.0 were investigated on both the naked and metal ion chelated columns. Four proteins were quickly separated in 2.0 min with linear gradient elution at a flow rate of 3.0 mL·min^?1 by using the synthesized Ni²⁺‐CM‐Asp‐P_GMA/EDMA packings. The separation time was shorter than other immobilized metal affinity chromatography reported in the literature. The Ni²⁺‐CM‐Asp‐P_GMA/EDMA column was further investigated for the rapid separation and purification of copper‐zinc superoxide dismutase (Cu,Zn‐SOD) from the blood of pig in 3.0 min with only one step. The results obtained were satisfactory.