Competitive Elution of Proteins in Metal Chelate Affinity Chromatography

The retention behavior of proteins on strong affinity metal chelate column(IDA-Cu(10) was studied using cytochrome-C(Cyt-C) and lysozyme (Lys) as typifiers. The effects of competitive agents and buffer systems on the retention of proteins on IDA-Cu (10 column were investigated. In potassium dihydrogen phosphate(PB) and sodium acetate-acetic acid(NaAc-HAc) buffer systems, the better separation for proteins could be obtained by using imidazole(Imid) and glycine(Gly) as competitive agents. The effects of pH on the retention of proteins on IDA-Cu(I) column were examined in Imid and Gly eluting systems, respectively. For effective isolation of proteins and less leakage of Cu(2+), the eluting method of competitive displace combined with decreasing pH was a better choice for Gly system. As for Imid, the pH value should be controlled at 6.0. Proteins could not be easily eluted at the low pH. The results indicate that the relationship between the concentration of competitive agent and the retention of proteins on IDA-Cu(II) column conforms to the stoichiometric displacement theory for retention(SDT-R). The retention time decreased with increasing competitive agent concentration. The retention factor (k') and the reciprocal of competitive agent concentration (1/[D]) were well logarithmic linear relationship. The linear correlation coefficients were above 0.984 and 0.986 respectively.