IMMOBILIZED METAL AFFINITY-CHROMATOGRAPHY - DISPLACER CHARACTERISTICSOF TRADITIONAL MOBILE-PHASE MODIFIERS

Imidazole was examined as a potential displacer for protein purificati on in metal affinity chromatographic systems. A dynamic affinity plot was developed for predicting the elution order and efficacy of displac ers in metal affinity displacement chromatography. Theoretical predict ions and experimental results indicate that small molecular weight com pounds, such as imidazole, with a single coordination site can indeed displace proteins with multiple coordination sites to the surface. In addition, the theory predicts the concentration dependence of imidazol e displacement behavior observed in the experiments. In the presence o f additional mobile phase modifiers, imidazole was shown to induce rel atively high modifier gradients due to its high affinity and negligibl e steric factor. N-protected histidines and tryptophan were also shown to act as protein displacers in IMAC systems. While the N-protected h istidines resulted in relatively sharp displacement boundaries, trypto phan was a less effective displacer, producing significant protein tai ling during the separation. The results in this manuscript are expecte d to have major implications for downstream processing of proteins usi ng IMAC in the displacement mode of operation.