PROTEIN DENATURATION IN NONLINEAR ISOCRATIC AND GRADIENT ELUTION CHROMATOGRAPHY

Protein denaturation, common in hydrophobic adsorption systems, causes misinterpretation of adsorption mechanisms, interferes with analysis in analytical chromatography, and complicates the design of large-scal e adsorption processes. A detailed adsorption model isolates the effec ts due to denaturation from those due to mass transfer and intrinsic a dsorption kinetics. The model is verified using protein gradient eluti on data. Simulations establish that typical symptoms of denaturation i n frontal and elution chromatograms include sensitivity to changes in feed composition, column length, particle size, and operating conditio ns (feed size, flow rate, and column history). When a denatured specie s adsorbs irreversibly, the elution chromatogram shows decreasing peak area with increasing incubation time and apparent adsorption hysteres is over repeated cycles. In gradient elution, the peak elution order, resolution, and relative peak height depend highly on modulator proper ties and operating conditions. Interfering species limit solid-phase i nduced denaturation by competing for binding sites. Strategies for det ecting and minimizing denaturation are proposed.