Experimental verification of sample-solvent induced modifier–solute peak interactions in biochromatography

In a previous theoretical analysis based on equilibrium theory it has been shown how differences in the sample and elution modifier concentrations can lead to unexpected behavior of the solute eluted peaks such as retention time distortion, peak deformation and peak doubling. All these features are verified experimentally in this work using the polypeptide calcitonin and a variant of a specific monoclonal antibody as chromatographic model systems. For both experimental systems, the retention time distortion can be predicted with high accuracy by the solution of the equilibrium theory model. For the polypeptide, the predictions from the theory about the occurrence of peak deformation and double peaks has been successfully verified by a series of tailored experiments with positive as well as negative modifier perturbations.