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Isoelectric chromatofocusing (ICF) is a powerful and flexible method for separating complex protein mixtures that is finding increasing use in industry. Here, Choy et al. report physico-chemical and chromatographic data that serve to define the various mechanisms that influence protein retention and elution during ICF and account for observed deviations between a protein's elution pH and isoelectric point. A new model that captures these mechanisms quantitatively is derived and shown to accurately predict protein elution during ICF. Using the model, the authors demonstrate how elution pH values and separation efficiencies can be tuned by modulating the strengths of these elution mechanisms. This feature can potentially be used to formulate custom ICF separations such that a protein product and contaminants elute at distinct pH values required to meet performance and process compatibility criteria.