High-resolution chromatography of proteins in short columns and adsorptive membranes.

Short chromatographic columns prepared from stacks of microporous adsorptive membranes are promising for preparative-scale fractionation of even rather closely related proteins, but careful selection of operating conditions is needed for success. It has been shown that existing devices exhibit very low internal diffusional resistance, and that resolution is almost totally independent of percolation velocity. Total column length is short, however, and the number of plates exhibited under isocratic low-loading conditions is small, on the order of 100. Simulations using a large survey of protein thermodynamic data show that one can frequently obtain excellent protein separations in these short columns by using the sensitivity of protein adsorption equilibria to eluting solvent composition. In fact, some proteins can be separated in a single stage utilizing this 'on-off' behavior and properly selected solvent gradients. Solely on-off, or differential elution, behavior cannot often be depended upon under the mild conditions need for preparative operations. Carefully programmed gradient elution can frequently produce acceptable purification by maximizing both differential elution and differential migration, resulting in protein separations in columns of less than fifty plates. Means for doing this are described for some simple situations, and criteria are provided for selecting modulator gradient schedules.