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.

Comparing steady counterflow separation with differential chromatography.

Separation of closely related solutes by steady solid-fluid counterflow is compared with differential separation in a fixed chromatographic bed. Analogous expressions for exit concentration and mean residence time in the two systems are presented. A counterpart to chromatographic resolution is derived for binary steady counterflow separations. Estimated counterflow savings in product-concentration dilution, solvent volume requirement and solid-phase volume requirement obtained with these expressions relative to comparable chromatographic operations are compared with experimental results from adsorptive, simulated moving beds. Analysis of a size-exclusion protein separation suggests counterflow substantially decreases solvent and resin usage relative to conventional, batch operation.