Preparative Chromatography

Report by Ramesh Vunnum, 1992

The three major modes of operation in preparative LC are elution (isocratic and gradient), frontal and displacement chromatography.  Due to the ease and familiarity of operation in analytical LC, elution chromatography continues to be the most widely employed mode for preparative separations.  Elution chromatography can be carried out under isocratic (constant mobile phase composition), gradient (continuous change in mobile phase composition) or step elution conditions.  Under these conditions, a feed mixture is injected into the column inlet as a finite volume pulse.  The feed components then migrate through the column inlet as a finite volume pulse.  The feed components then migrate through the column at different speeds, which are a function of the mobile phase velocity and the distribution of the compounds between the mobile and stationary phases.

Preparative elution chromatography is generally carried out under mass and/or volume overloaded conditions in order to increase the product throughput.  In volume overloading, the sample concentration is maintained in the linear region of the isotherm and the volume is increased until the throughput is optimized.  A fundamental problem with this technique is the under-utilization of the column and the corresponding low throughputs.  In mass overloading, the sample concentration is increased beyond the linear adsorption region, resulting in asymmetric band profiles, with self-sharpening fronts and tailing rear boundaries for Langmuirian adsorption systems.  A combination of volume and mass overloading is commonly used to maximize throughput in preparative elution chromatography.

In recent years displacement chromatography has received considerable attention as a promising preparative chromatographic technique for protein separations.  The key operational feature which distinguishes displacement from step elution chromatography is the use of the displacer compound.  The displacer is selected such that it has a higher affinity for the stationary phase than any of the feed components.  A large volume of the feed mixture is then loaded onto the column, followed by a constant front of the displacer solution.  The displacement process is based on the competition of solutes for adsorption sites on the stationary phase according to their relative binding affinities and mobile phase concentrations.  The action of the displacer causes the feed components to migrate through the column at velocities greater than that dictated solely by their individual adsorption isotherms.  The product components exit from the column as adjacent "square wave" zones of higher concentrated pure material, in the order of increasing affinity of adsorption.