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Loading Step
Purification starts with a column equilibrated with a loading buffer. Then the feed mixture comprising impure molecule mixture in buffer is loaded onto the column at a fairly slow rate, under conditions where the materials are well retained. After the feed mixture has been loaded, approximately one column volume of loading buffer is passed through the column. The purpose of these steps is to help the displacement train start to form during the sample loading. This is shown in the graphic as the mixture of proteins begins an early separation into yellow and purple components.
Displacement Step
After the sample has been loaded, the column is fed with a solution of a displacer at a fairly low concentration (typically 5 mM) in the same buffer used to load the sample. The displacer is designed to bind more tightly to the matrix than any of the biomolecules and thus "pushes" all components of the mixture off the matrix ahead of it. How does this happen? As long as each sample component and the displacer is not irreversibly adsorbed on the column matrix, there is some of each continually adsorbing to, and desorbing from, the matrix.
In elution mode, optimal results are obtained when concentrations are so low that individual components act independently and do not compete for binding sites on the matrix. In displacement mode, sample components are introduced in much more concentrated form, and so it is possible for a stronger binding component —either the displacer or one of the biomolecules in the sample mixture —to compete for binding sites. The stronger binding components (initially, the displacer itself) then displace by successfully competing for binding sites.
Based on their individual binding strengths, each component in the original sample then becomes a "displacer" for the next less tightly bound component. Thus, a displacement train is established as adjacent, focused bands with little overlap (yellow and purple in the graphic). In a successful run, the train is fully established before the components of interest arrive at the bottom of the column. Unlike Elution chromatography components are not separated into widely spaced, Gaussian-shaped bands. They emerge from the column in concentrated, adjacent, trapezoid nearly square zones. The steepness of the trapezoid sides are dependent on such factors such as flow rate and specific binding interactions of the components with the matrix and each other. Fractions, can be collected and the desired product cuts made. Weakly bound components may elute off the column in advance of the displacement train, which is composed of the more tightly bound components
Regeneration Step
When the displacer breaks through, i.e., begins to emerge from the column, the run is complete and column regeneration can begin. Regeneration is accomplished by using a buffer that can remove the displacer from the matrix, followed by an equilibration with loading buffer in preparation for the next run. Since displacers must bind more strongly than any protein being purified, it is to be expected that their removal from the column should require some special conditions. A non-trivial part of the design of a good displacer, then, is the incorporation of some structural feature that allows for complete removal from the matrix under some conditions.
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