Chemical Sciences: A Manual for CSIR-UGC National Eligibility Test for Lectureship and JRF/Displacement chromatography

Discovery
The advent of displacement chromatography can be attributed to Tiselius, who in 1943 first classified the modes of chromatography as frontal, elution, and displacement. Displacement chromatography has since found a variety of applications from isolation of transuranic elements to biochemical entities. Displacement chromatography is a chromatographic method in which the components are resolved into consecutive “rectangular” zones of highly concentrated pure substances rather than solvent separated “peaks”. The molecules are forced to migrate down the column by an advancing wave of a displacer molecule that has a higher affinity for the stationary phase than the feed solute. Because of this forced migration, higher product concentrations and purities may be obtained compared to other modes of chromatography. The technique was rediscovered by Horvath and has since found many applications, particularly in the realm of biological macromolecule purification.

Principle
The basic principle of displacement chromatography is: A molecule with a high affinity for the chromatography matrix (the displacer) will compete effectively for binding sites, and thus displace all molecules with lesser affinities. There are distinct differences between displacement and elution chromatography. In elution mode, substances typically emerge from a column in narrow, Gaussian peaks. Wide separation of peaks, preferably to baseline, is desired in order to achieve maximum purification. The speed at which any component of a mixture travels down the column in elution mode depends on many factors. But for two substances to travel at different speeds, and thereby be resolved, there must be substantial differences in some interaction between the biomolecules and the chromatography matrix. Operating parameters are adjusted to maximize the effect of this difference. In many cases, baseline separation of the peaks can be achieved only with gradient elution and low column loadings. Thus, two drawbacks to elution mode chromatography, especially at the preparative scale, are operational complexity, due to gradient solvent pumping, and low throughput, due to low column loadings. Displacement chromatography has advantages over elution chromatography in that components are resolved into consecutive zones of pure substances rather than “peaks”. Because the process takes advantage of the nonlinearity of the isotherms, a larger column feed can be separated on a given column with the purified components recovered at significantly higher concentrations.

Theory and method
As shown above, a displacement chromatography process can be broken down into three distinct phases: loading, displacement, and regeneration. For the sake of simplicity, examples of biomolecule purification given here will be restricted to proteins. In principle, all biomolecules of commercial interest today – including oligonucleotides and antibodies – can be purified in displacement mode, with the right choice of column matrix and displacer.

Loading phase

The purification starts with a column equilibrated with a loading buffer similar to elution chromatography methods. The feed mixture containing the impure proteins in buffer is loaded onto the column at a fairly slow rate, under conditions where the materials are well retained. The purpose of this step is to initiate the binding of the biomolecules in a semiequilibrium state. This is shown in the graphic as the mixture of proteins begins an early separation into yellow and purple components.

Displacement phase

After the sample has been loaded, the column is fed a solution of 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. 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 proteins 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. Fractions can be collected and the desired product cuts made.

Regeneration phase

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.

Advantages of displacement chromatography

An important distinction between displacement and step gradient chromatography is that the displacer front always remains behind the adjacent feed zones in the displacement train whereas desorbents (e.g., salts in ion exchange, organic modifiers in reversed-phase chromatography) move through the feed zones. The displacement mode of chromatography takes advantage of the thermodynamic characteristics of the chromatographic system to overcome many of the shortcomings of preparative elution and gradient chromatography. This characteristic of displacement makes it less sensitive to the feed loads than the elution modes of operation, thus enabling it to deliver higher process throughputs. Another advantage is the high resolution that displacement can deliver as compared to elution processes. Displacement chromatography exploits the nonlinear, multicomponent competition among the components to be separated, resulting in higher resolution, particularly among closely related species. In contrast, in elution processes (e.g., linear gradient, step gradient), the separation takes place under relatively weaker binding conditions (which are essential to get the solutes off the column). The separation factors among solutes are thus lower in elution than in displacement, leading to poorer resolution in the elution modes of operation. Other advantages of displacement include a better control over the product concentrations and the emergence of the product in relatively low concentrations of the mobile phase modifier. The combination of high throughputs and high resolutions in a single process makes displacement an attractive mode of operation for protein purification.

Applications
The chromatographic purification of proteins from complex mixtures can be quite challenging, particularly when the mixtures contain similarly retained proteins or when it is desired to enrich trace components in the feed. Further, column loading is often limited when high resolutions are required using traditional modes of chromatography (e.g. linear gradient, isocratic chromatography). In these cases, displacement chromatography is an efficient technique for the purification of proteins from complex mixtures at high column loadings in a variety of applications. An important advance in the state of the art of displacement chromatography was the development of low molecular mass displacers for protein purification in ion exchange systems. . This research was significant in that it represented a major departure from the conventional wisdom that large polyelectrolyte polymers are required to displace proteins in ion exchange systems. Low molecular mass displacers have significant operational advantages as compared to large polyelectrolyte displacers. For example, if there is any overlap between the displacer and the protein of interest, these low molecular mass materials can be readily separated from the purified protein during post-displacement processing using standard size-based purification methods (e.g. size exclusion chromatography, ultrafiltration). In addition, the salt dependent adsorption behavior of these low MW displacers greatly facilitates column regeneration. These displacers have been employed for a wide variety of high resolution separations in ion exchange systems. In addition, the utility of displacement chromatography for the purification of recombinant growth factors, antigenic vaccine proteins and antisense oligonucleotides has also been demonstrated. There are several examples in which displacement chromatography has been applied to the purification of proteins using ion exchange, hydrophobic interaction, as well as reversed phase chromatography. Displacement chromatography is well suited for obtaining mg quantities of purified proteins from complex mixtures using standard analytical chromatography columns at the bench scale. It is also particularly well suited for enriching trace components in the feed. Displacement chromatography can be readily carried out using a variety of resin systems including, ion exchange, HIC and RPLC , Two-dimensional chromatography represents the most thorough and rigorous approach to evaluation of the proteome. While previously accepted approaches have utilized elution mode chromatographic approaches such as cation exchange to reversed phase HPLC, yields are typically very low requiring analytical sensitivities in the picomolar to femptomolar range. As Displacement chromatography offers the advantage of concentration of trace components, two dimensional chromatography utilizing displacement rather than elution mode in the upstream chromatography step represents a potentially powerful tool for analysis of trace components, modifications, and identification of minor expressed components of the proteome.