Displacement chromatography is a chromatography

Chromatography

Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures....

 technique in which a sample is placed onto the head of the column and is then displaced by a solute

Solute

Solute may refer to:* Solute, UMIK or UBOOK desolving in a substance,forming INT/INTY* Solute , a group of Paleozoic echinoderms...

 that is more strongly sorbed than the components of the original mixture. The result is that the components are resolved into consecutive “rectangular” zones of highly concentrated pure substances rather than solvent-separated “peaks”. It is primarily a preparative technique; higher product concentration, higher purity, and increased throughput may be obtained compared to other modes of chromatography.

Discovery

The advent of displacement chromatography can be attributed to Arne Tiselius

Arne Tiselius

Arne Wilhelm Kaurin Tiselius was a Swedish biochemist who won the Nobel Prize in Chemistry in 1948.- Biography:Tiselius was born in Stockholm...

, who in 1943 first classified the modes of chromatography

Chromatography

Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures....

 as frontal, elution

Elution

Elution is a term used in analytical and organic chemistry to describe the process of extracting one material from another by washing with a solvent ....

, and displacement. Displacement chromatography has since found a variety of applications from isolation of transuranic elements  to biochemical entities.
The technique was redeveloped by Csaba Horváth

Csaba Horváth (chemical engineer)

Csaba Horváth was a Hungarian-American chemical engineer, particularly noted for building the first high performance liquid chromatograph.-Life:...

, who employed modern high-pressure columns and equipment. It has since found many applications, particularly in the realm of biological macromolecule purification.

Principle

The basic principle of displacement chromatography is: there are only a finite number of binding sites for solutes on the matrix (the stationary phase), and if a site is occupied by one molecule, it is unavailable to others. As in any chromatography, equilibrium is established between molecules of a given kind bound to the matrix and those of the same kind free in solution. Because the number of binding sites is finite, when the concentration of molecules free in solution is large relative to the dissociation constant

Dissociation constant

In chemistry, biochemistry, and pharmacology, a dissociation constant is a specific type of equilibrium constant that measures the propensity of a larger object to separate reversibly into smaller components, as when a complex falls apart into its component molecules, or when a salt splits up into...

 for the sites, those sites will mostly be filled. This results in a downward-curvature in the plot of bound vs free solute, in the simplest case giving a Langmuir isotherm. A molecule with a high affinity

Chemical affinity

In chemical physics and physical chemistry, chemical affinity is the electronic property by which dissimilar chemical species are capable of forming chemical compounds...

 for the matrix (the displacer) will compete more effectively for binding sites, leaving the mobile phase enriched in the lower-affinity solute. Flow of mobile phase through the column preferentially carries off the lower-affinity solute and thus at high concentration the higher-affinity solute will eventually displace all molecules with lesser affinities.

Loading

At the beginning of the run, a mixture of solutes to be separated is applied to the column, under conditions selected to promote high retention. The higher-affinity solutes are preferentially retained near the head of the column, with the lower-affinity solutes moving farther downstream. The fastest moving component begins to form a pure zone downstream. The other components also begin to form zones, but the continued supply of the mixed feed at head of the column prevents full resolution.

Displacement

After the entire sample is loaded, the feed is switched to the displacer, chosen to have higher affinity than any sample component. The displacer forms a sharp-edged zone at the head of the column, pushing the other components downstream. Each sample component now acts as a displacer for the lower-affinity solutes, and the solutes sort themselves out into a series of contiguous bands (a "displacement train"), all moving downstream at the rate set by the displacer. The size and loading of the column are chosen to let this sorting process reach completion before the components reach the bottom of the column. The solutes appear at the bottom of the column as a series of contiguous zones, each consisting of one purified component, with the concentration within each individual zone effectively uniform.

Regeneration

After the last solute has been eluted, it is necessary to strip the displacer from the column. Since the displacer was chosen for high affinity, this can pose a challenge. On reverse-phase materials, a wash with a high percentage of organic solvent may suffice. Large pH shifts are also often employed. One effective strategy is to remove the displacer by chemical reaction; for instance if hydrogen ion was used as displacer it can be removed by reaction with hydroxide, or a polyvalent metal ion can be removed by reaction with a chelating agent. For some matrices, reactive groups on the stationary phase can be titrated to temporarily eliminate the binding sites, for instance weak-acid ion exchangers or chelating resins can be converted to the protonated form. For gel-type ion exchangers, selectivity reversal at very high ionic strength can also provide a solution. Sometimes the displacer is specifically designed with a titratable functional group to shift its affinity. After the displacer is washed out, the column is washed as needed to restore it to its initial state for the next run.

Common fundamentals

In any form of chromatography, the rate at which the solute moves down the column is a direct reflection of the percentage of time the solute spends in the mobile phase. To achieve separation in either elution or displacement chromatography, there must be appreciable differences in the affinity of the respective solutes for the stationary phase. Both methods rely on movement down the column to amplify the effect of small differences in distribution between the two phases. Distribution between the mobile and stationary phases is described by the binding isotherm, a plot of solute bound to (or partitioned into) the stationary phase as a function of concentration in the mobile phase. The isotherm is often linear, or approximately so, at low concentrations, but commonly curves (concave-downward) at higher concentrations as the stationary phase becomes saturated.

Characteristics of elution mode

In elution mode, solutes are applied to the column as narrow bands and, at low concentration, move down the column as approximately Gaussian peaks. These peaks continue to broaden as they travel, in proportion to the square root of the distance traveled. For two substances to be resolved, they must migrate down the column at sufficiently different rates to overcome the effects of band spreading. Operating at high concentration, where the isotherm is curved, is disadvantageous in elution chromatography because the rate of travel then depends on concentration, causing the peaks to spread and distort.

Retention in elution chromatography is usually controlled by adjusting the composition of the mobile phase (in terms of solvent composition, pH, ionic strength, and so forth) according to the type of stationary phase employed and the particular solutes to be separated. The mobile phase components generally have lower affinity for the stationary phase than do the solutes being separated, but are present at higher concentration and achieve their effects due to mass action

Mass action

In Chemistry, the law of mass action is a mathematical model that explains and predicts behaviors of solutions in dynamic equilibrium. It can be described with two aspects: 1) the equilibrium aspect, concerning the composition of a reaction mixture at equilibrium and 2) the kinetic aspect...

. Resolution in elution chromatography is generally better when peaks are strongly retained, but conditions that give good resolution of early peaks lead to long run-times and excessive broadening of later peaks unless gradient elution is employed. Gradient equipment adds complexity and expense, particularly at large scale.

Advantages and disadvantages of displacement mode

In contrast to elution chromatography, solutes separated in displacement mode form sharp-edged zones rather than spreading peaks. Zone boundaries in displacement chromatography are self-sharpening: if a molecule for some reason gets ahead of its band, it enters a zone in which it is more strongly retained, and will then run more slowly until its zone catches up. Furthermore, because displacement chromatography takes advantage of the non-linearity of the isotherms, loadings are deliberately high; more material can be separated on a given column, in a given time, with the purified components recovered at significantly higher concentrations. Retention conditions can still be adjusted, but the displacer controls the migration rate of the solutes. The displacer is selected to have higher affinity for the stationary phase than does any of the solutes being separated, and its concentration is set to approach saturation of the stationary phase and to give the desired migration rate of the concentration wave. High-retention conditions can be employed without gradient operation, because the displacer ensures removal of all solutes of interest in the designed run time.

Because of the concentrating effect of loading the column under high-retention conditions, displacement chromatography is well suited to purify components from dilute feed streams. However, it is also possible to concentrate material from a dilute stream at the head of a chromatographic column and then switch conditions to elute the adsorbed material in conventional isocratic or gradient modes. Therefore this approach is not unique to displacement chromatography, although the higher loading capacity and less dilution allow greater concentration in displacement mode.

A disadvantage of displacement chromatography is that non-idealities always give rise to an overlap zone between each pair of components; this mixed zone must be collected separately for recycle or discard to preserve the purity of the separated materials. The strategy of adding spacer molecules to form zones between the components (sometimes termed "carrier displacement chromatography") has been investigated and can be useful when suitable, readily removable spacers are found. Another disadvantage is that the raw chromatogram, for instance a plot of absorbance or refractive index

Refractive index

In optics the refractive index or index of refraction of a substance or medium is a measure of the speed of light in that medium. It is expressed as a ratio of the speed of light in vacuum relative to that in the considered medium....

 vs elution volume, can be difficult to interpret for contiguous zones, especially if the displacement train is not fully developed. Documentation and troubleshooting may require additional chemical analysis to establish the distribution of a given component. Another disadvantage is that the time required for regeneration limits throughput.

According to John C. Ford's article in the Encyclopedia of Chromatography, theoretical studies indicate that at least for some systems, optimized overloaded elution chromatography offers higher throughput than displacement chromatography, though limited experimental tests suggest that displacement chromatography is superior (at least before consideration of regeneration time).

Applications

Historically, displacement chromatography was applied to preparative separations of amino acids and rare earth elements and has also been investigated for isotope separation.

Proteins

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

Molecular mass

The molecular mass of a substance is the mass of one molecule of that substance, in unified atomic mass unit u...

 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

Polyelectrolyte

Polyelectrolytes are polymers whose repeating units bear an electrolyte group. These groups will dissociate in aqueous solutions , making the polymers charged. Polyelectrolyte properties are thus similar to both electrolytes and polymers , and are sometimes called polysalts. Like salts, their...

 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

Size exclusion chromatography

Size-exclusion chromatography is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight . It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers...

, ultrafiltration

Ultrafiltration

Ultrafiltration is a variety of membrane filtration in which hydrostatic pressure forces a liquid against a semipermeable membrane. Suspended solids and solutes of high molecular weight are retained, while water and low molecular weight solutes pass through the membrane...

). In addition, the salt-dependent adsorption

Adsorption

Adsorption is the adhesion of atoms, ions, biomolecules or molecules of gas, liquid, or dissolved solids to a surface. This process creates a film of the adsorbate on the surface of the adsorbent. It differs from absorption, in which a fluid permeates or is dissolved by a liquid or solid...

 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

Recombinant DNA

Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...

 growth factors, antigen

Antigen

An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...

ic vaccine

Vaccine

A vaccine is a biological preparation that improves immunity to a particular disease. A vaccine typically contains an agent that resembles a disease-causing microorganism, and is often made from weakened or killed forms of the microbe or its toxins...

 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

Ion exchange

Ion exchange is an exchange of ions between two electrolytes or between an electrolyte solution and a complex. In most cases the term is used to denote the processes of purification, separation, and decontamination of aqueous and other ion-containing solutions with solid polymeric or mineralic 'ion...

, hydrophobic interaction, as well as reversed-phase chromatography

Reversed-phase chromatography

Reversed-phase chromatography includes any chromatographic method that uses a non-polar stationary phase. The name "reversed phase" has a historical background. In the 1970s most liquid chromatography was done on non-modified silica or alumina with a hydrophilic surface chemistry and a stronger...

.

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

Two-dimensional chromatography

Two-dimensional chromatography

Two-dimensional chromatography is a type of chromatographic technique in which the injected sample is separated by passing through two different separation stages. This is done by injecting the eluent from the first column onto a second column...

 represents the most thorough and rigorous approach to evaluation of the proteome

Proteome

The proteome is the entire set of proteins expressed by a genome, cell, tissue or organism. More specifically, it is the set of expressed proteins in a given type of cells or an organism at a given time under defined conditions. The term is a portmanteau of proteins and genome.The term has been...

. 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 femtomolar 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.