Displacement chromatography

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Displacement chromatography is a chromatography technique in which a sample is placed onto the head of the column [n 1] and is then displaced by a solute 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". [1] It is primarily a preparative technique; higher product concentration, higher purity, and increased throughput may be obtained compared to other modes of chromatography.

Contents

Discovery

The advent of displacement chromatography can be attributed to Arne Tiselius, [2] who in 1943 first classified the modes of chromatography as frontal, elution, and displacement. Displacement chromatography found a variety of applications including isolation of transuranic elements [3] and biochemical entities. [4] The technique was redeveloped by Csaba Horváth, [5] who employed modern high-pressure columns and equipment. It has since found many applications, particularly in the realm of biological macromolecule purification.

Principle

Example of a Langmuir isotherm, for a population of binding sites having uniform affinity. In this case the vertical axis represents the amount bound per unit of stationary phase, the horizontal axis the concentration in the mobile phase. In this case, the dissociation constant is 0.5 and the capacity 10; units are arbitrary. Langmuir sorption isotherm.svg
Example of a Langmuir isotherm, for a population of binding sites having uniform affinity. In this case the vertical axis represents the amount bound per unit of stationary phase, the horizontal axis the concentration in the mobile phase. In this case, the dissociation constant is 0.5 and the capacity 10; units are arbitrary.

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 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. [n 2] A molecule with a high affinity 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.

Mode of operation

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. [n 3] 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. [n 4] 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. [6] [7] [8]

Comparison with elution chromatography

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. 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. [6] [7] [8]

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 [9] 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 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). [7]

Applications

Historically, displacement chromatography was applied to preparative separations of amino acids and rare earth elements and has also been investigated for isotope separation. [9] [10] [11] [12]

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 displacers for protein purification in ion exchange systems. [13] [14] [15] 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. [16] [17] [18] [19] [20] [21] [22] In addition, the utility of displacement chromatography for the purification of recombinant growth factors, [23] antigenic vaccine proteins [24] and antisense oligonucleotides [25] 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. [26]

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. [27]

Two-dimensional chromatography

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 femtomolar range. [28] 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.

Notes

  1. ↑ For simplicity, this article is written using the terminology of column liquid chromatography. Examples of other types of displacement chromatography are known.
  2. ↑ In some forms of chromatography, including gel permeation chromatography and some liquid-liquid partition systems, distinct binding sites are not involved and the isotherm remains essentially linear even at high concentrations. These forms are not suitable for displacement-mode operation.
  3. ↑ It is possible to set the retention too high; the rate of desorption must be appreciable for displacement to occur.
  4. ↑ Sometimes a short rinse is interposed before starting the displacer.

Related Research Articles

In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent called the mobile phase, which carries it through a system on which a material called the stationary phase is fixed. Because the different constituents of the mixture tend to have different affinities for the stationary phase and are retained for different lengths of time depending on their interactions with its surface sites, the constituents travel at different apparent velocities in the mobile fluid, causing them to separate. The separation is based on the differential partitioning between the mobile and the stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus affect the separation.

<span class="mw-page-title-main">Size-exclusion chromatography</span> Chromatographic method in which dissolved molecules are separated by their size & molecular weight

Size-exclusion chromatography, also known as molecular sieve 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. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography, versus the name gel permeation chromatography, which is used when an organic solvent is used as a mobile phase. The chromatography column is packed with fine, porous beads which are commonly composed of dextran, agarose, or polyacrylamide polymers. The pore sizes of these beads are used to estimate the dimensions of macromolecules. SEC is a widely used polymer characterization method because of its ability to provide good molar mass distribution (Mw) results for polymers.

<span class="mw-page-title-main">High-performance liquid chromatography</span> Technique in analytical chemistry

High-performance liquid chromatography (HPLC), formerly referred to as high-pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify specific components in mixtures. The mixtures can originate from food, chemicals, pharmaceuticals, biological, environmental and agriculture, etc, which have been dissolved into liquid solutions.

Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the specification of the function, structure and interactions of the protein of interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Ideally, to study a protein of interest, it must be separated from other components of the cell so that contaminants will not interfere in the examination of the protein of interest's structure and function. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps usually exploit differences in protein size, physico-chemical properties, binding affinity and biological activity. The pure result may be termed protein isolate.

Affinity chromatography is a method of separating a biomolecule from a mixture, based on a highly specific macromolecular binding interaction between the biomolecule and another substance. The specific type of binding interaction depends on the biomolecule of interest; antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid binding interactions are frequently exploited for isolation of various biomolecules. Affinity chromatography is useful for its high selectivity and resolution of separation, compared to other chromatographic methods.

<span class="mw-page-title-main">Column chromatography</span> Method to isolate a compound in a mixture

Column chromatography in chemistry is a chromatography method used to isolate a single chemical compound from a mixture. Chromatography is able to separate substances based on differential adsorption of compounds to the adsorbent; compounds move through the column at different rates, allowing them to be separated into fractions. The technique is widely applicable, as many different adsorbents can be used with a wide range of solvents. The technique can be used on scales from micrograms up to kilograms. The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process. The latter prevents cross-contamination and stationary phase degradation due to recycling. Column chromatography can be done using gravity to move the solvent, or using compressed gas to push the solvent through the column.

<span class="mw-page-title-main">Ion chromatography</span> Separates ions and polar molecules

Ion chromatography is a form of chromatography that separates ions and ionizable polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule—including small inorganic anions, large proteins, small nucleotides, and amino acids. However, ion chromatography must be done in conditions that are one pH unit away from the isoelectric point of a protein.

<span class="mw-page-title-main">Solid-phase extraction</span> Process to separate compounds by properties

Solid-phase extraction (SPE) is a solid-liquid extractive technique, by which compounds that are dissolved or suspended in a liquid mixture are separated, isolated or purified, from other compounds in this mixture, according to their physical and chemical properties. Analytical laboratories use solid phase extraction to concentrate and purify samples for analysis. Solid phase extraction can be used to isolate analytes of interest from a wide variety of matrices, including urine, blood, water, beverages, soil, and animal tissue.

<span class="mw-page-title-main">Fast protein liquid chromatography</span>

Fast protein liquid chromatography (FPLC) is a form of liquid chromatography that is often used to analyze or purify mixtures of proteins. As in other forms of chromatography, separation is possible because the different components of a mixture have different affinities for two materials, a moving fluid and a porous solid. In FPLC the mobile phase is an aqueous buffer solution. The buffer flow rate is controlled by a positive-displacement pump and is normally kept constant, while the composition of the buffer can be varied by drawing fluids in different proportions from two or more external reservoirs. The stationary phase is a resin composed of beads, usually of cross-linked agarose, packed into a cylindrical glass or plastic column. FPLC resins are available in a wide range of bead sizes and surface ligands depending on the application.

Reversed-phase liquid chromatography (RP-LC) is a mode of liquid chromatography in which non-polar stationary phase and polar mobile phases are used for the separation of organic compounds. The vast majority of separations and analyses using high-performance liquid chromatography (HPLC) in recent years are done using the reversed phase mode. In the reversed phase mode, the sample components are retained in the system the more hydrophobic they are.

Mixed-mode chromatography (MMC), or multimodal chromatography, refers to chromatographic methods that utilize more than one form of interaction between the stationary phase and analytes in order to achieve their separation. What is distinct from conventional single-mode chromatography is that the secondary interactions in MMC cannot be too weak, and thus they also contribute to the retention of the solutes.

<span class="mw-page-title-main">Hydrophilic interaction chromatography</span> Type of chromatography

Hydrophilic interaction chromatography is a variant of normal phase liquid chromatography that partly overlaps with other chromatographic applications such as ion chromatography and reversed phase liquid chromatography. HILIC uses hydrophilic stationary phases with reversed-phase type eluents. The name was suggested by Andrew Alpert in his 1990 paper on the subject. He described the chromatographic mechanism for it as liquid-liquid partition chromatography where analytes elute in order of increasing polarity, a conclusion supported by a review and re-evaluation of published data.

Chromatography is a physical method of separation that distributes the components you want to separate between two phases, one stationary, the other moving in a definite direction. Cold ethanol precipitation, developed by Cohn in 1946, manipulates pH, ionic strength, ethanol concentration and temperature to precipitate different protein fractions from plasma. Chromatographic techniques utilise ion exchange, gel filtration and affinity resins to separate proteins. Since the 1980s it has emerged as an effective method of purifying blood components for therapeutic use.

Micellar liquid chromatography (MLC) is a form of reversed phase liquid chromatography that uses an aqueous micellar solutions as the mobile phase.

Aqueous normal-phase chromatography (ANP) is a chromatographic technique that involves the mobile phase compositions and polarities between reversed-phase chromatography (RP) and normal-phase chromatography (NP), while the stationary phases are polar.

Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) is a form of chromatography that is used to separate or purify biomolecules from complex mixtures. It was developed at the Swiss Federal Institute of Technology Zürich by Aumann and Morbidelli. The process consists of two to six chromatographic columns which are connected to one another in such a way that as the mixture moves through the columns the compound is purified into several fractions.

<span class="mw-page-title-main">Elution</span> Extraction of a material by washing with a solvent

In analytical and organic chemistry, elution is the process of extracting one material from another by washing with a solvent; as in washing of loaded ion-exchange resins to remove captured ions.

<span class="mw-page-title-main">Countercurrent chromatography</span>

Countercurrent chromatography is a form of liquid–liquid chromatography that uses a liquid stationary phase that is held in place by inertia of the molecules composing the stationary phase accelerating toward the center of a centrifuge due to centripetal force and is used to separate, identify, and quantify the chemical components of a mixture. In its broadest sense, countercurrent chromatography encompasses a collection of related liquid chromatography techniques that employ two immiscible liquid phases without a solid support. The two liquid phases come in contact with each other as at least one phase is pumped through a column, a hollow tube or a series of chambers connected with channels, which contains both phases. The resulting dynamic mixing and settling action allows the components to be separated by their respective solubilities in the two phases. A wide variety of two-phase solvent systems consisting of at least two immiscible liquids may be employed to provide the proper selectivity for the desired separation.

Solvent impregnated resins (SIRs) are commercially available (macro)porous resins impregnated with a solvent/an extractant. In this approach, a liquid extractant is contained within the pores of (adsorption) particles. Usually, the extractant is an organic liquid. Its purpose is to extract one or more dissolved components from a surrounding aqueous environment. The basic principle combines adsorption, chromatography and liquid-liquid extraction.

Thermoresponsive polymers can be used as stationary phase in liquid chromatography. Here, the polarity of the stationary phase can be varied by temperature changes, altering the power of separation without changing the column or solvent composition. Thermally related benefits of gas chromatography can now be applied to classes of compounds that are restricted to liquid chromatography due to their thermolability. In place of solvent gradient elution, thermoresponsive polymers allow the use of temperature gradients under purely aqueous isocratic conditions. The versatility of the system is controlled not only through changing temperature, but through the addition of modifying moieties that allow for a choice of enhanced hydrophobic interaction, or by introducing the prospect of electrostatic interaction. These developments have already introduced major improvements to the fields of hydrophobic interaction chromatography, size exclusion chromatography, ion exchange chromatography, and affinity chromatography separations as well as pseudo-solid phase extractions.

References

  1. ↑ N. Tugcu. Purification of proteins using displacement chromatography. pp 71–89 in M. Zachariou (Ed.) Methods in Molecular Biology: Vol 421 Affinity Chromatography: Methods and Protocols. 2nd edition. Humana Press, Totowa NJ.
  2. ↑ A. Tiselius. Displacement development in adsorption analysis. Ark. Kemi. Mineral Geol. 16A: 1–18 (1943).
  3. ↑ G. T. Seaborg. The Transuranium Elements. Science 104(2704):379-386 (1946).
  4. ↑ J. Frenz and C.S. Horvath. High performance displacement chromatography. pp 212-314 in C. Horvath (Ed.) High Performance Liquid Chromatography-advances and perspectives. Vol. 5, Academic Press, San Diego, CA.
  5. ↑ C.S. Horvath, A. Nahum, and J. Frenz. High performance displacement chromatography. J. Chromatogr. 218, 365–393(1981).
  6. 1 2 Little, Charles Displacement Chromatography Comes of Age. An indispensable tool for the purification of biomacromolecules Archived February 22, 2011, at the Wayback Machine Chromatography Techniques (Date of original not given on website but Google shows Nov 15, 2008; accessed Feb. 2011)
  7. 1 2 3 Ford, J.C. "Displacement chromatography" in Jack Cazes, Encyclopedia of Chromatography, pp255-257 Marcel Dekker 2001
  8. 1 2 Helfferich, F. Ion Exchange McGraw Hill, NY, 1962
  9. 1 2 Buchanan, D.C. Preparative isolation of amino acids by carrier displacement chromatography on ion exchange resins Journal of Biological Chemistry 229, 211-229, 1957
  10. ↑ Partridge, S.M., and R.C. Brimley. Displacement chromatography on ion exchange resins. 8. A systematic method for the separation of Amino Acids. Biochemical Journal 51, 628-639, 1952
  11. ↑ Spedding, F.H., J.E. Powell, and E.J. Wheelwright. The use of copper as the retaining ion in the elution of rare earths with ammonium ethylenediamine tetraacetate solutions Journal of the American Chemical Society 76,2557-60, 1954,
  12. ↑ Phillips, T.R., D.R. Owens, and A.G. Hamlin. Purification of hydrogen isotopes by self-displacement chromatography at low temperatures Nature 192 1067-8, 1961
  13. ↑ S. M. Cramer and G. Jayaraman, Current Opinions in Biotechnology 4: 217-225, (1993)
  14. ↑ G. Jayaraman, S. Gadam, and S. M. Cramer. J. Chromatogr. A 630:53-68. (1993)
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  21. ↑ A. Kundu, and S. M. Cramer. Anal. Biochem., 248:111-116. ( 1997)
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  23. ↑ K. A. Barnthouse, W. Trompeter, R. Jone, P. Inampudi, R.Rupp, and S. M. Cramer. J. Biotechnol. 66:125-136 (1998)
  24. ↑ A. A. Shukla, R. L. Hopfer, D. N. Chakravarti, E. Bortell, and S. M. Cramer. Biotechnol. Prog. 14: 91-101(1998)
  25. ↑ N. Tugcu, R. R. Deshmukh, Y. S. Sangvic, J. A. Moored, and S. M. Cramer J. Chromatogr. A 923:65-73(2001)
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  27. ↑ N. Tugcu, R. R. Deshmukh, Y. S. Sanghvi, and S. M. Cramer. Reactive and Functional Polymers 54, 37–47(2003).
  28. ↑ . E. Nagele, M. Vollmer, P. Horth, and C. Vad. 2D-LC/MS techniques for the identification of proteins in highly complex mixtures. Expert Reviews in Proteomics. Vol. 1, No. 1, Pages 37-46 (2004).
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