Last updated
Chromatography column Chromatography.png
Chromatography column

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.


In a liquid chromatography experiment, for example, an analyte is generally adsorbed, or "bound to", an adsorbent in a liquid chromatography column. The adsorbent, a solid phase (stationary phase), is a powder which is coated onto a solid support. Based on an adsorbent's composition, it can have varying affinities to "hold" onto other molecules—forming a thin film on the surface of its particles. Elution then is the process of removing analytes from the adsorbent by running a solvent, called an "eluent", past the adsorbent/analyte complex. As the solvent molecules "elute", or travel down through the chromatography column, they can either pass by the adsorbent/analyte complex or they can displace the analyte by binding to the adsorbent in its place. After the solvent molecules displace the analyte, the analyte can be carried out of the column for analysis. This is why as the mobile phase passes out of the column, it typically flows into a detector or is collected for compositional analysis.

Predicting and controlling the order of elution is a key aspect of column chromatographic methods.

Eluotropic series

An eluotropic series is listing of various compounds in order of eluting power for a given adsorbent. The "eluting power" of a solvent is largely a measure of how well the solvent can "pull" an analyte off the adsorbent to which it is attached. This often happens when the eluent adsorbs onto the stationary phase, displacing the analyte. Such series are useful for determining necessary solvents needed for chromatography of chemical compounds. Normally such a series progresses from non-polar solvents, such as n-hexane, to polar solvents such as methanol or water. The order of solvents in an eluotropic series depends both on the stationary phase as well as on the compound used to determine the order.

Adsorption strength (least strongly adsorbed → most strongly adsorbed)
Saturated hydrocarbons; alkyl halidesUnsaturated hydrocarbons; alkenyl halidesAromatic hydrocarbons; aryl halidesPolyhalogenated hydrocarbonsEthersEstersAldehydes and ketonesAlcoholsAcids and bases (amines)
Eluting power (least eluting power → greatest eluting power)
Hexane or pentaneCyclohexaneBenzeneDichloromethaneChloroformEther (anhydrous)Ethyl acetate (anhydrous)Acetone (anhydrous)MethanolEthanolPyridineAcetic acidWater


The eluent or eluant is the "carrier" portion of the mobile phase. It moves the analytes through the chromatograph. In liquid chromatography, the eluent is the liquid solvent; in gas chromatography, it is the carrier gas. [1]


The eluate is the analyte material that emerges from the chromatograph. It specifically includes both the analytes and solutes passing through the column, while the eluent is only the carrier.

Elution time and elution volume

The "elution time" of a solute is the time between the start of the separation (the time at which the solute enters the column) and the time at which the solute elutes. In the same way, the elution volume is the volume of eluent required to cause elution. Under standard conditions for a known mix of solutes in a certain technique, the elution volume may be enough information to identify solutes. For instance, a mixture of amino acids may be separated by ion-exchange chromatography. Under a particular set of conditions, the amino acids will elute in the same order and at the same elution volume.

Antibody elution

Antibody elution is the process of removing antibodies that are attached to their targets, such as the surface of red blood cells. Techniques include using heat, a freeze/thaw cycle, ultrasound, acids or organic solvents. No single method is best in all situations. [2]

See also

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 (SEC), 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 used to separate components of a liquid mixture

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 each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out of the column.

Gel permeation chromatography (GPC) is a type of size-exclusion chromatography (SEC), that separates analytes on the basis of size, typically in organic solvents. The technique is often used for the analysis of polymers. As a technique, SEC was first developed in 1955 by Lathe and Ruthven. The term gel permeation chromatography can be traced back to J.C. Moore of the Dow Chemical Company who investigated the technique in 1964. The proprietary column technology was licensed to Waters Corporation, who subsequently commercialized this technology in 1964. GPC systems and consumables are now also available from a number of manufacturers. It is often necessary to separate polymers, both to analyze them as well as to purify the desired product.

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

Gas chromatography (GC) is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture. In preparative chromatography, GC can be used to prepare pure compounds from a mixture.

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

Paper chromatography is an analytical method used to separate coloured chemicals or substances. It is now primarily used as a teaching tool, having been replaced in the laboratory by other chromatography methods such as thin-layer chromatography (TLC).

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>

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>

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

<span class="mw-page-title-main">Thin-layer chromatography</span> Technique used to separate non-volatile mixtures

Thin-layer chromatography (TLC) is a chromatography technique used to separate non-volatile mixtures.

<span class="mw-page-title-main">Solid-phase extraction</span>

Solid-phase extraction (SPE) is an extractive technique by which compounds that are dissolved or suspended in a liquid mixture are separated from other compounds in the 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.

Reversed-phase chromatography includes any chromatographic method that uses a hydrophobic stationary phase. RPC refers to liquid chromatography.

<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.

Supercritical fluid chromatography (SFC) is a form of normal phase chromatography that uses a supercritical fluid such as carbon dioxide as the mobile phase. It is used for the analysis and purification of low to moderate molecular weight, thermally labile molecules and can also be used for the separation of chiral compounds. Principles are similar to those of high performance liquid chromatography (HPLC), however SFC typically utilizes carbon dioxide as the mobile phase; therefore the entire chromatographic flow path must be pressurized. Because the supercritical phase represents a state in which liquid and gas properties converge, supercritical fluid chromatography is sometimes called convergence chromatography.

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 region between reversed-phase chromatography (RP) and organic normal-phase chromatography (ONP).

Displacement chromatography is a chromatography technique in which a sample is placed onto the head of the column 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”. It is primarily a preparative technique; higher product concentration, higher purity, and increased throughput may be obtained compared to other modes of chromatography.

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

Two-dimensional chromatography is a type of chromatographic technique in which the injected sample is separated by passing through two different separation stages. Two different chromatographic columns are connected in sequence, and the effluent from the first system is transferred onto the second column. Typically the second column has a different separation mechanism, so that bands that are poorly resolved from the first column may be completely separated in the second column. Alternately, the two columns might run at different temperatures. During the second stage of separation the rate at which the separation occurs must be faster than the first stage, since there is still only a single detector. The plane surface is amenable to sequential development in two directions using two different solvents.

<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.

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.


  1. "IUPAC Gold Book: eluent". International Union of Pure and Applied Chemistry. doi:10.1351/goldbook.E02040 . Retrieved 2008-09-28.{{cite journal}}: Cite journal requires |journal= (help)
  2. George H. Roberts (2006). "Elution Techniques in Blood Bank" (PDF). American Medical Technologists (AMT).