Chromatography

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Pictured is a sophisticated gas chromatography system. This instrument records concentrations of acrylonitrile in the air at various points throughout the chemical laboratory.

Chromatography (from Greek χρώμα:chroma, colour and γραφειν:"grafein" to write) is the collective term for a family of laboratory techniques for the separation of mixtures. It involves passing a mixture dissolved in a "mobile phase" through a stationary phase, which separates the analyte to be measured from other molecules in the mixture and allows it to be isolated.

For those new to separation science, an analogy which is sometimes useful is to suppose a mixture of bees and wasps passing over a flower bed. The bees would be more attracted to the flowers than the wasps, and would become separated from them. If one were to observe at a point past the flower bed, the wasps would pass first, followed by the bees. In this analogy, the bees and wasps represent the analytes to be separated, the flowers represent the stationary phase, and the mobile phase could be thought of as the air. The key to the separation is the differing affinities among analyte, stationary phase, and mobile phase. The observer could represent the detector used in some forms of analytical chromatography. A key point is that the detector need not be capable of discriminating between the analytes, since they have become separated before passing the detector.

Chromatography may be preparative or analytical. Preparative chromatography seeks to separate the components of a mixture for further use (and is thus a form of purification). Analytical chromatography normally operates with smaller amounts of material and seeks to measure the relative proportions of analytes in a mixture. The two are not mutually exclusive.

[edit] History

It was the Russian botanist Mikhail Semyonovich Tsvet who invented the first chromatography technique in 1900 during his research on chlorophyll. He used a liquid-adsorption column containing calcium carbonate to separate plant pigments. The method was described on December 30, 1901 at the 11th Congress of Naturalists and Doctors (XI съезд естествоиспытателей и врачей) in St. Petersburg. The first printed description was in 1903, in the Proceedings of the Warsaw Society of Naturalists, section of biology. He first used the term chromatography in print in 1906 in his two papers about chlorophyll in the German botanical journal, Berichte der Deutschen Botanischen Gesellschaft. In 1907 he demonstrated his chromatograph for the German Botanical Society. Interestingly, Mikhail's surname "Tsvet" means "color" in Russian, so there is the possibility that his naming the procedure chromatography (literally "color writing") was a way that he could make sure that he, a commoner in Tsarist Russia, could be immortalized.

In 1952 Archer John Porter Martin and Richard Laurence Millington Synge were awarded the Chemistry Nobel Prize for their invention of partition chromatography.^[1] Since then, the technology has advanced rapidly. Researchers found that the principles underlying Tsvet's chromatography could be applied in many different ways, giving rise to the different varieties of chromatography described below. Simultaneously, advances continually improved the technical performance of chromatography, allowing the separation of increasingly similar molecules.

[edit] Chromatography terms

• The analyte is the substance which is to be purified or isolated during chromatography.
• Analytical chromatography is used to determine the identity and concentration of molecules in a mixture.
• A bonded phase is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing.
• A chromatogram is the visual output of the chromatograph. Different peaks or patterns on the chromatogram correspond to different components of the separated mixture.

Plotted on the x-axis is the retention time and plotted on the y-axis a signal (for example obtained by UV spectroscopy) corresponding to the amount of analyte exiting the system.

• A chromatograph takes a chemical mixture carried by liquid or gas and separates it into its component parts as a result of differential distributions of the solutes as they flow around or over the stationary phase.
• Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction.
• The effluent is the mobile phase leaving the column.
• An immobilized phase is a stationary phase which is immobilized on the support particles, or on the inner wall of the column tubing.
• The mobile phase is the analyte and solvent mixture which travels through or along the the stationary phase, in a definite direction. It may be a liquid (LC), a gas (GC), or a supercritical fluid (supercritical-fluid chromatography).
• Preparative chromatography is used to nondestructively purify sufficient quantities of a substance for further use, rather than analysis.
• The retention time is the characteristic time it takes for a particular molecule to pass through the system under set conditions.
• The sample is the mixture consisting of a number of components the separation of which is attempted on the chromatographic system as they are carried or eluted by the mobile phase.
• The solute refers to the sample components in partition chromatography.
• The solvent refers to the liquid stationary phase in partition chromatography.
• The stationary phase is the substance which is fixed in place for the chromatography procedure and is the phase to which solvents and the analyte travels through or binds to. Examples include the silica layer in thin layer chromatography.

[edit] Chromatography theory

Chromatography is a separation method that exploits the differences in partitioning behavior between a mobile phase and a stationary phase to separate the components in a mixture. Components of a mixture may be interacting with the stationary phase based on charge, relative solubility or adsorption. There are two theories of chromatography, the plate and rate theories.

[edit] Retention

The retention is a measure of the speed at which a substance moves in a chromatographic system. In continuous development systems like HPLC or GC, where the compounds are eluted with the eluent, the retention is usually measured as the retention time R_t or t_R, the time between injection and detection. In interrupted development systems like TLC the retention is measured as the retention factor R_f, the run length of the compound divided by the run length of the eluent front:

The retention of a compound often differs considerably between experiments and laboratories due to variations of the eluent, the stationary phase, temperature, and the setup. It is therefore important to compare the retention of the test compound to that of one or more standard compounds under absolutely identical conditions.

During the chromatographic process the analyte experiences zone broadening as a result of diffusion. Two analytes with different retention times yet with large broadening do not resolve and this is why in any chromatographic system broadening needs to be minimized. This is done by selecting the proper stationary and mobile phase, the eluent velocity, the track length and temperature. The Van Deemter's equation gives an ideal eluent velocity taking into account several physical parameters.

[edit] Plate theory

The plate theory of chromatography was developed by Archer John Porter Martin and Richard Laurence Millington Synge. The plate theory describes the chromatography system, the mobile and stationary phases, as being in equilibrium. The partition coefficient K is based on this equilibrium, and is defined by the following equation:

K is assumed to be independent of concentration, and can change if experimental conditions are changed, for example temperature is increased or decreased. As K increases, it takes longer for solutes to separate. For a column of fixed length and flow, the retention time (t_R) and retention volume (V_r) can be measured and used to calculate K.

[edit] Techniques by chromatographic bed shape

[edit] Column chromatography

A diagram of a standard column chromatography and a flash column chromatography setup

Column chromatography is a separation technique in which the stationary bed is within a tube. the particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube (packed column) or be concentrated on or along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube (open tubular column). Differences in rates of movement through the medium are calculated to different retention times of the sample.^[2]

In 1978, W. C. Still introduced a modified version of column chromatography called flash column chromatography (flash).^[3] The technique is very similar to the traditional column chromatography, except for that the solvent is driven through the column by applying positive pressure. This allowed most separations to be performed in less than 20 minutes, with improved separations compared to the old method. Modern flash chromatography systems are sold as pre-packed plastic cartridges, and the solvent is pumped through the cartridge. Systems may also be linked with detectors and fraction collectors providing automation. The introduction of gradient pumps resulted in quicker separations and less solvent usage.

[edit] Planar Chromatography

Thin layer chromatography is used to separate components of chlorophyll

Planer chromatography is a separation technique in which the stationary phase is present as or on a plane. The plane can be a paper, serving as such or impregnated by a substance as the stationary bed (paper chromatography) or a layer of solid particles spread on a support such as a glass plate (thin layer chromatography).

[edit] Paper Chromatography

Paper chromatography is a technique that involves placing a small dot of sample solution onto a strip of chromatography paper. The paper is placed in a jar containing a shallow layer of solvent and sealed. As the solvent rises through the paper it meets the sample mixture which starts to travel up the paper with the solvent. Different compounds in the sample mixture travel different distances according to how strongly they interact with the paper. This allows the calculation of an R_f value and can be compared to standard compounds to aid in the identification of an unknown substance.

[edit] Thin layer chromatography

Thin layer chromatography (TLC) is a widely-employed laboratory technique and is similar to paper chromatography. However, instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate. Compared to paper, it has the advantage of faster runs, better separations, and the choice between different adsorbents. Different compounds in the sample mixture travel different distances according to how strongly they interact with the adsorbent. This allows the calculation of an R_f value and can be compared to standard compounds to aid in the identification of an unknown substance.

[edit] Techniques by physical state of mobile phase

[edit] Gas chromatography

Gas chromatography (GC) is a separation technique in which the mobile phase is a gas. Gas chromatography is always carried out in a column.

Gas chromatography (GC) is based on a partition equilibrium of analyte between a solid stationary phase and a mobile gas. The stationary phase is adhered to the inside of a small-diameter glass tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column). It is widely used in analytical chemistry; though the high temperatures used in GC make it unsuitable for high molecular weight biopolymers, frequently encountered in biochemistry, it is well suited for use in the petrochemical, environmental monitoring, and industrial chemical fields. It is also used extensively in chemistry research.

[edit] Liquid chromatography

Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. Liquid chromatography can be carried out ieither in a column or a plane. Present day liquid chromatography generally utilizes very small particles and a relatively high pressure is characterized by high performance liquid chromatography (HPLC).

In the HPLC technique, the analyte is forced through a column (stationary phase) by a liquid (mobile phase) at high pressure, which decreases the time the separated components remain on the stationary phase and thus the time they have to diffuse within the column. Specific techniques which come under this broad heading are listed below. It should also be noted that the following techniques can also be considered fast protein liquid chromatography if no pressure is used to drive the mobile phase through the stationary phase. See also Aqueous Normal Phase Chromatography.

[edit] Supercritical fluid chromatography

Supercritical fluid chromatography is a separation technique in which the mobile phase is a a fluid above and relatively close to its critical temperature and pressure.

[edit] Techniques by separation mechanism

[edit] Ion exchange chromatography

Ion exchange chromatography is a column chromatography that uses a charged stationary phase. It is used to separate charged compounds including amino acids, peptides, and proteins. The stationary phase is usually an ion exchange resin that carries charged functional groups which interact with oppositely charged groups of the compound to be retained. Ion exchange chromatography is commonly used to purify proteins using FPLC.

[edit] Size exclusion chromatography

Size exclusion chromatography (SEC) is also known as gel permeation chromatography or gel filtration chromatography and separates particles on the basis of size. Smaller molecules enter a porous media and take longer to exit the column, whereas larger particles leave the column earlier. It is generally a low resolution chromatography and thus it is often reserved for the final, "polishing" step of a purification. It is also useful for determining the tertiary structure and quaternary structure of purified proteins, especially since it can be carried out under native solution conditions.

[edit] Affinity chromatography

Affinity chromatography is based on selective non-covalent interaction between an analyte and specific molecules. It is very specific, but not very robust. It is often used in biochemistry in the purification of proteins bound to tags. These fusion proteins are labelled with compounds such as His-tags, biotin or antigens, which bind to the stationary phase specifically. After purification, some of these tags are usually removed and the pure protein is obtained.

[edit] Special techniques

[edit] Reversed-phase chromatography

Reversed-phase chromatography is an elution procedure used in liquid chromatography in which the mobile phase is significantly more polar then the stationary phase.

[edit] Two-dimensional chromatography

[edit] Pyrolysis gas chromatography

[edit] Fast protein liquid chromatography

Fast protein liquid chromatography (FPLC) is a term applied to several chromatography techniques which are used to purify proteins. Many of these techniques are identical to those carried out under high performance liquid chromatography.

[edit] Countercurrent chromatography

Countercurrent chromatography (CCC) is a type of liquid-liquid chromatography, where both the stationary and mobile phases are liquids. It involves mixing a solution of liquids, allowing them to settle into layers and then separating the layers.

[edit] See also

• Paper chromatography of amino acids on Wikibooks
• Aqueous Normal Phase Chromatography
• MCSGP = Multicolumn Countercurrent Solvent Gradient Purification Process

[edit] References

1. ^ Nobelprize.org: The Nobel Prize in Chemistry 1952
2. ^ IUPAC Nomenclature for ChromatographyIUPAC Nomenclature for Chromatography, Pure & Appl. Chem., Vol. 65, No. 4, pp.819-872, 1993.
3. ^ Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43(14), 2923-2925. (DOI:10.1021/jo00408a041)

[edit] External links

• IUPAC Nomenclature for Chromatography
• LCGC North America
• Library 4 Science: Chrom-Ed Series
• GC Help
• Overlapping Peaks Program - Learning by Simulations
• Chromatography Videos - MIT OCW - Digital Lab Techniques Manual
• GC Video - University of Colorado
• Chromatography Equations Calculators - MicroSolv Technology Corporation