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Chromatography is a laboratory process that occurs in several steps and is used to separate mixtures of various chemicals into their individual components. The governing principle ofÂ chromatographyÂ is that different chemicals in a mixture have different degrees of dissolving in a liquid or sticking to a solid surface. In other words, chromatography can identify a chemical and separate it from a dense mixture of other chemicals and show it on a surface.Â
Various chemicals in a mixture have different sticking ability on a surface. By varying this process in many ways, the chromatography technique can be used to separate any amount of quantities ranging from micrograms (in laboratories) to tons (in chemical plants). There are various chromatography procedures that have become popular since the invention of chromatography by Russian botanist, Mikhail Semyonovich Tsvet, in 1901.
In the chromatography processes a stream of liquid, which is called as mobile phase, is made to flow through a tube known as column, and it is packed with porous solid material, called as the stationary phase. The sample of the mixture that is to be analyzed is sent through the mobile phase and as the mixture proceeds in the tube, the compounds are separated. Chromatography is preferred over many other techniques as it doesn't cause any molecular changes in the composition of the chemicals involved. Read more onÂ paper chromatography.
Chromatography may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture for further use (and is thus a form of purification). Analytical chromatography is done normally with smaller amounts of material and is for measuring the relative proportions of analytes in a mixture. The two are not mutually exclusive.
TheÂ history of chromatographyÂ begins during the mid-19th century. Chromatography, literally "colour writing", was used-and named- in the first decade of the 20th century, primarily for the separation of plantÂ pigmentsÂ such asÂ chlorophyll. New types of chromatography developed during the 1930s and 1940s made the technique useful for many types ofÂ separation process.
Some related techniques were developed during the 19th century (and even before), but the first true chromatography is usually attributed to Russian botanistÂ Mikhail SemyonovichTsvet, who used columns of calcium carbonate for separating plant pigments during the first decade of the 20th century during his research ofÂ chlorophyll.
Chromatography became developed substantially as a result of the work ofÂ Archer John Porter MartinÂ andÂ Richard Laurence Millington SyngeÂ during the 1940s and 1950s. They established the principles and basic techniques ofÂ partition chromatography, and their work encouraged the rapid development of several types of chromatography method:Â paper chromatography,Â gas chromatography, and what would become known asÂ high performance liquid chromatography. Since then, the technology has advanced rapidly. Researchers found that the main principles of Tsvet's chromatography could be applied in many different ways, resulting in the different varieties of chromatography described below. Simultaneously, advances continually improved the technical performance of chromatography, allowing the separation of increasingly similar molecules.
SOME IMPORTANT CHROMATOGRAPHY TERMS
TheÂ analyteÂ is the substance to be separated during chromatography.
Analytical chromatographyÂ is used to determine the existence and possibly also the concentration of analyte(s) in aÂ sample.
AÂ bonded phaseÂ is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing.
AÂ chromatogramÂ is the visual output of the chromatograph. In the case of an optimal separation, 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 aÂ spectrophotometer,Â mass spectrometerÂ or a variety of other detectors) corresponding to the response created by the analytes exiting the system. In the case of an optimal system the signal is proportional to the concentration of the specific analyte separated.
AÂ chromatographÂ is equipment that enables a sophisticated separation e.g. gas chromatographic or liquid chromatographic separation.
ChromatographyÂ 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.
TheÂ eluateÂ is the mobile phase leaving the column.
TheÂ eluentÂ is the solvent that will carry the analyte.
AnÂ eluotropic seriesÂ is a list of solvents ranked according to their eluting power.
AnÂ immobilized phaseÂ is a stationary phase which is immobilized on the support particles, or on the inner wall of the column tubing.
TheÂ mobile phaseÂ is the phase which moves in a definite direction. It may be a liquid (LC and CEC), a gas (GC), or a supercritical fluid (supercritical-fluid chromatography, SFC). The mobile phase consists of the sample being separated/analyzed and the solvent that moves the sample through the column. In the case ofÂ HPLCÂ the mobile phase consists of a non-polar solvent(s) such as hexane in normal phase or polar solvents in reverse phase chromotagraphy and the sample being separated. The mobile phase moves through the chromatography column (the stationary phase) where the sample interacts with the stationary phase and is separated.
Preparative chromatographyÂ is used to purify sufficient quantities of a substance for further use, rather than analysis.
TheÂ retention timeÂ is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions. See also:Â KovatsHYPERLINK "http://en.wikipedia.org/wiki/Kovats_retention_index"'HYPERLINK "http://en.wikipedia.org/wiki/Kovats_retention_index" retention index
TheÂ sampleÂ is the matter analyzed in chromatography. It may consist of a single component or it may be a mixture of components. When the sample is treated in the course of an analysis, the phase or the phases containing the analytes of interest is/are referred to as the sample whereas everything out of interest separated from the sample before or in the course of the analysis is referred to as waste.
TheÂ soluteÂ refers to the sample components in partition chromatography.
TheÂ solventÂ refers to any substance capable of solubilizing other substance, and especially the liquid mobile phase in LC.
TheÂ stationary phaseÂ is the substance which is fixed in place for the chromatography procedure. Examples include theÂ silicaÂ layer inÂ thin layer chromatography
VARIOUS KINDS OF CHROMATOGRAPHY TECHNIQUES
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.
In 1978, W. C.Still introduced a modified version of column chromatography calledÂ flash column chromatographyÂ (flash).Â 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.
InÂ expanded bed adsorption, a fluidized bed is used, rather than a solid phase made by a packed bed. This allows omission of initial clearing steps such as centrifugation and filtration, for culture broths or slurries of broken cells.
Planar chromatographyÂ 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). DifferentÂ compoundsÂ in the sample mixture travel different distances according to how strongly they interact with the stationary phase as compared to the mobile phase. The specificÂ Retention factorÂ (Rf) of each chemical can be used to aid in the identification of an unknown substance.
Paper chromatography is a technique that involves placing a small dot or line of sample solution onto a strip ofÂ chromatography paper. The paper is placed in a jar containing a shallow layer ofÂ solventÂ and sealed. As the solvent rises through the paper, it meets the sample mixture which starts to travel up the paper with the solvent. This paper is made of cellulose, a polar substance, and the compounds within the mixture travel farther if they are non-polar. More polar substances bond with the cellulose paper more quickly, and therefore do not travel as far.
Thin layer chromatography
Thin layer chromatography is used to separate components of chlorophyll
Thin layer chromatography (TLC) is a widely employed laboratory technique and is similar toÂ paper chromatography. However, instead of using a stationary phase of paper, it involves a stationary phase of a thin layer ofÂ adsorbentÂ likeÂ silica gel,Â alumina, orÂ celluloseÂ on a flat, inertÂ substrate. Compared to paper, it has the advantage of faster runs, better separations, and the choice between different adsorbents. For even better resolution and to allow for quantification,Â high-performance TLCÂ can be used.
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.
Pictured is a sophisticated gas chromatography system. This instrument records concentrations of acrylonitrile in the air at various points throughout the chemical laboratory.
Gas chromatography (GC), also sometimes known as Gas-Liquid chromatography, (GLC), is a separation technique in which the mobile phase is a gas. Gas chromatography is always carried out in a column, which is typically "packed" or "capillary" (see below) .
Gas chromatography (GC) is based on aÂ partition equilibriumÂ of analyte between a solid stationary phase (often a liquid silicone-based material) and a mobile gas (most often Helium). 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 inÂ analytical chemistry; though the high temperatures used in GC make it unsuitable for high molecular weight biopolymers or proteins (heat will denature them), frequently encountered inÂ biochemistry, it is well suited for use in thepetrochemical,Â environmental monitoringÂ andÂ remediation, andÂ industrial chemicalÂ fields. It is also used extensively in chemistry research.
Preparative HPLC apparatus
Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. Liquid chromatography can be carried out either in a column or a plane. Present day liquid chromatography that generally utilizes very small packing particles and a relatively high pressure is referred to asÂ high performance liquid chromatographyÂ (HPLC).
In the HPLC technique, the sample is forced through a column that is packed with irregularly or spherically shaped particles, a porous monolithic layer (stationary phase) or a porous membrane by a liquid (mobile phase) at high pressure. HPLC is historically divided into two different sub-classes based on the polarity of the mobile and stationary phases. Methods in which the stationary phase is more polar than the mobile phase (e.g. toluene as the mobile phase, silica as the stationary phase) are termed normal phase liquid chromatography (NPLC) and the opposite (e.g. water-methanol mixture as the mobile phase and C18 = octadecylsilyl as the stationary phase) is termed reversed phase liquid chromatography (RPLC). Ironically the "normal phase" has fewer applications and RPLC is therefore used considerably more.
Specific techniques which come under this broad heading are listed below. It should also be noted that the following techniques can also be consideredÂ fast protein liquid chromatographyÂ if no pressure is used to drive the mobile phase through the stationary phase.
Affinity chromatographÂ is based on selective non-covalent interaction between an analyte and specific molecules. It is very specific, but not veryÂ robust. It is often used in biochemistry in the purification ofÂ proteinsÂ bound to tags. TheseÂ fusion proteinsÂ are labeled with compounds such asÂ His-tags,Â biotinÂ orÂ antigens, which bind to the stationary phase specifically. After purification, some of these tags are usually removed and the pure protein is obtained.
Affinity chromatography often utilizes a biomolecule's affinity for a metal (Zn, Cu, Fe, etc.). Columns are often manually prepared. Traditional affinity columns are used as a preparative step to flush out unwanted biomolecules.
However, HPLC techniques exist that do utilize affinity chromatogaphy properties. Immobilized Metal Affinity Chromatography (IMAC) is useful to separate aforementioned molecules based on the relative affinity for the metal (I.e. Dionex IMAC) . Often these columns can be loaded with different metals to create a column with a targeted affinity.
Supercritical Fluid Chromatography
Supercritical fluid chromatography is a separation technique in which the mobile phase is a fluid above and relatively close to its critical temperature and pressure.
Ion Exchange Chromatography
Ion exchange chromatography uses ion exchange mechanism to separate analytes. It is usually performed in columns but can also be useful in planar mode. Ion exchange chromatography uses a charged stationary phase to separate charged compounds includingÂ amino acids,Â peptides, andÂ proteins. In conventional methods the stationary phase is anÂ ion exchange resinÂ that carries charged functional groupsÂ which interact with oppositely charged groups of the compound to be retained. Ion exchange chromatography is commonly used to purify proteins usingÂ FPLC.
Size-exclusion chromatography (SEC) is also known asÂ gel permeation chromatographyÂ (GPC) orÂ gel filtration chromatographyÂ and separates molecules according to their size (or more accurately according to their hydrodynamic diameter or hydrodynamic volume). Smaller molecules are able to enter the pores of the media and, therefore, molecules are trapped and removed from the flow of the mobile phase. The average residence time in the pores depends upon the effective size of the analyte molecules. However, molecules that are larger than the average pore size of the packing are excluded and thus suffer essentially no retention; such species are the first to be eluted. It is generally a low-resolution chromatography technique and thus it is often reserved for the final, "polishing" step of a purification. It is also useful for determining theÂ tertiary structureÂ andÂ quaternary structureÂ of purified proteins, especially since it can be carried out under nativeÂ solutionÂ conditions.
Reversed Phase Chromatography
Reversed-phase chromatography is an elution procedure used in liquid chromatography in which the mobile phase is significantly more polar than the stationary phase.
In some cases, the chemistry within a given column can be insufficient to separate some analytes. It is possible to direct a series of unresolved peaks onto a second column with different physico-chemical (Chemical classification) properties. Since the mechanism of retention on this new solid support is different from the first dimensional separation, it can be possible to separate compounds that are indistinguishable by one-dimensional chromatography. The sample is spotted at one corner of a square plate,developed, air-dried, then rotated by 90Â° and usually redeveloped in a second solvent system.
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, however use of FPLC techniques are typically for preparing large scale batches of a purified product.
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.
Chiral chromatography involves the separation of stereoisomers. In the case of enantiomers, these have no chemical or physical differences apart from being three-dimensional mirror images. Conventional chromatography or other separation processes are incapable of separating them. To enable chiral separations to take place, either the mobile phase or the stationary phase must themselves be made chiral, giving differing affinities between the analytes.Â Chiral chromatography HPLC columnsÂ (with a chiral stationary phase) in both normal and reversed phase are commercially available.
USES and APPLICATIONS
Chromatography of many kinds is widely used throughout the chemical industry. Environmental testing laboratories look for trace quantities of contaminants such as PCBs in waste oil, andÂ pesticidesÂ such as DDT inÂ groundwater. The Environmental Protection Agency uses chromatography to testÂ drinkingÂ waterÂ and to monitor air quality.Â Pharmaceutical c mpaniesÂ use chromatography both to prepare large quantities of extremely pure materials, and also to analyze the purified compounds for trace contaminants.
A growing use of chromatography in theÂ pharmaceuticalÂ industryÂ is for the separation of chiral compounds. These compounds have molecules that differ slightly in the way theirÂ atomsÂ are oriented in space. Although identical in almost every other way, includingÂ molecular weight, element composition, and physical properties, the two different forms-called optical isomers, or enantiomers-can have enormous differences in their biological activity. The compoundÂ thalidomide, for example, has two optical isomers. One causesÂ birth defectsÂ when women take it early in pregnancy; the otherÂ isomerÂ does not. Because this compound looks promising for the treatment of certain drug-resistant illnesses, it is important that the benign form be separated completely from the dangerous isomer.
Chromatography is used for quality control in the food industry, by separating and analyzing additives,Â vitamins, preservatives,Â proteins, and amino acids. It can also separate and detect contaminants such as aflatoxin, aÂ cancer-causing chemical produced by aÂ moldÂ on peanuts. Chromatography can be used for purposes as varied as finding drug compounds in urine or other body fluids, to looking for traces of flammableÂ chemicalsÂ in burned material from possible arson sites.
Chromatography has evolved to be one of the most widely used chemical techniques to separate particles and contaminates in chemical plants. For example, in the chemical industries, pesticides and insecticides like DDT in the groundwater and PCBs (Polychlorinated biphenyls) are removed by the process of chromatography. As a major testing tool, chromatography is used by government agencies to separate toxic materials from the drinking water and also to monitor air quality.Â
One of the significant chromatography uses is made in pharmaceutical companies, who specialize in making medicines. Chromatography is used byÂ pharmaceutical companiesÂ to prepare large amounts of pure materials that are further required in making medicines. Also, it is used to check the presence of any contamination in the manufactured compounds.
In the field of organic chemistry and pharmacy, chiral compounds are very close to each other in terms of atomic or molecular weight, element composition, and the physical properties. However, they exist in two different forms, called as the enantiomers and optical isomers. Both these compounds though may appear to be same, have very different chemical properties. So, in pharmacy, chromatography becomes crucial to analyze the exact chiral compound so that correct medicines can be manufactured. For instance, a compound called as thalidomide has two optical isomers and one of the isomers can cause birth defect if a pregnant women consumes it in early stages of pregnancy. So, it is important to carefully separate the isomers.
Other important chromatography uses are in the food industry where proper food maintenance is necessary to ensure quality. Chromatography is used as a technique to separate the additives, vitamins, preservatives, proteins and amino acids. Some other chromatography uses are in the detection of drugs or medications in the urine and the separation of traces of chemicals in the case of fire in houses or buildings. It is also very popular in forensic scienceÂ for investigative purposes.
Chromatography technology has gained immense industrial popularity in the past few decades as it can separate chemicals that just differ even in their atomic orientations in space. These were some of the chromatography uses that are used in various technological pursuits in chemical industries.
Chromatography has many applications in biology. It is used to separate and identify amino acids, carbohydrates, fatty acids, and other natural substances. Environmental testing laboratories use chromatography to identify trace quantities of contaminants such as PCBs in waste oil and pesticides such as DDT in groundwater. It is also used to test drinking water and test air quality. Pharmaceutical companies use chromatography to prepare quantities of extremely pure materials. The food industry uses chromatography to detect contaminants such as aflatoxin.