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Chromatography

Chromatography

Chromatography is a family of laboratory techniques for separating mixtures of chemicals into their individual compounds. The basic principle of chromatography is that different compounds will stick to a solid surface or dissolve in a film of liquid to different degrees. Chromatography is used extensively in forensics, from analyzing body fluids for the presence of illicit drugs , to fiber analysis, blood analysis from a crime scene, and at airports to detect residue from explosives .

When a gas or liquid containing a mixture of different compounds is made to flow over such a surface, the molecules of the various compounds will tend to stick to the surface. If the stickiness is not too strong, a given molecule will become stuck and unstuck hundreds or thousands of times as it is swept along the surface. This repetition exaggerates even tiny differences in the various molecules' stickiness, and they become spread out along the "track," because the stickier compounds move more slowly than the less sticky ones do. After a given time, the different compounds will have reached different places along the surface and will be physically separated from one another. Or, they can all be allowed to reach the far end of the separation surface and be detected or measured one at a time as they emerge.

Using variations of this basic phenomenon, chromatographic methods have become an extremely powerful and versatile tool for separating and analyzing a vast variety of chemical compounds in quantities from picograms (10-12 gram) to tons.

Chromatographic methods all share certain characteristics, although they differ in size, shape, and configuration. Typically, a stream of liquid or gas (the mobile phase) flows constantly through a tube (the column) packed with a porous solid material (the stationary phase). A sample of the chemical mixture is injected into the mobile phase at one end of the column, and the compounds separate as they move along. The individual separated compounds can be removed one at a time as they exit (or "elute from") the column.

Because it usually does not alter the molecular structure of the compounds, chromatography can provide a non-destructive way to obtain pure chemicals from various sources. It works well on very large and very small scales; chromatographic processes are used both by scientists studying micrograms of a substance in the laboratory, and by industrial chemists separating tons of material.

The technology of chromatography has advanced rapidly in the past few decades. It is now possible to obtain separation of mixtures in which the components are so similar they only differ in the way their atoms are oriented in space, in other words, they are isomers of the same compounds. It is also possible to obtain separation of a few parts per million of a contaminant from a mixture of much more concentrated materials.

In gas-liquid chromatography (now called gas chromatography), the material that separates components is chemically bonded to the solid support, which improves the temperature stability of the column's packing. Gas chromatographs can be operated at high temperatures, so even large molecules can be vaporized and progress through the column without the stationary phase vaporizing and bleeding off. Additionally, since the mobile phase is a gas, the separated compounds are very pure; there is no liquid solvent to remove.

The shapes of chromatographic columns, originally vertical tubes an inch or so (23 cm) in diameter, became longer and thinner when it was found that this increased the efficiency of separation. Eventually, chemists were using coiled glass or fused silica capillary tubes less than a millimeter in diameter and many yards long. Capillaries cannot be packed, but they are so narrow that the stationary phase can simply be a thin coat on the inside of the column.

A somewhat different approach is the set of techniques known as "planar" or "thin layer" chromatography (TLC), in which no column is used at all. The stationary phase is thinly coated on a glass or plastic plate. A spot of sample is placed on the plate, and the mobile phase migrates through the stationary phase by capillary action.

In the mid-1970s, interest in liquid mobile phases for column chromatography resurfaced when it was discovered that the efficiency of separation could be vastly improved by pumping the liquid through a short packed column under pressure, rather than allowing it to flow slowly down a vertical column by gravity alone. High-pressure liquid chromatography, also called high performance liquid chromatography (HPLC), is now widely used in industry. A variation on HPLC is super-critical fluid chromatography (SFC). Certain gases (carbon dioxide, for example), when highly pressurized above a certain temperature, become a state of matter intermediate between gas and liquid. These "supercritical fluids" have unusual solubility properties, some of the advantages of both gases and liquids, and appear very promising for chromatographic use.

All chromatographs must have a detection device attached, and some kind of recorder to capture the output of the detectorusually a chart recorder or its computerized equivalent. In gas chromatography, several kinds of detectors have been developed; the most common are the thermal conductivity detector, the flame ionization detector, and the electron capture detector. For HPLC, the UV detector is standardized to the concentration of the separated compound. The sensitivity of the detector is of special importance, and research has continually concentrated on increasing this sensitivity, because chemists often need to detect and quantify exceedingly small amounts of a material.

Within the last few decades, chromatographic instruments have been attached to other types of analytical instrumentation so that the mixture's components can be identified as well as separated (this takes the concept of the "detector" to its logical extreme). Most commonly, this second instrument has been a mass spectrometer, which allows identification of compounds based on the masses of molecular fragments that appear when the molecules of a compound are broken up.

Absorption chromatography (the original type of chromatography) depends on physical forces such as dipole attraction to hold the molecules onto the surface of the solid packing. In gas chromatography and HPLC, however, the solubility of the mixture's molecules in the stationary phase coating determines which ones progress through the column more slowly. Polarity can have an influence here as well. In gel filtration (also called size-exclusion or gel permeation) chromatography, the relative sizes of the molecules in the mixture determine which ones exit the column first. Large molecules flow right through; smaller ones are slowed down because they spend time trapped in the pores of the gel. Ion exchange chromatography depends on the relative strength with which ions are held to an ionic resin. Ions that are less strongly attached to the resin are displaced by more strongly attached ions. Hence the name ion exchange: one kind of ion is exchanged for another. This is the same principle upon which home water softeners operate. Affinity chromatography uses a stationary phase composed of materials that have been chemically altered. In this type of chromatography, the stationary phase is attached to a compound with a specific affinity for the desired molecules in the mobile phase. This process is similar to that of ion exchange chromatography, and is used mainly for the recovery of biological compounds. Hydrophobic interaction chromatography is used for amino acids that do not carry a positive or negative charge. In this type of chromatography, the hydrophobic amino acids are attracted to the solid phase, which is composed of materials containing hydrophobic groups.

Chemists choose the mobile and stationary phases carefully because it is the relative interaction of the mixture's compounds with those two phases that determines how efficient the separation can be. If the compounds have no attraction for the stationary phase at all, they will flow right through the column without separating. If the compounds are too strongly attracted to the stationary phase, they may stick permanently inside the column.

see also Analytical instrumentation; Gas chromatograph-mass spectrometer.

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Chromatography

Chromatography

Chromatography works by separating the individual parts of a mixture so that each one can be analyzed and identified. In the decades since its invention, the chromatograph has become an essential piece of equipment in bio-chemical laboratories. Using the analytical technique of chromatography, scientists can tell what chemical compounds are present in complex mixtures. These mixtures include such diverse things as smog, cigarette smoke, petroleum products, or even coffee aroma. Without chromatography, chemists might not have been able to synthesize proteins such as insulin or understand how plants use the sun's energy to make food.

The First Chromatograph

The first chromatograph was invented by Russian botanist Mikhail Semenovich Tsvett (1872-1919). While working in Poland, Tsvett was looking for a method of separating a mixture of plant pigments (tints) which are chemically very similar to each other. To isolate different types of chlorophyll, he trickled a mixture of dissolved pigments through a glass tube packed with calcium carbonate powder. As the solution washed downward, each pigment stuck to the powder with a different degree of strength, creating a series of colored bands. Each band of color represented a different substance. Tsvett referred to the colored bands as a chromatogram. He also suggested that the technique (now called adsorption chromatography) could be used to separate colorless substances.

Although Tsvett published a report of his work in the early 1900s, chemists paid very little attention to it. There were a few reasons for ignoring the work. First, the report was written in Russian, which few Western chemists of the time read. Second, the technique may have seemed too simple to chemists who were used to relying on lengthy extraction, crystallization, or distillation processes to separate mixtures. Within a few years, Tsvett's technique was rediscovered. The rediscovery was by the German organic chemist Richard Martin Willstatter (1872-1942), who was also studying chlorophyll. By introducing chromatography to Western European scientists, Willstatter helped establish one of the most versatile analytical techniques known to chemistry.

Ion-Exchange Chromatography

Chromatography was found to work on almost all kinds of mixtures, including colorless ones, just as Tsvett had predicted. Absorbing powders were discovered that perform better than calcium carbonate for separating ordinary molecules. Also, compounds known as "zeolites" were introduced to separate individual ions, or electrically charged particles, in a process called ion-exchange chromatography. American chemist Frank Harold Spedding adapted this technique to the separation of rare-earth metals. In the 1930s, synthetic resins were developed for complex ion-exchange processes. During World War II (1939-1945), life rafts were equipped with survival kits that contained resins for removing most salts from seawater.

Paper chromatography

The most dramatic advance in the history of chromatography took place in 1944. It was then that scientists discovered that a strip of porous (full of small holes) filter paper could substitute for the column of absorbing powder. The technique was called paper chromatography. A drop of the mixture to be separated is placed on the paper, then one edge is dipped into a solvent (a substance that dissolves). The solvent spreads across the paper, carrying the mixture's components with it.

When the components are finished spreading, the paper is dried and sprayed with a reagent that reveals a change in color. Because the components move at different speeds, they show up as distinct, physically separated spots that can be cut out with scissors and further analyzed. The paper method is a type of partition chromatography, which is based on differences in solubility (the measured rate at which one substance will dissolve in another) rather than differences in adsorption. One of its advantages is that it requires only a small sample of material.

Martin and Synge

Paper chromatography was invented by two British biochemists, Archer John Porter Martin (1910-) and Richard Laurence Millington Synge (1914-). In 1941 Martin and Synge began working together on proteins, which are made up of chains of amino acids. The duo was trying to characterize a particular protein by determining the precise numbers of each amino acid present. Amino acids are so similar to each other, however, that the problem of separating them had defeated a whole generation of biochemists. Martin and Synge's development of paper chromatography to solve this problem was an instant success. It worked not only on amino acids but also on various other mixtures. The two scientists were awarded the 1952 Nobel Prize in chemistry for their work.

Martin and Synge's research led to a number of other important scientific advances. After Synge determined the structure of an antibiotic peptide called "Gramicidin-S," Frederick Sanger (1918-) used paper chromatography to figure out the structure of the insulin molecule. He determined the number of amino acids in it as well as the order in which they occurred. Insulin is now used to control blood sugar levels in people afflicted with diabetes.

The same technique was used by Melvin Calvin (1911-) during the 1950s. Calvin discovered the complex series of reactions that enable green plants to convert solar energy into the chemical energy stored in food. Working with green algal (algae) cells, Calvin interrupted the photosynthetic process (process by which plants that contain chlorophyll use light to change carbon dioxide and water to carbohydrates) at different stages by plunging the cells into alcohol. Then he crushed them and separated their components via paper chromatography. Calvin was thus able to identify at least ten different intermediate products that had been created within a few seconds.

Paper chromatography was also used by Austrian-American biochemist Erwin Chargaff (1905-), who modified the technique to study the components of the nucleic acid molecule. His research revealed four components, or nitrogenous bases, that occur in pairs. British biochemists James Dewey Watson and Francis Harry Compton Crick later used these results to work out the structure of DNA (deoxyribonucleic acid).

Gas chromatography

In addition to inventing paper chromatography, Martin developed another technique called gas chromatography. The process allows chemists to separate mixtures of gases, or substances that can be vaporized or gasified by heat. Instead of a liquid solvent, helium gas is usually used to force the mixture through a column and separate the gaseous components. Martin and his colleague A. T. James first used gas chromatography to micro-analyze fatty acids.

The widespread acceptance of gas chromatography is unique in the laboratory instrumentation field. Today it is used in almost every branch of the chemical industry, particularly in the production of petrochemicals from oil and natural gas. One of the most common fixtures in biochemical laboratories is "GCMS" (gas chromatography, mass spectrometry) analytical equipment. This equipment uses gas chromatography to separate individual components from complex organic mixtures, then uses mass spectrometry to identify each component.

Thin-Layer Chromatography

Recently, chromatography has evolved into even more sophisticated analytical techniques. In thin-layer chromatography, for example, an alumina gel, silica gel, or other finely divided solid is spread onto a glass plate in a thin, uniform layer. This takes the place of filter paper in the chromatographic process. The technique is not only faster than paper chromatography, but it can also separate smaller quantities of pure components. It is often used in the pharmaceutical industry to isolate penicillin and other antibiotics.

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chromatography

chromatography (krō´mətŏg´rəfē), resolution of a chemical mixture into its component compounds by passing it through a system that retards each compound to a varying degree; a system capable of accomplishing this is called a chromatograph. The retarding system can be a surface adsorbant, such as silica, alumina, cellulose, or charcoal, capable of reversibly adsorbing the compounds (see adsorption). The earliest use of this technique, by the Russian botanist Mikhail Tsvett (c.1903), involved the separation of highly colored compounds, hence the name chromatography [Gr.,=color recording].

Column Chromatography

In column chromatography the adsorbant is packed into a column and a solution of the mixture is added at the top. An appropriate solvent is passed through the column, washing, or eluting, the compounds down the column. A polar substance that is adsorbed very tightly to the surface will be efficiently retarded by the column, while a nonpolar substance will elute (dissolve in the solvent) very rapidly. By varying the nature of the solid adsorbant and the eluting solvent, a wide variety of resolutions, even of very similar substances, can be carried out.

Gas Chromatography

The gas chromatograph (GC) is a system consisting of a liquid with a high boiling point impregnated on an inert solid support as the stationary phase and helium gas as the mobile phase. The stationary phase is packed into a thin metal column and helium gas is allowed to flow through it. The column is attached to an injection port, and the entire system is heated in an oven. A solution of the mixture is injected into the column through the injection port by means of a syringe and is immediately volatilized. The helium gas then sweeps the components out of the column and past a detector. The polarity of the compounds and their volatility determines how long they are retained by the column. When each component passes the detector, a peak is registered on a recorder. The relative quantities of the components can be determined from the relative areas under the peaks. By varying the polarity of the column and its temperature, many different resolutions can be carried out. Since the capacity of GC columns is very low, the gas chromatograph is used chiefly as an analytical tool, although it can be used for preparative purposes as well. Miniaturized GC instruments have been employed in space probes to analyze the atmospheres of other planets.

Liquid Chromatography

For compounds that cannot be volatilized readily, the liquid chromatograph (LC) can be used instead of the gas chromatograph. The stationary phase consists of a finely powdered solid adsorbant packed into a thin metal column and the mobile phase consists of an eluting solvent forced through the column by a high-pressure pump. The mixture to be analyzed is injected into the column and monitored by a detector. Many different LC packings and eluting solvents are available to achieve the desired resolution.

Gel-Permeation Chromatography

In gel-permeation chromatography, compounds are separated on the basis of their molecular size. Porous beads of the gel are packed into a column and the mixture is added at the top in an appropriate solvent. Large molecules move straight down the column, while small molecules stick in the pores and are retarded.

Ion-Exchange Chromatography

For compounds that can exist as ions, ion-exchange chromatography can be used to separate them from neutral or oppositely charged compounds. The mixture is added to a column packed with a porous, insoluble resin which has a negatively charged (anionic) group attached to it and an unattached, positively charged (cationic) counterion. A cation from the mixture will exchange with the positive counterion of the resin and will be retarded while neutral and anionic substances are not affected. Ion-exchange resins with exchangeable anions work in a similar manner.

Thin-Layer and Paper Chromatography

A layer of adsorbant also can be spread on a glass plate, instead of packed into a column, for analytical purposes. By means of a thin capillary tube, the plate is spotted with a solution of the mixture that is to be resolved, and the solvent is allowed to evaporate. An eluting solvent is then allowed to move up the plate by capillary action, drawing the components of the mixture along by varying degrees. The plate is developed by spraying it with an oxidizing agent, so that each component becomes charred and appears as a dark spot on the plate. The location and size of the spots serve to identify and measure the relative quantities of the components. As in column chromatography, polar substances will not elute as well and will remain nearer the bottom of the plate, while nonpolar substances will elute to the top. This process is called thin-layer chromatography (TLC). In paper chromatography a procedure similar to TLC is used except that the cellulose in the paper acts as the adsorbant.

Electrophoresis

Electrophoresis, like ion-exchange chromatography, can be used as an effective tool for analyzing mixtures of ions. A strip of paper or a column of polymeric gel, saturated with an electrolyte, is set up so that it spans two solutions containing electrodes. The mixture to be analyzed is spotted onto the paper or gel and the two electrodes are connected to a high-energy power source (about 5,000 volts). Positive ions will migrate in one direction and negative ions in the other. The greater the charge on the ion, the farther it will migrate. This method is especially useful for the resolution of mixtures of proteins.

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chromatography

chromatography A technique for analysing or separating mixtures of gases, liquids, or dissolved substances, such as mixtures of amino acids or chlorophyll pigments. The original technique (invented by the Russian botanist Mikhail Tsvet (1872–1919) in 1906) is a good example of column chromatography. A vertical glass tube is packed with an adsorbing material, such as alumina. The sample is poured into the column and continuously washed through with a solvent (a process known as elution). Different components of the sample are adsorbed to different extents and move down the column at different rates. In Tsvet's original application, plant pigments were used and these separated into coloured bands in passing down the column (hence the name chromatography). The usual method is to collect the liquid (the eluate) as it passes out from the column in fractions.

In general, all types of chromatography involve two distinct phases – the stationary phase (the adsorbent material in the column in the example above) and the moving phase (the solution in the example). The separation depends on competition for molecules of sample between the moving phase and the stationary phase. The form of column chromatography above is an example of adsorption chromatography, in which the sample molecules are adsorbed on the alumina. In partition chromatography, a liquid (e.g. water) is first absorbed by the stationary phase and the moving phase is an immiscible liquid. The separation is then by partition between the two liquids. In ion-exchange chromatography the process involves competition between different ions for ionic sites on the stationary phase (see ion exchange). Gel filtration is another chromatographic technique in which the size of the sample molecules is important.

See also affinity chromatography; gas–liquid chromatography; paper chromatography; thin-layer chromatography.

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chromatography

chromatography An analytical technique used for the separation of the components of complex mixtures, which is based on their repetitive distribution between a mobile phase (of gas or liquid) and a stationary phase (of solids or liquid-coated solids). The distribution of the different component molecules between the two phases is dependent on the method of chromatography used (e.g. gel-filtration, or ion-exchange), and on the movement of the mobile phase (which results in the differential migration and therefore separation of the components along the stationary phase).

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chromatography

chromatography An analytical technique used for the separation of the components of complex mixtures, that is based on their repetitive distribution between a mobile phase (of gas or liquid) and a stationary phase (of solids or liquid-coated solids). The distribution of the different component molecules between the two phases is dependent on the method of chromatography used (e.g. gel-filtration, or ion-exchange), and on the movement of the mobile phase (which results in the differential migration and therefore separation of the components along the stationary phase).

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chromatography

chromatography Analytical technique for the separation of the components of complex mixtures, based on their repetitive distribution between a mobile phase (of a gas or liquid) and a stationary phase (of solids or liquid-coated solids). The distribution of the different component molecules between the two phases is dependent on the method of chromatography used (e.g. gel-filtration, or ion-exchange), and on the movement of the mobile phase (which results in the differential migration and therefore separation of the components along the stationary phase).

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chromatography

chromatography An analytical technique for separating the components of complex mixtures, based on their repetitive distribution between a mobile phase (of gas or liquid) and a stationary phase (of solids or liquid-coated solids). The distribution of the different component molecules between the two phases is dependent on the method of chromatography used (e.g. gel-filtration, or ion-exchange), and on the movement of the mobile phase (which results in the differential migration and therefore separation of the components along the stationary phase).

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chromatography

chromatography (kroh-mă-tog-răfi) n. any of several techniques for separating the components of a mixture by selective absorption. In two such techniques, widely used in medicine, a sample of the mixture is placed at the edge of a sheet of filter paper (paper c.) or a column of a powdered absorbent (column c.). The components of the mixture are absorbed to different extents and thus move along the paper or column at different rates.

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chromatography

chromatography Techniques of chemical analysis by which substances are separated from one another, identified and measured. All involve a mobile phase consisting of a liquid or gaseous mixture of the substances to be separated, and a stationary phase consisting of a material that differentially absorbs the substances in the mixture. The two major types are gas and paper chromatography. See also electrophoresis

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chromatography

chro·ma·tog·ra·phy / ˌkrōməˈtägrəfē/ • n. Chem. the separation of a mixture by passing it in solution or suspension or as a vapor (as in gas chromatography) through a medium in which the components move at different rates. DERIVATIVES: chro·mat·o·graph·ic / krōˌmatəˈgrafik/ adj.

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chromatography

chromatographydaffy, taffy •Amalfi •Cavafy, Gaddafi •Effie •beefy, Fifi, leafy •cliffy, iffy, jiffy, Liffey, niffy, sniffy, spiffy, squiffy, stiffy, whiffy •salsify •coffee, toffee •wharfie •Sophie, strophe, trophy •Dufy, goofy, Sufi •fluffy, huffy, puffy, roughie, roughy, scruffy, snuffy, stuffy, toughie •comfy • atrophy •anastrophe, catastrophe •calligraphy, epigraphy, tachygraphy •dystrophy, epistrophe •autobiography, bibliography, biography, cardiography, cartography, chirography, choreography, chromatography, cinematography, cosmography, cryptography, demography, discography, filmography, geography, hagiography, historiography, hydrography, iconography, lexicography, lithography, oceanography, orthography, palaeography (US paleography), photography, pornography, radiography, reprography, stenography, topography, typography •apostrophe •gymnosophy, philosophy, theosophy •furphy, murphy, scurfy, surfy, turfy

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