chromatography, 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.

Type of chromatography using as the stationary phase a thin layer (0.01 inch [0.25 mm]) of a special finely ground matrix (silica gel, alumina, or similar material) coated on a glass plate or incorporated in a plastic film. Solutions of the mixtures to be analyzed are spotted near one edge. Solutions of reference compounds are similarly applied. The edge of the plate is then dipped in a solvent. The solvent travels up the matrix by capillarity, moving the components of the samples at various rates because of their different degrees of attachment to the matrix and solubility in the developing solvent. The components, visible as separated spots, are identified by comparing the distances they have traveled with those of the known reference materials. TLC is useful for biological mixtures, especially lipids in animal or vegetable tissues and isoprenoids and essential oils found in flowers and other parts of plants. The matrices withstand strong solvents and developers better than the paper used in paper chromatography.

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Type of chromatography using filter paper or other special paper as the stationary phase. Spots of sample and reference materials are applied, usually as liquids, near one edge (or corner, for two-dimensional PC) of the paper. The edge of the paper is dipped in a solvent, which travels along it by capillarity, moving the components of the sample at rates depending on their relative solubilities in the solvent. In two-dimensional PC, the paper is then turned 90° and the new edge dipped in a different solvent. The components of the sample mixture, visible as separated spots, are identified by comparing the distances they have traveled with those of the known reference materials. PC is especially useful for complex mixtures of amino acids, peptides, carbohydrates, steroids, and many other organic compounds and inorganic ions.

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Type of chromatography with a gas mixture as the mobile phase. In a packed column, the packing or solid support (held in a tube) serves as the stationary phase (vapour-phase chromatography, or VPC) or is coated with a liquid stationary phase (gas-liquid chromatography, or GLC). In capillary columns, the stationary phase coats the walls of small-diameter tubes. The sample of gas or volatile liquid to be analyzed is injected into the inlet; its components move through with a carrier gas (usually hydrogen, helium, or argon) at rates influenced by their degree of interaction with the stationary phase. The temperature, nature of the stationary phase, and column length can be varied to improve separation. The gas stream issuing from the column's end may pass through a thermal conductivity detector or a flame ionization detector, where its properties are compared with those of known reference substances. GC is used to measure air pollutants, essential oils, gases or alcohol in blood, and composition of industrial process streams.

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Method first described in 1903 by Mikhail S. Tsvet for separating mixed chemical substances. Tsvet's neglected work, rediscovered in the 1930s, uses the different affinities of substances in a solution in a mobile phase (a moving stream of gas or liquid) for adsorption onto a stationary phase (a fine-grained solid, a sheet of filtering material, or a thin film of a liquid on a solid surface). Choices of materials for these phases allow enormous versatility for separating substances including biological fluids (e.g., amino acids, steroids, carbohydrates, pigments), chemical mixtures, and forensic samples. In the original technique, an organic solvent flowed through a column of powdered alumina (see aluminum), sodium carbonate, or even powdered sugar to separate mixed plant pigments. Among current adaptations are paper chromatography (PC), thin-layer chromatography (TLC), liquid chromatography (LC, including high-performance liquid chromatography, or HPLC), and gas chromatography (GC). Some remain laboratory techniques, but others (especially HPLC) can be used on an industrial scale. They require different methods for detecting and identifying the separated components, including colorimetry, spectrophotometry, mass spectrometry, and measurement of fluorescence, ionization potential, or thermal conductivity. A.J.P. Martin shared a 1952 Nobel Prize for developing LC and PC, and in his Nobel lecture announced the development (with his cowinner R.L.M. Synge and other colleagues) of GC.

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