column chromatography

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chromatography

 [kro″mah-tog´rah-fe]
a technique for analysis of chemical substances. The term chromatography literally means color writing, and denotes a method by which the substance to be analyzed is poured into a vertical glass tube containing an adsorbent, the various components of the substance moving through the adsorbent at different rates of speed, according to their degree of attraction to it, and producing bands of color at different levels of the adsorption column. The term has been extended to include other methods utilizing the same principle, although no colors are produced in the column. adj., adj chromatograph´ic.

The mobile phase of chromatography refers to the fluid that carries the mixture of substances in the sample through the adsorptive material. The stationary or adsorbent phase refers to the solid material that takes up the particles of the substance passing through it. Kaolin, alumina, silica, and activated charcoal have been used as adsorbing substances or stationary phases.

Classification of chromatographic techniques tends to be confusing because it may be based on the type of stationary phase, the nature of the adsorptive force, the nature of the mobile phase, or the method by which the mobile phase is introduced.

The technique is a valuable tool for the research biochemist and is readily adaptable to investigations conducted in the clinical laboratory. For example, chromatography is used to detect and identify in body fluids certain sugars and amino acids associated with inborn errors of metabolism.
adsorption chromatography that in which the stationary phase is an adsorbent.
affinity chromatography that based on a highly specific biologic interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand. Any of these substances, covalently linked to an insoluble support or immobilized in a gel, may serve as the sorbent allowing the interacting substance to be isolated from relatively impure samples; often a 1000-fold purification can be achieved in one step.
column chromatography the technique in which the various solutes of a solution are allowed to travel down a column, the individual components being adsorbed by the stationary phase. The most strongly adsorbed component will remain near the top of the column; the other components will pass to positions farther and farther down the column according to their affinity for the adsorbent. If the individual components are naturally colored, they will form a series of colored bands or zones.

Column chromatography has been employed to separate vitamins, steroids, hormones, and alkaloids and to determine the amounts of these substances in samples of body fluids.
exclusion chromatography that in which the stationary phase is a gel having a closely controlled pore size. Molecules are separated based on molecular size and shape, smaller molecules being temporarily retained in the pores.
gas chromatography a type of automated chromatography in which the mobile phase is an inert gas. Volatile components of the sample are separated in the column and measured by a detector. The method has been applied in the clinical laboratory to separate and quantify steroids, barbiturates, and lipids.
gas-liquid chromatography gas chromatography in which the substances to be separated are moved by an inert gas along a tube filled with a finely divided inert solid coated with a nonvolatile oil; each component migrates at a rate determined by its solubility in oil and its vapor pressure.
gel-filtration chromatography (gel-permeation chromatography) exclusion chromatography.
ion exchange chromatography that utilizing ion exchange resins, to which are coupled either cations or anions that will exchange with other cations or anions in the material passed through their meshwork.
molecular sieve chromatography exclusion chromatography.
paper chromatography a form of chromatography in which a sheet of blotting paper, usually filter paper, is substituted for the adsorption column. After separation of the components as a consequence of their differential migratory velocities, they are stained to make the chromatogram visible. In the clinical laboratory, paper chromatography is employed to detect and identify sugars and amino acids.
partition chromatography a process of separation of solutes utilizing the partition of the solutes between two liquid phases, namely the original solvent and the film of solvent on the adsorption column.
thin-layer chromatography that in which the stationary phase is a thin layer of an adsorbent such as silica gel coated on a flat plate. It is otherwise similar to paper chromatography.

col·umn chro·ma·tog·ra·phy

a form of partition, adsorption, ion exchange, or affinity chromatography in which one phase is liquid (aqueous) flowing down a column packed with the second phase, a solid; the dissolved substances form a partition between the solid and liquid phases depending on the chemical and physical conditions of each phase; the more strongly adsorbed solutes reach the bottom of the column later than the less strongly adsorbed.

column chromatography

Etymology: L, columna + Gk, chroma, color, graphein to record
the process of separating and analyzing a group of substances according to the differences in their absorption affinities for a given absorbent as evidenced by pigments deposited during filtration through the same absorbent contained in a glass cylinder or tube. The substances are dissolved in a liquid that is passed through the absorbent. The absorbates move down the column at different rates and leave behind a band of pigments that is subsequently washed with a pure solvent to develop discrete pigmented bands that constitute a chromatograph. The cylinder of absorbent is then pushed from the tube, and the individual bands are either separated with a knife or further diluted with the pure solvent and collected in the bottom of the tube for analysis. Effective column chromatography depends on the selection of the appropriate absorbent and solvent and a flow rate that is slow enough to allow complete diffusion of the absorbates from the solvent to the absorbent and the retardation of the absorbates according to their different affinities for the absorbent. Compare gas chromatography, ion exchange chromatography.

col·umn chro·ma·tog·ra·phy

(kol'ŭm krō'mă-tog'ră-fē)
A form of partition, adsorption, ion exchange, or affinity chromatography in which one phase is liquid (aqueous) flowing down a column packed with the second phase, a solid.

chromatography

a technique for analysis of chemical substances. The term chromatography literally means color writing, and denotes a method by which the substance to be analyzed is poured into a vertical glass tube containing an adsorbent, the various components of the substance moving through the adsorbent at different rates, according to their degree of attraction to it, and producing bands of color at different levels of the adsorption column. The term has been extended to include other methods utilizing the same principle, although no colors are produced in the column.
The mobile phase of chromatography refers to the fluid that carries the mixture of substances in the sample through the adsorptive material. The stationary phase (or adsorbent) refers to the solid material that takes up the particles of the substance passing through it. Kaolin, alumina, silica and activated charcoal have been used as adsorbing substances or stationary phases.
Classification of chromatographic techniques tends to be confusing because it may be based on the type of stationary phase, the nature of the adsorptive force, the nature of the mobile phase, or the method by which the mobile phase is introduced.
The technique is a valuable tool for the research biochemist and is readily adaptable to investigations conducted in the clinical laboratory. For example, chromatography is used to detect and identify in body fluids certain sugars and amino acids associated with inborn errors of metabolism.

adsorption chromatography
that in which the stationary phase is an adsorbent.
affinity chromatography
a method of chromatography that utilizes the biologically important binding interactions that occur on protein surfaces. For example, an enzyme substrate is covalently coupled to an inert matrix such as a polysaccharide bead. The enzyme can be bound to the bead and thereby separated when present in very low concentration in a very complex mixture of other macromolecules.
column chromatography
the technique in which the various solutes of a solution are allowed to travel down a column, the individual components being adsorbed by the stationary phase. The most strongly adsorbed component will remain near the top of the column; the other components will pass to positions farther and farther down the column according to their affinity for the adsorbent. If the individual components are naturally colored, they will form a series of colored bands or zones.
Column chromatography has been employed to separate vitamins, steroids, hormones and alkaloids and to determine the amount of these substances in samples of body fluids.
exclusion chromatography
that in which the stationary phase is a gel having a closely controlled pore size. Molecules are separated based on molecular size and shape, smaller molecules being temporarily retained in the pores.
gas chromatography
a type of chromatography in which the mobile phase is an inert gas. Volatile components of the sample are separated in the column and measured by a detector. The method has been applied in the clinical laboratory to separate and quantify steroids, barbiturates and lipids.
gas-liquid chromatography
gas chromatography in which the substances to be separated are moved by an inert gas along a tube filled with a finely divided inert solid coated with a nonvolatile substance; each component migrates at a rate determined by its solubility in the stationary phase and its vapor pressure.
gel-filtration chromatography, gel-permeation chromatography
exclusion chromatography.
high performance liquid chromatography (HPLC)
a miniaturized method in which the solution to be analyzed is passed, under high pressure, through a long, thin column packed with tiny beads such that analyses are completed in minutes rather than hours and with improved resolution.
ion-exchange chromatography
that utilizing resins to which are coupled either cations or anions that will exchange with other cations or anions in the material passed through their meshwork.
molecular sieve chromatography
exclusion chromatography.
paper chromatography
a form of chromatography in which a sheet of special paper is substituted for the adsorption column. After separation of the components as a consequence of their differential migratory velocities, they are stained to make the chromatogram visible. In the clinical laboratory paper chromatography is employed to detect and identify sugars and amino acids.
partition chromatography
a form of separation of solutes utilizing the partition of the solutes between two liquid phases, namely the original solvent and the film of solvent on the adsorption column.
thin-layer chromatography
that in which the stationary phase is a thin layer of an adsorbent such as silica gel coated on a flat plate. It is otherwise similar to paper chromatography.

column

an anatomical part in the form of a pillar-like structure; anything resembling a pillar.

anal c's
longitudinal folds of mucous membrane at the cranial half of the anal canal; called also rectal columns.
column of Bertin
extensions of renal cortex between the renal pyramids.
column chromatography
dorsal column
the dorsal portion of the gray substance of the spinal cord, in transverse section seen as a horn.
gray column
the longitudinally oriented parts of the spinal cord in which the nerve cell bodies are found, comprising the gray matter of the spinal cord.
lateral column
the lateral portion of the gray substance of the spinal cord, in transverse section seen as a horn; present only in the thoracic and anterior lumbar regions.
rectal c's
anal columns.
spinal column
the rigid structure in the midline of the back, composed of the vertebrae. Called also vertebral column. See also spine.
ventral column
the ventral portion of the gray substance of the spinal cord, in transverse section seen as a horn.