Chromatography analysis is used to determine the presence and concentration of analytes in a sample.
Chromatography refers to a set of laboratory methods and techniques for the separation of mixtures. It involves passing a mixture that dissolved in a mobile phase through a medium known as the stationary phase. This separates the analyte to be measured from other components of the mixture and allows it to be isolated.
Chromatography may be preparatory or analytical in nature. Preparatory chromatography is performed to separate the components of a mixture for further analysis as well as for cleansing and purification applications. Analytical chromatography is usually done with smaller amounts of material and is used to measure the relative proportions of analytes in a mixture.
In chromatography analysis, chemical substances are introduced into a vertical glass tube containing an adsorbent. The various components of the substance move through the adsorbent material at different rates of speed according to their degree of attraction to it. This produces bands of color at different levels of the adsorption column.
Analysis techniques by physical state of the mobile phase fall into several categories. Gas chromatography (sometimes called gas-liquid chromatography) is a separation technique in which the mobile phase is a gas. Gas chromatography is always performed in a column, typically packed or capillary. Liquid chromatography is a separation methodology in which the mobile phase is a liquid and can be performed either in a column or a plane. Present day liquid chromatography analysis generally utilizes very small packing particles and a relatively high pressure; a method referred to as high performance liquid chromatography or HPLC.
Affinity chromatography is based on selective non-covalent interaction between an analyte and particular molecules. It is frequently used in biochemistry in the purification of proteins bound to tags.
Other techniques use a variety of separation mechanisms. Ion exchange chromatography employs the 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 including amino acids, peptides, and proteins.
Size exclusion chromatography analysis (also known as gel permeation chromatography or gel filtration chromatography) separates molecules according to their size (or more accurately according to hydrodynamic diameter or volume). Smaller molecules are able to enter the pores of the media and take longer to elute, while larger molecules are excluded from the pores and elute more rapidly.
Special chromatography methodologies are sometimes needed. Reversed-phase chromatography is an elution procedure used in liquid chromatography analysis, using a mobile phase which is significantly more polar than the stationary phase.
If the chemistry within a given column is insufficient to separate some analytes, two-dimensional chromatography can be used, making it possible to direct a series of unresolved peaks onto a second column with different properties. This method allows for the separation of compounds which are indistinguishable from one another when using one-dimensional chromatography methods.
Additional specialized chromatography analysis techniques include simulated moving-bed chromatography, pyrolysis gas chromatography, fast protein liquid chromatography, countercurrent chromatography and chiral chromatography.
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