Chromatography
In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent called the mobile phase, which carries it through a system on which a material called the stationary phase is fixed. As the different constituents of the mixture tend to have different affinities for the stationary phase and are retained for different lengths of time depending on their interactions with its surface sites, the constituents travel at different apparent velocities in the mobile fluid, causing them to separate. The separation is based on the differential partitioning between the mobile and the stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus affect the separation.
Chromatography may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture for later use, and is thus a form of purification. This process is associated with higher costs due to its mode of production. Analytical chromatography is done normally with smaller amounts of material and is for establishing the presence or measuring the relative proportions of analytes in a mixture. The two types are not mutually exclusive.
Etymology and pronunciation
Chromatography, pronounced, is derived from Greek χρῶμα chrōma, which means "color", and γράφειν gráphein, which means "to write". The combination of these two terms was directly inherited from the invention of the technique first used to separate biological pigments.History
The method was developed by botanist Mikhail Tsvet in 1901–1905 in universities of Kazan and Warsaw. He developed the technique and coined the term chromatography in the first decade of the 20th century, primarily for the separation of plant pigments such as chlorophyll, carotenes, and xanthophylls. Since these components separate in bands of different colors they directly inspired the name of the technique. New types of chromatography developed during the 1930s and 1940s made the technique useful for many separation processes.Chromatography technique developed substantially as a result of the work of Archer John Porter Martin and Richard Laurence Millington Synge during the 1940s and 1950s, for which they won the 1952 Nobel Prize in Chemistry. They established the principles and basic techniques of partition chromatography, and their work encouraged the rapid development of several chromatographic methods: paper chromatography, gas chromatography, and what would become known as 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. Advances are continually improving the technical performance of chromatography, allowing the separation of increasingly similar molecules.
Terms
- Analyte – the substance to be separated during chromatography. It is also normally what is needed from the mixture.
- Analytical chromatography – the use of chromatography to determine the existence and possibly also the concentration of analyte in a sample.
- Bonded phase – a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing.
- Chromatogram – 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 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.
- Chromatograph – an instrument that enables a sophisticated separation, e.g. gas chromatographic or liquid chromatographic separation.
- Chromatography – a physical method of separation that distributes components to separate between two phases, one stationary, the other moving in a definite direction.
- Eluent – the solvent or solvent fixture used in elution chromatography and is synonymous with mobile phase.
- Eluate – the mixture of solute and solvent exiting the column.
- Effluent – the stream flowing out of a chromatographic column. In practise, it is used synonymously with eluate, but the term more precisely refers to the stream independent of separation taking place.
- Eluite – a more precise term for solute or analyte. It is a sample component leaving the chromatographic column.
- Eluotropic series – a list of solvents ranked according to their eluting power.
- Immobilized phase – a stationary phase that is immobilized on the support particles, or on the inner wall of the column tubing.
- Mobile phase – the phase that moves in a definite direction. It may be a liquid, a gas, or a supercritical fluid. The mobile phase consists of the sample being separated/analyzed and the solvent that moves the sample through the column. In the case of HPLC the mobile phase consists of a non-polar solvent such as hexane in normal phase or a polar solvent such as methanol in reverse phase chromatography and the sample being separated. The mobile phase moves through the chromatography column where the sample interacts with the stationary phase and is separated.
- Preparative chromatography – the use of chromatography to purify sufficient quantities of a substance for further use, rather than analysis.
- Retention time – the characteristic time it takes for a particular analyte to pass through the system under set conditions. See also: Kovats' retention index
- Sample – 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.
- Solute – the sample components in partition chromatography.
- Solvent – any substance capable of solubilizing another substance, and especially the liquid mobile phase in liquid chromatography.
- Stationary phase – the substance fixed in place for the chromatography procedure. Examples include the silica layer in [|thin-layer chromatography]
- Detector – the instrument used for qualitative and quantitative detection of analytes after separation.
Chromatography is based on the concept of partition coefficient. Any solute partitions between two immiscible solvents. When one make one solvent immobile and another mobile it results in most common applications of chromatography. If the matrix support, or stationary phase, is polar it is forward phase chromatography. Otherwise this technique is known as reversed phase, where a non-polar stationary phase is used.
Techniques by chromatographic bed shape
Column chromatography
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 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. Differences in rates of movement through the medium are calculated to different retention times of the sample.In 1978, W. Clark Still introduced a modified version of column chromatography called flash column chromatography. The technique is very similar to the traditional column chromatography, except 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.
In 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.
Phosphocellulose chromatography utilizes the binding affinity of many DNA-binding proteins for phosphocellulose. The stronger a protein's interaction with DNA, the higher the salt concentration needed to elute that protein.