Chromatography is a common laboratory technique used to separate a mixture into its individual components, or fractions. This can allow the components to be identified and quantified. The first stage in chromatography is to dissolve the mixture in a fluid solvent, which may be either a liquid or a gas. The fluid solvent is known as the mobile phase. When a liquid solvent is used as the mobile phase, the technique is known as liquid chromatography.
Once the mixture has been dissolved into the mobile phase, it is passed through a system which may consist of a column, a capillary tube, a plate, or some combination of such items. This system is known as the stationary phase and is designed to present surface sites at which the constituents of the mixture interact. Because of differences in the way constituents in the mixture will interact with the surface sites, they move a different speeds, causing separation along the stationary phase. Typically these differences are based on the affinity of substances for the surface material, but separation may also be based on particle size (size exclusion) or charge (ion exchange).
Liquid Chromatography may be classified according to the shape and configuration of the stationary phase:
- Column chromatography uses a tube as the stationary bed, typically a vertically orientated cylinder with a narrow opening at the bottom so that the liquid flows slowly through the tube due to gravity. The lower opening may be further restricted by packing with a matrix such as cottonwool and a layer of sand. Because the mixture separates as it moves through the column, each fraction can be collected in turn by swopping test tubes under the opening.
- Open tubular column chromatography is a form of column chromatography in which the liquid simply flows through an open tube, interacting with the walls of the tube.
- Packed column chromatography is a form of column chromatography in which the actual stationary phase is made up of particle which fill the tube. In this case the primary sites of interaction are the surfaces of the particles and the tube simply functions as a container for the particles and a guide for the liquid. Silica is a commonly used stationary phase for packed column chromatography.
- Flash column chromatography is a form of packed column chromatography in which a pump is used to increase the pressure on the column, speeding up the rate of separation.
- Paper chromatography a sample of the mixture is placed at one end of a strip of paper and the mobile phase carries it up through the matrix of the paper by capillary attraction, causing the mixture to separate as it moves through the paper. This is a cheap and easy technique but it is not very precise.
- Thin-layer chromatography is similar to paper chromatography but instead of using cellulose as the stationary phase, it uses a thin layer of aluminum oxide or silicon dioxide on an inert glass or plastic support.
Multiple stationary phases may be used sequentially. So, for example, column chromatography may be initially used for bulk separation and the obtained fractions then applied to thin-layer chromatography.