Chromatography can be used to separate complex mixtures containing very small quantities of different substances. There are many different types of chromatography, but in each case there are two phases, a mobile phase and a stationary phase. The different components of the mixture become partitioned or adsorbed to different extents between the two phases.
A sheet of paper consists largely of cellulose fibres. Cellulose is a polysaccharide composed of glucose molecules, which have a large number of hydroxyl groups. Water molecules become hydrogen bonded to these groups. so that even a sheet of 'dry' paper contains about 10% water by mass. This water acts as the stationary phase for the technique of paper chromatography. The mobile phase is a solvent consisting of an aqueous solution or an organic liquid such as ethanol. The mixture to be separated is dissolved in this mobile phase, which moves along the paper via the mechanism of capillary action resulting from the forces between the solvent and the solid fibres of the paper.
Thin-layer chromatography (TLC) is a technique similar to paper chromatography; the stationary phase is a solid while the mobile phase is a liquid. TLC uses a thin layer of material such as silica (silicon dioxide, SiO2) or alumina (aluminium oxide, AlO3) coated onto a glass, plastic or aluminium plate. The separated substances may be recovered for further analysis or reaction by selectively scraping patches from the plate and dissolving them ina suitable solvent. The detection of pregnanediol in urine is a positive test for pregnancy.
Column chromatography is a convenient technique for physically separating the components of a mixture for further use, rather than for identification. The stationary phase is a powder packed into a vertical column of quite large diameter (about 2cm) and wetted with a solvent. The mixture is applies to the top of the column, followed by the solvent which passes downwards under the influence of gravity. The components of the mixture adsorb onto the surface of the solid to different extents and hence emerge from the base of the tube at different times.
High-performance liquid chromatography
Although it is useful for demonstrating liquid chromatography, column chromatography operating by the force of gravity is no longer used much in laboratories. Instead chemists use high-performance liquid chromatography. In this technique, the stationary phase is held in a column and the mobile phase is forced through under pressure. Separation is much faster. A widely used stationary phase consists of silica particles with long-chain alkanes adsorbed onto their surfaces. A common mobile phase is methanol.
The separated components of the mixture are usually detected by UV spectroscopy as they pass through a flow cell between a UV source and detector. The output of the detector is recorded as a chromatogram.
HPLC can be used for identification as well as for separation. The components of a mixture are identified by the time they take to pass through the system. The time between injection and the appearance of a peak on the chromatogram is called the retention time. Identical substances will have the same retention times under the same circumstances.
In common with all the chromatographic techniques described so far, gas-liquid chromatography (GLC) uses a stationary phase and a mobile phase. This method is used to separate and identify volatile liquids that do not decompose at temperatures around their boiling points. GLC is generally used for identifying the components of a mixture and measuring their concentrations. For example, evidence given in Court during prosecutions of drunk drivers usually comes from a gas chromatogram of the defendant's breath.
The stationary phase in a GLC apparatus consists of a liquid coated onto the walls of a long, thin capillary tube. The mobile phase is an unreactive gas such as helium or nitrogen. The sample is injected into a heated entrance port, where it immeadiately vapourises. The vapour is carried into the column by the mobile phase, which is usually refered to as the carrier gas. The carrier gas does not play any part in the separation except to carry the sample along. At the end of the column, the separated components of the mixture pass through a detector, typically a mass spectrometer. As in HPLC, the components are identified by their retention times. The relative quantity of each constituent is proportional to the area under its peak in the chromatogram.