An experimental technique for separating small organic molecules. Originally done with coloured substances (as with inks on filter paper), it now includes High-Performance Liquid Chromatography to separate compounds that can't be stained. Of course, the old paper and solvent stuff is still useful - Plant Biochemistry involves a lot of colourful natural dyes. Where the metabolites are not visible, radiolabelling enables the spots to be visualised.

Chromatography can be used to seperate out dyes in a sample of ink.

  1. Spots of dye are dropped onto a baseline on a square of filter paper.
  2. The sheet is rolled up and placed in a beaker containing shallow solvent.
  3. The solvent seeps up the paper, taking the dyes with it.
  4. Each different dye in the ink samples will form a distinct spot on the paper.
  5. It is now possible to compare the unknown ink to known dyes.

Chromatography is the name of a wide category of methods for separating components of mixtures. Chromatography was invented by a Russian botanist named Mikhail Tswett around the beginning of the twentieth century. He separated plant pigments by putting solutions of these compounds through glass columns packed with calcium carbonate. The separated pigments formed different bands of color as they moved through the column, explaining the use of chroma (color) in the name he gave to the technique.

All chromatographic methods involve introducing a sample into a mobile phase (usually a liquid or gas, but more exotic things such as supercritical fluid can also be used) which is forced through an immiscible stationary phase which is either bonded to a solid surface or packed into a column. As the mobile phase flows through or over the stationary phase, different compounds will flow through at varying rates based on how strongly or weakly they are retained by interactions with the stationary phase. Separations are made possible because of the different properties of substances in a mixture, such as different ionic strengths, affinities, adsorption properties, or even size. If the mobile phase and/or stationary phase is not chosen carefully it is possible for a compound to be "unretained", that is, it flows through the stationary phase at the same rate as the mobile phase. It is also possible for a compound to be completely retained on the stationary phase, never eluting no matter how much mobile phase is used.

Some of the more common types of chromatography include column chromatography, high-performance liquid chromatography (also known as high pressure liquid chromatography or HPLC), gas chromatography (GC) and thin-layer chromatography (TLC).

Chromatography can be used in biochemistry to identify amino acids. In essence, the process goes like this:

  1. Apply small spots of amino acid solution to chromatography paper (a type of filter paper used as the adsorbent), and mark their position.
  2. Roll the paper into a cylinder and stand in a solvent inside a glass beaker. Mark the position of the solvent. (There are a number of appropriate solvents, based around simple organic substances such as butyl alcohol and acetic acid.)
  3. Wait until the solvent level has nearly reached the top of the paper.
  4. Dry the paper, but do not apply a direct flame.
  5. Apply ninhydrin spray (which is nasty stuff, so take care).
  6. Heat in an oven for around 10 minutes.
  7. Measure the distances moved by the solvent and the acid(s).
  8. Calculate the Rf value for each acid, which is the distance it has moved divided by the distance the solvent has moved.

Rf values for amino acids in different solvents have been determined, so these can be used to determine which ones you've got in your sample.


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.

Paper Chromatography

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

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

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.

Gas-liquid Chromatography

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.

Chro`ma*tog"ra*phy (?), n. [Gr. , , color + -graphy.]

A treatise on colors


© Webster 1913.

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