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Chromatography was originally introduced by Tswett in 1906, a Polish Botanist, for separation of different colour pigments present in the plant extract. Amino acids can also be separated from one another by partition chromatography.
Chromatography might be of different types, such as:
(a) Paper,
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(b) Thin-layer,
(c) Column, and
The amino acids are separated between a stationary and a mobile phase. In paper chromatography (Fig. 2.2), a drop of amino acid mixture is placed on a filter paper and allowed to dry.
The paper is then kept in contact with a suitable solvent which is allowed to flow over the dried drop slowly either by capillary action alone (ascending chromatography) or in combination with gravitational force (descending chromatography).
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As the solvent moves, it carries along with it the individual amino acids. Suitable tests are then applied to localise the individual amino acids which have been found to be carried away to a characteristic distance from the original place of application. The ratio of the distance travelled by the compound to the distance covered by the solvent on paper is called RF value of the compound.
To have a clearer separation, an improved method, the two-dimensional chromatography (Fig. 2.3) has been developed. Here after suitable chromatographic procedure, the paper is allowed to re-chromatograph at a right angle to the first one.
In thin-layer chromatography (TLC), a suitable adsorbent like alumina, cellulose powder, etc., is spread on glass plates which are then used as ‘paper in chromatographic separation. This technique has some special advantages in separation.
In column chromatography or ion-exchange chromatography, various solid adsorbents like starch, cellulose or an ion- pig. 2.4 exchange resins are placed as columns in glass tubes (Fig. 2.4). Different amino acids (or other substances) are adsorbed on the column are then eluted with suitable solvents.
