It is a known fact that the rate of adsorption of different materials on a given adsorbent varies from material to material. Some substances are adsorbed more readily on an adsorbent than others. This phenomenon of selective adsorption is utilized in column chromatography. The mixture to be separated is dissolved in a suitable solvent and is allowed to pass through a straight tube or a column containing adsorbent.
The constituent of the mixture which has greater adsorbing power is adsorbed first in the upper part of the column. Those with lower adsorbing capacity are retained in lower part of the column. In this way the constituents of the mixture are separated and retained in the column in order of decreasing capacity of adsorption to the particles of adsorbent packed in the column.
Once the separation of different constituents of the mixture has been achieved, the column is pushed out, different zones cut out with the help of knife and extracted with the help of a suitable solvent. Once in solution the different components of the mixture can be analysed by any of the suitable analytical method. Another way to remove different fractions of the mixture is to pass more solvent through the column.
These results in gradual movement of different zones downwards – the last fraction (lowermost in the column) is first to flow out from the column which is collected. In this way different fractions of the mixture are collected separately one after the other and analysed by a suitable analytical technique.
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Preparation of the apparatus for column chromatography is simple. A tube about 1 cm in diameter and 30-40 cm in length is taken. A wider tube can be taken but in such cases the length of the column should be increased in ratio of 1 : 25.
At the lower end of the column a rubber cork with a stopper tube passing through it for the flow of the solvent, is fitted. Over the rubber cork a glass wool plug is placed and small particles of uniform size of the adsorbent which may be alumina, activated magnesium silicate, lime, sodium carbonate, calcium carbonate, calcium phosphate, magnesium carbonate etc. as the case may, be is packed into the column.
The column is first washed with the solvent to be used so as to remove extraneous material and settle the particles. The choice of the solvent depends largely on the mixture and its constituents to be analysed. Common solvents used are light petroleum, cyclohexane, carbon disulphide, carbon tetrachloride, ethyl alcohol etc. As compared to paper chromatography or thin layer chromatography larger quantities of samples may be handled by column chromatography. Substances present in very small fraction in a mixture may be separated and analysed by this technique by taking a larger sample to begin with.