CHROMATOGRAPHY

CHROMATOGRAPHY

Chromatography: “A method, often used in laboratories, which enables the easy and efficient separation of mixtures of chemical compounds using the phenomenon of adsorption. The technique’s strengths are especially manifested in the separation of isomers and natural materials.

In 1910, Tswett first used this method in the separation of leaf pigments, and the technique achieved rapid growth later when it was applied to the separation of carotinoid pigments, among other uses.

The separation method called ‘column chromatography’ uses a glass column filled with adsorbent though which passes a composite liquid mixture. The technique operates such that each component will form in a different section of the column arranged by color according to the adsorption affinity of each material.

In performing column chromatography, a vertical glass column is filled evenly with the proper amount of adsorbent. Next, a liquid mixture is poured into the column and the liquid passes through the adsorbent. Each compound is absorbed into beds at different heights depending on the component’s individual adsorption affinity.

At this stage, the sections into which each of the components have been absorbed are still not completely separated. However, if the appropriate desorbent is poured into the column, the components which have been adsorbed on the adsorbent dissolve into the desorbent and start moving downwards in the column. Each component moves towards the lower part of the column, but the migration rates again differ according to each component’s adsorption affinity. The components in the lower layers move faster, and in the end, each components will be clearly separated. This stage of the column chromatography process is called ‘development.’

If the material being separated is a mixture of pigments, there will be colored zones at different heights in the column filled with adsorbent. These colored zones are called a chromatogram.

When the development stage is over and the pigments appear, the adsorbent is pushed out of the column, divided into each zone and the absorbates are extracted separately using a desorbent. This stage of the process is called ‘elution.’ Alternately, without removing the adsorbent from the column, a desorbent may be successively poured in from above, and each zone preferentially eluted by the desorbent will dissolve into it and trickle down the column one at a time. The liquids can then be collected from the bottom of the column as they drip out.”