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How to do Column Chromatography

Column Chromatography: A Step-by-Step Guide on How to do it

Invented by the American chemist, D.T. Davy in 1897, Column chromatography is a technique to study the chemical composition of different natural petroleum products.

Based on the principle of selective adsorption, the chemical mixtures are passed through a column of an adsorbent material and separated into its individual components.

A typical column chromatography system consists of a solid stationary phase like glass, silica gel, alumina or cellulose, and a liquid mobile phase.

Here are the steps involved in Column Chromatography:

1) Preparation / Packing of an adsorbent column

Sucrose, cellulose, starch, calcium carbonate, calcium phosphate, calcium oxide, silica gel, charcoal, magnesium and alumina are the common materials used as adsorbents. However, alumina is the strongest adsorbent giving best and accurate results.

Glass wool or cotton plug is used to support the column which is kept in a vertical position. The chromatography column is filled 1/3rd with the solvent and the adsorbent material is added slowly. It is carefully poured down with a glass rod to prevent air bubble formation.

Wait till the suspension settles down and discard the excess solvent. Repeat the process until you get the column of 5 cm of height.

2) Solvent system / Mobile phase

Column chromatography uses a single solvent. The commonly used ones are petroleum ether, benzene, carbon tetrachloride, ethyl acetate, acetone, cyclohexane, ether, chloroform, etc.

The role of the solvent in column chromatography is:

  • Separate the mixture into different zones/bands.
  • Introduce the mixture into the column
  • Remove the pure components of each separated bands as eluents

3) Application of samples

The study sample can be applied to the top of the prepared column in various ways:

  • Remove most of the mobile phase from above the column using a substrate and drain the remaining solvent into the column bed. Apply the sample carefully with a pipette and allow it to run in the column. To wash the final traces of the sample into the column bed, apply a small volume of the mobile phase. Carefully add the mobile phase to the column to the height of 2-5 cm and maintain it by connecting the column to a suitable reservoir of mobile phase.
  • Add sucrose to a concentration of 1% to increase the density of the sample when the solution is layered into the liquid above the column bed; it will automatically sink to the surface of the column.
  • Use capillary tubing or roller pumps to pass the sample directly to the column surface.

However, in all cases, avoid overloading the column with the sample, otherwise irregular separation will occur.

4) Sample elution

After sample application, the next step is to remove the material from the column using an appropriate solvent. This can be carried out in following ways:

  1. Frontal development: A large volume of the sample mixture is passed through a column of adsorbent. Pure solvent appears first followed by the least strongly held component of the mixture and so on. The components which are strongly held appear in pure form.
  2. Displacement development: A solvent which has the higher affinity for the stationary phase than any of the components of the mixture is used. As a result, all the components get displaced down of the column.
  3. Elution development: Here, the components of the applied sample are separated by the continuous flow of the mobile phase through the column. The interaction between the solvent and the column is weaker than the solute molecules. As a result, the bound molecule gets eluted from the column.

5) Collection and analysis of fraction

Collect the fractions from the column into a series of test tubes, either manually or with a fraction collector. Analyse each fraction for the presence of the compound. Coloured compounds can be detected by visual observation, but colourless compounds are detected by spectrophotometer or colorimetry or UV adsorption method.

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