Paper chromatography is a biochemical technique used to separate amino acids. Chromatography means to write in colours. Originally this method was used to Separate coloured substances. Hence the name. This method was devised by Tswett in 1906. It is a partition chromatography.
Procedure of paper chromatography
- The procedure of separation is called chromato-graphy. The preparation is called Chromatogram.
- The mixture to be separated is called solute.
- In paper chromatography two phases or media are used for the separation of amino acids. The phases are stationary phase and mobile phase.
- In paper chromatography stationary and mobile phases are liquids.
- The amino acids are partitioned between the liquids.
- The components are separated by their differential migration. A Whatmann filter paper is used as a supporting medium.
- The liquid remaining in the meshes of the filter paper functions as the stationary Phase.
- The solvent used is the mobile phase.
- The chromatography apparatus includes a rectangular jar, a lid and a Whatmann Filter paper.
- A strip of rectangular filter paper is suspended vertically. The lower end is allowed To dip into the solvent. The mixture to be separated is spotted at the lower end above the surface level of the solvent. It is called origin.
- The solvent moves up the filter paper.
- The components of the mixture are also carried along with the solvent.
- The components migrate at different rates depending upon their solubility on the solvent to certain distances on the filter paper. Afterwards they stop migrating.
- But the solvent rises up leaving behind the components of the mixture.
- The run is stopped as the solvent reaches near the upper edge of the filter paper.
- The strip of the filter paper is removed and the position of the solvent, called solvent front is marked on the filter paper.
- The filter paper is dried and stained with ninhydrin. The positions of the different spots (components) are observed.
- Individual components are identified by their Rf (resolution front) values. Rf value Is the migration rate. It is defined as the ratio of distance of the spot from the spotted line to the distance of the solvent front from the spotted line.
- Rf values are constant for any amino acid when chromatographed under the same experimental condition.
Types of Paper Chromatography
Paper chromatography is of the following types:
- Ascending Chromatography: In ascending chromatography, the solvent moves up the filter paper.
- Descending Chromatography: In descending chromatography the solvent moves down the filter paper.
- Circular Chromatography: In circular chromatography, a circular filter paper is used and the solvent moves radially from the centre.
- One Dimensional Chromatography: The solvent is allowed to move in one direction only, ascending or descending.
- Two Dimensional Chromatography: The solvent is allowed to move in two directions, namely vertically and horizontally.
- Ascending and Descending Chromatography: It is a combination of ascending and descending chromatography.
Principles of Paper Chromatography
In paper chromatography, the substances of a mixture are separated by two principles.
1. Different rates of migration of the components of a mixture on the solvent system.
2. Different rates of adsorption of the components in the stationary phase.
The stationary phase is water (liquid) and is held in between the fibres of filter paper. The Filter paper acts as a supporting medium to hold the stationary phase. The mobile phase is a solvent (liquid). The components are partitioned (separated) by two liquids. Hence it is a partition chromatography. The components migrate up at different rates on the filter paper and stop at different places.
Uses of Paper Chromatography
1. chromatography is used for the separation of amino acids from plant tissues.
2. It is used for the separation of proteins.
3. It is used for the identification of amino acids.
4. It is used for the quantitative estimation of amino acids.
What is Chromatography?
