Paper Chromatography | Principle and Procedure

Paper chromatography is a planar type of chromatography used for the separation of mixture of substances. It is a simple, inexpensive and a powerful technique that can separate the components by using small quantities of samples.

It is a two-dimensional technique which was discovered by Synge and Martin in the year 1943.

Principle of Paper Chromatography (PC)

Paper chromatography(PC) is based on the principle of both adsorption as well as partition chromatography with a stationary phase (either liquid or solid) and a mobile phase (either gas or liquid).

Paper Chromatography is generally based on the principle of partition rather than adsorption wherein the sample solution travels up through the stationary phase separating the components of the mixture based on their migration rate.

1. Paper adsorption Chromatography:- In this principle, the stationary phase is the silica or alumina while solvent is used as the mobile phase.
2. Paper partition Chromatography:- In this principle, the stationary phase is the liquid (because the cellulose paper has water in its pores) while the mobile phase is the solvent.

This technique is similar to Thin-Layer Chromatography. The only difference lies in the use of stationary phase. In TLC, a layer of silica gel on aluminium or glass slide while a special paper made of Cellulose is used in PC.

Method/Procedure of Paper Chromatography

The following steps used in the PC technique include:-

1. Selecting stationary and mobile phase:- The stationary phase include a paper which is decided based on the pores, quality of sample which also favours good rate of movement of solvent. The mobile phase is of different combinations of organic and inorganic solvents such as mixture of Butanol, Acetic Acid, and Water for separation of Amino acids.
2. Saturation of Development Chamber:- After the mobile phase is selected, it is kept in the development chamber for 15-20 minutes for saturation of solvent molecules in the chamber.
3. Preparation and Loading of Sample:- The sample is prepared and spotted on the stationary phase with the help of Capillary tube on the base line such that when the paper is dissolved in the chamber, the sample doesn’t dissolve with the solvent.
4. Paper Chromatogram development:- After the paper is immersed in the chamber, the solvent moves up the paper with the help of capillary action and carries the sample components along with it at velocities that are dependent on their solubilities in the stationary and flowing solvents.
5. Drying and Detection:- After the chromatogram is established, the stationary phase is taken out and dried for a couple of minutes. After drying, the detecting solution is sprayed on the paper chromatogram such as Ninhydrin or Iodine vapours. The paper is then dried again and detected for sample chromatogram spots.

Rf Value

All components of mixture travel different distances. Some may travel along with solvent all the way while some may stop. Thus, the distance travelled by the component relative to the solvent is called as Rf value.

Rf is also defined as the ratio of distance travelled by the component to the distance travelled by the solvent.