Paper Chromatography

This technique is commonly used for the separation of amino acids, sugars, sugar derivatives and peptides. In paper chromatography, stationary phase is a paper, usually cellulose acetate and the mobile phase is a solvent in which the solute in the mixture are soluble. In this technique, a few drops of solution containing a mixture of the compounds to be separated is applied at one end of the paper (Whatman No. 1 or No. 3 Filter paper), usually above 2 to 3 cm. The paper is dried and dipped into a solvent mixture consisting of butanol, acetic acid and water in 4:1:5 ratio. The aqueous component of the solvent system binds to the paper and forms a stationary phase. The organic component that migrates on the paper is the mobile phase. The filter paper is hung vertically into the solvent, the migration of the solvent is upwards by capillary action is referred to as ascending chromatography. In descending chromatography, the solvents moves downwards. As the solvents flows, it takes along with it the unknown substances. The rate of migration of the molecules depends on the relative solubilities in the stationary phase and mobile phase. 

Remove the paper after a sufficient migration of the solvent, dried and sprayed with a chemical of colour development. Identifies the specific spots at different sites. Ninhydrin, which forms purple complex with ∝-amino acids, is frequently used as a coloring reagent. The chemical nature of the individual spots can be identified by running known standards with the unknown mixture.