Paper Chromatography


To identify the mixtures of given amino acids and compared with standard using ascending paper chromatography technique and calculate Rf value.


Whatman filter paper No:1, chromatographic chamber, capillary tube separating funnel n-butanol, acetic acid, distilled water, a standard sample of amino acid, and 0.1% w/v of Ninhydrin reagent.


The separation of a mixture of amino acids depends upon differences in the partition coefficient of the component between the aqueous stationary phase and mobile organic phase. The aqueous phase is held on the fibers of the chromatographic paper. The components of the mixture to be separated migrate at different speeds depending on the difference in partition coefficient of component between 2 phases and appear as distinct spots at different points on paper.

Paper Chromatography
Paper Chromatography


  1. The mobile phase was prepared by mixing n-Butanol: Acetic acid: Water (4:1:3) in a separating funnel and organic layer were separated.
  2. The ingredients of the organic layer were poured into the chromatographic chamber and set aside for saturation
  3. A paper suitable for chromatography was prepared, drew a line above the base and the standard, and samples were  spotted side by side using a capillary tube 
  4. The chromatogram was run by keeping the paper inside the chamber and when the solvent level reached 90% paper was removed and the solvent front was marked with pencil and air-dried the paper
  5. Paper was sprayed with 0.1% Ninhydrine reagent and again dried paper.
  6. The Rf values were calculated for each component of the mixture and identified the given amino acids by comparing them with the standard.


The calculated   Rf value was

  • Phenylalanine = 0.51
  • Glycine = 0.48
  • Tryptophan = 0.24  
Make sure you also check our other amazing Article on : Thin Layer Chromatography
Sharing Is Caring:

Leave a Comment