Paper Chromatography


Our objective is to separate and study plant pigments by paper chromatography.


Before going into detail, let’s understand the role of pigments in plants.

Photosynthetic plants convert light energy from the sun to chemical food energy.  During photosynthesis, molecules referred to as pigments are used to capture light energy.  Pigments are chemical compounds which reflect only certain wavelengths of visible light.  Plant leaves contain four primary pigments: chlorophyll a (dark green), chlorophyll b (yellowish-green), xanthophylls (yellow) and carotenoids (orange). 

To separate and visualize the four primary pigments of green plants, we can use a simple technique called chromatography.

What is Chromatography?

Chromatography is a technique used to separate molecules on the basis of differences in size, shape, mass, charge, solubility and adsorption properties. The term chromatography is derived from Greek words Chroma-colour and Graphe-write. There are many types of chromatography: paper chromatography, column chromatography, thin layer chromatography and partition chromatography. These techniques involve the interaction between three components: the mixture to be separated, a solid phase and a solvent.

How does paper chromatography work?

In paper chromatography, the mixture is spotted onto the paper, dried and the solvent is allowed to flow along the sheet by capillary attraction.  As the solvent slowly moves through the paper, the different compounds of the mixture separate into different coloured spots. The paper is dried and the position of different compounds is visualized. The principle behind the paper chromatography is that the most soluble substances move further on the filter paper than the least soluble substances. Different plant pigments can be separated by using the technique of paper chromatography.

What is Retention Factor or Rf value?

Retention factor or R_f value is applied in chromatography to make the technique more scientific than a mere analysis. The retention factor or Rf is defined as the distance travelled by the compound divided by the distance traveled by the solvent.
R_f=(Distance travelled by the compound)/(Distance travelled by the solvent)

Diagrammatic example that demonstrates Rf value:

Learning Outcomes

  • Students will understand the principle behind chromatography techniques.
  • Students will learn about different types of pigments occurring in a plant leaves.
  • Students will learn how to calculate the Retention factor.
  • Students will be able to do the experiment more accurately in the real lab once they understand the steps through the animation and simulation.