Separation of Pigments of Leaves and Flowers using Chromatography Technique

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The Chromatography Technique

The chromatography technique can be widely used to separate, purify, and also identify the compounds. The substance balances in the chromatography between a stationary phase and a mobile phase. 


To separate the pigments that are present in flowers and leaves by the paper chromatography technique and determine their Rf values.


Paper chromatography is mainly a partition chromatography. In this paper chromatography, the stationary phase is defined as paper. The paper contains 22 percent of water molecules absorbed on about 78 percent of cellulose.

The components mixture separation takes place by partitioning of the components between the mobile phase and stationary phase. The mobile phase travels via paper by the capillary action. Depending on the ways the solvent travels on the paper, there exist three types of chromatography, given as follows.

  • Ascending Paper Chromatography

  • Descending Paper Chromatography

  • Circular Paper Chromatography

The distribution takes place in a definite ratio that represents the solution's characteristic distribution coefficient.

The Rf Coefficient Ration is Given as Follows:

Rf=distance travelled by solute, distance travelled by the solvent

Various substances possess various Rf values. Rf depends on a number of factors; where a few of them are listed below.

  • Nature of the solvent

  • Nature of the substance

  • Presence of impurities

  • Temparature

  • Quality of the filter paper

If any compound is coloured, it can be easily located on a chromatographic paper. If the same substance is colourless, however, a reagent can be used to treat it, which produces a characteristic colour. The term developer is given to this reagent. And iodine is the most frequently used paper chromatography developer.

Materials Required

  • Extract of leaves and extract of flowers

  • Whatman's filter paper

  • Acetone/Methanol

  • Acetone/Chloroform

  • Glass jar

  • Rubber cork fixed with a hook in the centre

  • Distilled water

  • Test tubes

  • Petroleum ether

Apparatus Setup

(Image to be added soon)


  • Take the Whatman filter paper and then draw a line using a pencil above 4cm from one end.

  • Then, grind the flowers and leaves in a motor and transfer the paste into the test tube.

  • In the crushed material, add methanol or acetone, and shake well and filter the respective mixture.

  • The filtrate is collected in a test tube to perform experiments.

  • With the help of a capillary tube, add one drop of the filtrate on the filter paper and allow it to dry.

  • After that, hang the filter paper in a jar containing 20ml of chloroform and petroleum ether.

  • Keep the same jar till the mobile phase rises up to 2/3rd of the length of the paper.

  • Then, remove the filter paper from the jar and mark the solvent front.

  • Outline the spots using a pencil and allow the filter paper to dry.

  • Now, measure the distance between the centre of different spots and the solvent front in relation to the reference line as indicated

  • Then, determine the pigment number in the flowers and leaves extract.

  • Finally, calculate the Rf value of different spots using the expression.

Observations and Inference

The noticed Observations and Inferences can be recorded in the respective fields of the table, which is given below.

S. No

Name of the Extract

Colour of the Spot

Distance travelled by spot from the original line

Distance travelled by solvent from the original line

Rf values





Results and Discussions

  • The Rf value of the components of the leaves is _____.

  • The Rf value of the components of the flowers is _____.


  • Always choose a fine capillary tube.

  • Do not allow the spots to spread while spotting the test solution available on the paper.

  • Do not disturb the jar once the experiment is arranged as long as the development of the chromatogram has been completed.

  • Use the capillary finely drawn to place the spot on the same paper.

  • Prior to developing the spots, allow the paper strip to become dry perfectly.

  • Handle the organic solvents carefully.

Finding the Number of Pigments in Leaves with the Help of Chromatography

If there exist more samples, it's spots are bigger on the TLC plate.

Like when we spill coffee on a white shirt- the more coffee, the larger the spot! In general, on a shirt, the water carries the solute, which is considered a poor example.

Remember that a sample is applied in a tiny spot that goes to thin-layer chromatography (TLC) glass or near the bottom of the paper or the plastic plate. Then, the plate is placed carefully in the appropriate solvent and moves in an upward direction uniformly. Solutes, which are otherwise called pigment molecules, dissolve. After that, move upward at various rates with the solvent while the paper or the TLC plate adsorbent wants to "hold on" to the molecules. This is the main reason why the molecules having different polarities are separated and hence generate an Rf value.

FAQ (Frequently Asked Questions)

1. What is an iodine spot?

The spot present on the chromatography paper strip where the acetone extract of leaf is to be loaded, is referred to as the iodine spot.

2. What is chromatography?

Chromatography is one of the processes of mixture separation laboratory technique. Then, the mixture is dissolved in a mobile phase fluid that carries it via a structure that holds another material, which is known as the stationary phase.

3. Give the advantages of chromatography compared to other techniques

Chromatography can be used in a wide variety of applications. This process is primarily used to separate complex mixtures. It works on various samples, including food particles, drugs, plastics, samples of air and water, pesticides, and extracts of tissue.

4. List some chromatographic techniques.

Column chromatography, paper chromatography, gas chromatography and thin-layer chromatography are a few chromatography techniques.

5. Mention the factors that the Rf value of a compound depends on.

The value of Rf depends on the nature of the solvent, the nature of the compound, and the temperature.