Separation of Pigments of Leaves and Flowers by Chromatography

The chromatography technique is widely used to separate, purify and identify compounds. Substance balances in chromatography between a mobile phase and a stationary phase. The more its movement is the interaction of the substance with the stationary phase.

Aim:

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

Theory:

Paper chromatography is essentially a partition chromatography. In paper chromatography the stationary phase is paper. Paper contains 22% of water molecules absorbed on about 78% of cellulose.

The separation of the components of the mixture takes place by partitioning of the components between the stationary phase and mobile phase. The mobile phase travels through the paper by capillary action. Based upon the ways the solvent travels on the paper there are three types of chromatography

  1. Ascending Paper Chromatography
  2. Descending Paper Chromatography
  3. Circular Paper Chromatography

The distribution occurs in a definite ratio which represents the characteristic distribution coefficient of the solution.

The Rf coefficient ration is given by

Rf = \(\frac{distance\, travelled\, by\, solute}{distance\, travelled\, by\, solvent}\)

Different substances possess different Rf values. Rf depends upon a number of factors.

  • Nature of the substance
  • Nature of the solvent
  • Temperature
  • Presence of impurities
  • Quality of the filter paper

If the compound is colored, it can be easily located on the chromatographic paper. If the substance is colorless, however, a reagent may be used to treat it, which gives it a characteristic color. The name developer is given to this reagent. Iodine is the most frequently used paper chromatography developer.

Materials Required:

  1. Whatman’s filter paper
  2. Extract of leaves and extract of flowers
  3. Chloroform/acetone
  4. Methanol/Acetone
  5. Rubber cork fixed with hook in the centre
  6. Glass jar
  7. Rubber cork fixed with hook in the centre
  8. Test tubes
  9. Distilled water
  10. Petroleum ether

Apparatus Setup:

Separation of Pigments of Leaves and Flowers by Chromatography

Procedure:

  1. Take the whatman filter paper and draw a line with the help of a pencil above 4cm from one end.
  2. Grind the leaves and flowers in a motor and transfer the paste into a test tube.
  3. This crushed material add acetone or methanol, shake well and filter the mixture.
  4. The filtrate is collected in a test tube for performing experiments.
  5. Using a capillary put one drop of the filtrate on the filter paper and allow it to dry.
  6. Now hang the filter paper in a jar containing 20ml of petroleum ether and chloroform.
  7. Keep this jar till the mobile phase rises up to 2/3th of the length of the paper.
  8. Remove the filter paper from the jar mark the solvent front.
  9. Outline the spots with the help of pencil and make the filter paper to dry.
  10. Measure the distance between the solvent front and the center of different spots in relation to the reference line as indicated
  11. Determine the number of pigments in the leaves and flowers extract.
  12. Using the expression, calculate the Rf value of different spots.

Observations and Inference:

S.No

Name of the extract

Colour of the spot

Distance travelled by the spot from the original line.

Distance travelled by the solvent from the original line

Rf Values

1

2

3

4

Results and Discussions:

1. The Rf value of the components of leaves are ________.

2. The Rf value of the components of flowers are ________.

Precautions:

  1. Always make use of a fine capillary tube.
  2. Do not allow spots to spread while spotting the test solution on the paper.
  3. Use the capillary finely drawn to place the spot on the paper.
  4. Do not disturb the jar once the experiment is set as long as the chromatogram is developed.
  5. Before developing the spots, make the paper strip perfectly dry.
  6. Carefully handle organic solvents.

Viva Questions:

1. What is chromatography?

Ans: Chromatography is a process of mixture separation laboratory technique. The mixture is dissolved in a mobile phase fluid that carries it through a structure that holds another material called the stationary phase.

2. What is iodine spot?

Ans: The spot on chromatography paper strip where the acetone extract of leaf is to be loaded called iodine spot.

3. Name some chromatographic techniques?

Ans: Paper chromatography, column chromatography, thin layer chromatography and gas chromatography are some of chromatography techniques.

4. What are the advantages of chromatography over other techniques?

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

5. On what factors does the Rf value of a compound depend?

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

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