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 R_f coefficient ration is given by

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

Different substances possess different R_f values. R_f 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 coloured, it can be easily located on the chromatographic paper. If the substance is colourless, however, a reagent may be used to treat it, which gives it a characteristic colour. 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:

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 centre 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 R_f value of different spots.

Observations and Inference:

Results and Discussions:

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

2. The R_f 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.

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