Separation of Plant Pigments Using Chromatography: Types & RF Factor

Paper chromatography is a technique by which there is separation and identification of different pigments of plants. The differentiation in this technique is based on the attributes of shape, size, charge, mass, adsorption and solubility. It is useful for identifying unknown compounds which is often used in crime scene investigations to match ink, lipstick, or colored fibers. In paper chromatography, paper sheets or strips are used as the adsorbent being the stationary phase through which a solution is made to pass. It is an inexpensive method that is used to separate dissolved chemical substances through their different migration rates across the paper sheets. It is a powerful analytical tool where very small quantities of material is used. Paper chromatography was discovered by Martin and Synge in the year 1943. The principle that is involved in paper chromatography is partition chromatography or adsorption chromatography.

Key terms: Chromatography, paper chromatography, Pigments, Cholorphyll, Solvent, Plants

What are Pigments?

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The pigments are the chemical compounds which reflect only a particular range of wavelengths of visible light. Various types of pigments are generally stored in the leaves of plants and their tissues.

There are 4 types of pigments which are mentioned below-

• Chlorophyll A - Dark green
• Chlorophyll B - Yellowish-green
• Xanthophylls - Yellow
• Carotenoids - Orange

Chromatography is a simple technique is used to view and distinguish the primary four plant pigments.

Leaves

What is Chromatography?

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Chromatography is the technique which is used to distinguish between two molecules. There are many types of chromatography.

These are –

• Column chromatography
• Paper chromatography
• Partition chromatography
• Thin-layer chromatography

Separation of Plant Pigments Using Chromatography Experiment

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Aim

The aim of the experiment is to distinguish and study the various pigments present in plants through the process of paper chromatography.

Theory

By the process of photosynthesis, plants convert light energy from the sun to chemical energy. These light waves are carried out by pigments which are present in the plants.

These pigments are present in the cells of the plants.

Mechanism of Paper chromatography

The interaction between three components is involved in this experiment which are solid phase, separation of a mixture and a solvent.

• Mixture is spotted on the paper and then is dried.
• The solvent is made to flow through capillary attraction.
• When the solvent moves through the paper, different colours are spotted due to the separation of different components.
• The paper is then allowed to dry and variations of the position of components are seen.
• The substance which is most soluble moves further on the paper as compared to other substances less soluble.

Material Required For the Experiment

• Chromatography chamber
• Spinach leaves
• Mortar and pestle
• Scissors
• Ether acetone solvent
• Acetone
• Capillary tube
• Pencil
• Spatula
• Scale
• Filter paper strips
• Stapler
• Thread
• Watch glass

Procedure

To separate different components, spinach leaves are used as the main subject for this experiment.

• We have to pick few fresh and green leaves of the spinach and wash it.
• Then we have to cut it using a scissors and add it to a mortar. We have to measure 5ml acetone correctly using measuring cyclinder and add it to the mortar.
• Now with the help of mortar and pestle we have to mix the acetone and spinach leaves in a smooth paste.
• Shifting the prepared paste of spinach into the watch glass with the help of a spatula, we have to then place it on a filter paper strip with a tapering notch. The tapering notch is towards one ending of the strip. We have to then horizontally trace a line with a scale and a pencil that is 2 to 3 cm apart from the notch’s tip.
• Now with the help of a capillary tube we have to add 1 drop of the extract of the pigment in the midsection of the line.
• Now we have to the drop dry and repeat the same process of adding a drop, allowing it to dry for 4-5 times.
• Then in the chromatographic chamber, we have to pour the ether acetone solvent and fold and staple an end side of the paper suspending the strip in the chamber.
• The loading spot remains approximately 1 cm above solvent level.
• We have to leave the chamber uninterrupted for a while. We will then notice that the solvent passes along the paper scattering various pigments of the blend to different distances.
• Once the solvent reaches 3/4th of the strip, we have to carefully take the strip off and allow the strip to dry.

Observation

The dried paper strip will displays four different bands and discrete pigments can be distinguished with the help of colours.

Conclusion

• At the topmost part, the Carotene pigment is observed as an orange-yellow band of pigments distinctively.
• A yellowish band which indicates the pigment Xanthophyll is just below the layer.
• The third band appearing dark green indicates chlorophyll-a pigment.
• The yellowish-green band that is present at the bottom is the chlorophyll b pigment.

Precautions

• The spinach leaves selected should be green and fresh.
• The loading spot needs to be 2 to 3 cm apart from the top of notch.
• While suspending the filter paper strips in the chamber, we need to ensure that the loading spot needs to be set up above 1 cm from the level of the solvent.

RF Factor

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A compound can be easily identified by calculating the distance travelled by a particular compound. The RF value is referred to as the ratio of the distance travelled by a compound to that of the solvent front. The unknown compounds may be identified by comparing their Rf's to the Rf's of known standards.

The Rf equation is:

Rf = distance traveled by compound/distance traveled by solvent

Things To Remember

• Paper chromatography is a technique by which there is separation and identification of different pigments of plants.
• The principle that is involved in paper chromatography is partition chromatography or adsorption chromatography. 
• The pigments are chemical compounds which reflect only a particular range of wavelengths of visible light.There are 4 types of pigments which are listed down below-Chlorophyll A Dark green, Chlorophyll B -Yellowish-green, Xanthophylls- Yellow, Carotenoids - Orange.
• There are many types of chromatography: Column chromatography, paper chromatography, partition chromatography, Thin-layer chromatography.