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Separation of Plant Pigments by Paper Chromatography

Plant Pigments – An Introduction

Last updated date: 21st Mar 2023
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A compound that absorbs light is called a pigment. Chlorophylls a and b are primary photosynthetic pigments that absorb light for photosynthesis. The accessory pigments carotenoids and xanthophyll absorb light and pass it to chlorophyll a. Even though chlorophyll is the primary pigment, the other pigments are essential to the plant's ability to produce colour and engage in photosynthesis because they absorb each light differently and effectively across the electromagnetic spectrum.

Paper Chromatography

Chromatography, which means "colour writing," is a Greek term that is formed from the words "chromo" and "graph". Chromatography enables the separation of the constituent parts of a given mixture, enabling scientists to observe and produce findings and theories. 

Paper chromatography is a method for classifying dissolved substances according to how soluble they are in a given solvent, such as chlorophyll, carotene, and xanthophyll. Paper chromatography can be used to separate the colours in plant cells. The stationary element in chromatography paper permits the reaction between the solute and solvent to take place and produce results.

Leaf Chromatography

The separation of leaf colours using chromatography is known as leaf chromatography. Leaf chromatography is an experiment that is conducted to determine the colour of the photosynthetic pigments.

The experiment is conducted to learn about the pigments in the leaf, and it is mostly done by using paper and thin-layer chromatography. Let’s discuss some brief points of leaf chromatography.

There is a procedure by which this experiment is conducted in labs.

  1. Sample leaves should be crushed into small pieces and put in a mortar for pestle grinding. Add solvent and keep using the pestle to crush.

  2. Then, carefully draw a pencil line 1 cm from the bottom of the chromatography paper, spot a little amount of leaf extract repeatedly onto the centre of the line, and let each spot dry. 

  3. Make sure the paper dips into the solvent but the spot of leaf extract doesn't by suspending it using a pin attached to a bung within a test tube with a 1 cm depth of solvent. 

  4. The solvent is allowed to run up the paper until it is close to the bung, at which point the paper is removed. The solvent's location is marked, and the paper is allowed to dry. 

  5. The final chromatography paper is known as a chromatogram, and it may be photographed to determine the exact position of each pigment. Next, determine the Rf value for each pigment spot on the chromatogram.

  6. The retention factor is pronounced Rf. The retention factor is calculated by dividing the component's travel distance by the solvent's travel distance.

The colour dissolves as the alcohol goes through the filter paper. Some pigments in the leaf travel more quickly than others because of their properties. 

The pigment's movement rate is measured by the Rf (retention factor) value. Rf value = distance transported by pigment from origin to centre of pigment spot/distance from the origin to the solvent front. By applying this formula, you can determine the Rf value.

Separation of Plant Pigments by Paper Chromatography

The pigments in the plant's leaf are separated by paper chromatography, i.e., separation chromatography. It is the same as a leaf chromatography experiment. The process of paper chromatography is also the same as the leaf chromatography experiment. 

Separation of Chlorophyll Pigments by Paper Chromatography

The chlorophyll molecule is present in the leaf and can be separated by using paper chromatography. The paper chromatography separates the pigments in the leaf based on the distance travelled by pigment molecules on the paper in a nonpolar solvent.

Separation of Plant Pigments by Paper Chromatography Diagram

The experimental setup of paper chromatography

The Experimental Setup of Paper Chromatography

Chromatogram Report

The final chromatography paper is known as a chromatogram, and it may be photographed to determine the exact position of each pigment. The pattern of pigment spots on the chromatography paper at the conclusion of the experiment is called a chromatogram. Along with the alcohol, the pigments also migrate along the strips of paper.

Chromatography Conclusion

Carotene is identified as having the lowest molecular weight by its yellow to orange tint near the top of the paper. In the pigment separation of chlorophyll, chlorophyll may be distinguished by its blue or dark green hue. When chlorophyll pigments are separated, the colour yellow-light green identifies chlorophyll B. In the chromatography solvent, xanthophyll is more soluble since it has gone up the paper. This describes the conclusion of paper chromatography.


The pigments are light-absorbing molecules and are separated by using paper chromatography techniques in the lab. The pigments move on the paper based on their solubility in the solvent. Along with the alcohol, pigments also migrate along the strips of paper. Some pigments in the leaf travel more quickly than others because of their properties.

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FAQs on Separation of Plant Pigments by Paper Chromatography

1. What is the spectrum of absorption and action?

Infrared and visible electromagnetic energy is absorbed by photochromic pigments. The term "absorption spectrum" refers to a graph that displays the degree of light absorption by various pigments in sunlight as a function of wavelength. These pigments in leaves absorb various light wavelengths. Chlorophyll is, especially, sensitive to the colours red and blue. The action spectrum is a graph that demonstrates the degree to which various light wavelengths may effectively catalyse a photochemical process. Green has the greatest detrimental impact on light absorption. The selectively permeable membrane does not absorb green, so the chloroplast reflects it.

2. What is chromatography and its different types?

Different molecules can be distinguished from one another using the process of chromatography. It enables the separation of a mixture into its constituent parts when it is passed through a stationary phase by a mobile phase. The phase that is fixed in place in a chromatography column or plate is known as the stationary phase. The analysed mixture is transported through the stationary phase by the mobile phase. These characteristics—shape, size, charge, mass, adsorption, and solubility—are the basis for this distinction. 

Column chromatography, paper chromatography, partition chromatography, and thin-layer chromatography are some examples of chromatography.

3. What role does pigment play in photosynthesis?

Molecules called photosynthetic pigments absorb some wavelengths of light while reflecting others. We perceive them to be the colour of the wavelength that they reflect. To absorb the lightest energy possible, a variety of pigments work together. They are kept in thylakoid membranes, arrayed in photosystems, which are funnel-shaped structures maintained in place by proteins. During photosynthesis, the free electrons in the pigments' chemical structures transform into high-energy electrons, releasing the energy that they had previously received from light and transferring it to other molecules.

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