Separation of pigments by paper chromatography method
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Objective
To separate and study plant pigments by paper chromatography.
Theory:
Pigments play a crucial role in the photosynthetic process of plants, as they are responsible for
capturing light energy from the sun and converting it into chemical food energy. These
pigments are chemical compounds that reflect only specific wavelengths of visible light. In
plant leaves, there are four main pigments: chlorophyll a (which appears dark green),
chlorophyll b (yellowish-green), xanthophylls (yellow), and carotenoids (orange).
To isolate and visualize these four primary pigments in green plants, we can use a simple
technique called chromatography.
What is Chromatography?
Chromatography is a scientific method that separates molecules based on their varying size,
shape, mass, charge, solubility, and adsorption properties. The term "chromatography" is
derived from the Greek words "chroma," which means color, and "graphein," which means to
write. These techniques rely on the interaction between three key components: the mixture that
needs to be separated, a solid phase, and a solvent.
How does paper chromatography work?
Paper chromatography is a widely-used technique for separating different pigments based on
their solubility in a specific solvent. In this method, the mixture is first spotted onto a sheet of
paper, dried, and then a solvent is allowed to flow through the sheet by capillary action. As the
solvent slowly moves through the paper, the various components of the mixture separate into
distinct coloured spots. The paper is then dried and the position of each compound is visualized.
The principle behind paper chromatography is that the more soluble substances will move
further on the filter paper than less soluble substances. This technique is particularly useful for
separating and identifying pigments found in plants or other biological samples.
Materials:
• Chromatography paper
• Solvent (such as petroleum ether, acetone, or water)
• Pigment extract or sample
• Pencil or pen
• Ruler
• Glass jar or beaker
• Filter paper
• Rubber bands
Procedure:
1. Cut a piece of chromatography paper to a suitable size. It is recommended to cut it to
the size of the glass jar or beaker you will be using.
2. Using a pencil or pen, draw a line approximately 1.5 cm from the bottom of the
chromatography paper. This is the baseline.
3. Apply the pigment sample or extract to the baseline using a capillary tube or a
micropipette. It is recommended to apply a small spot of the sample at the baseline.
4. Allow the spot to dry completely before proceeding to the next step.
5. Pour a small amount of solvent into the glass jar or beaker, enough to cover the bottom
of the jar to a depth of about 1 cm.
6. Place the chromatography paper into the jar, making sure that the baseline is above the
solvent level. It is important not to let the spot touch the solvent.
7. Cover the jar with a lid or a piece of filter paper to prevent the solvent from evaporating.
8. Wait for the solvent to travel up the paper, carrying the pigments with it. This process
is called developing the chromatogram. The time required for development may vary
depending on the solvent and the pigments being separated. Usually, it takes around 30
minutes.
9. Once the solvent front has reached the top of the paper, remove the chromatography
paper from the jar and let it dry.
10. Mark the positions of the pigment bands with a pencil or pen, taking care not to touch
the bands.
11. Measure the distance travelled by each pigment band from the baseline to the centre of
the band.
12. Calculate the Rf (retardation factor) value for each pigment band using the formula
Rf = distance travelled by pigment band / distance travelled by solvent front.
Note: Rf values are used to identify pigments and compare the relative polarities of the
pigments in the sample.
Observation Table:
Distance travelled by Distance travelled by
Pigment Rf value
different pigments the solvent
Carotene
Xanthophyll
Chlorophyll a
Chlorophyll b
Paper Chromatography Applications:
Paper chromatography has various applications across different fields. Some of the common
uses of this technique include:
• Separation of mixtures containing both polar and non-polar compounds
• Separation of amino acids
• Identification of organic compounds and biochemicals in urine
• Evaluation of inorganic compounds such as salts and complexes
• Checking the purity of pharmaceuticals
• Inspection of cosmetics
• Detection of adulterants in products
• Detection of contaminants in food and drinks
• Analysis of reaction mixtures in biochemical labs
• Determination of drugs and doping agents in humans and animals
Overall, paper chromatography is a versatile and useful tool for separating and identifying
various compounds in a range of applications.
Tips:
• If the solvent front reaches the top of the paper before all the pigments are separated, try using
a different solvent or increasing the distance between the baseline and the solvent level.
• To obtain accurate Rf values, ensure that the paper is fully dry before measuring the distances
travelled by the pigment bands and the solvent front.
Safety precautions:
• Be cautious when handling solvents, as they can be flammable and harmful if ingested or
inhaled.
• Wear gloves and protective eyewear when handling solvents or pigments.
• Perform the experiment in a well-ventilated area to prevent inhalation of solvent vapours.
