How to Separate Plant Pigments Using Paper Chromatography

Aim

To distinguish and study the various pigments present in plants through the process of paper chromatography.

Theory

Plants carry out the process of photosynthesis, during which light energy from the sun is converted into chemical energy (food). The capturing of light energy is carried out by molecules known as pigments, which are present within the plant cells.

Explore more: Plant cells.

What are Pigments?

Pigments are chemical compounds, which are able to reflect only a particular range of wavelengths of visible light. Leaves of plants primarily contain different types of pigments within their tissues. The four different types of pigments are listed below in a tabular column along with their colours.

In order to view and distinguish the primary four plant pigments, a simple technique known as chromatography can be used.

Read more: Pigments

What is Chromatography?

It is a technique that is used to distinguish between different molecules. This differentiation is based on these attributes-shape, size, charge, mass, adsorption and solubility.

Types of chromatography:

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

Mechanism of Paper Chromatography

In this technique, the interaction between three components is involved – solid phase, separation of a mixture and a solvent.

1. At first, the mixture is spotted onto the paper and is dried.
2. The solvent is made to flow through the capillary attraction.
3. While the solvent moves through the paper, the various components of the mixture differentiate into varied coloured spots.
4. Later the paper is allowed to dry and the position of various compounds is viewed.
5. The substance, which is the most soluble moves further on the paper as compared to the other substances that are less soluble.

Material Required

• 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

• In this experiment, spinach leaves are used to separate different pigments.
• Pick a few fresh and green leaves of spinach and wash it.
• Cut out small pieces of spinach using scissors. Add them to the mortar.
• Accurately measure 5ml acetone using a measuring cylinder and add it into the mortar.
• With the help of mortar and pestle, grind the spinach leaves into a smooth paste.
• Shift the prepared paste of spinach into the watch glass with the help of a spatula.
• Place a filter paper strip with a tapering notch towards one ending of the strip.
• Horizontally trace a line with a scale and a pencil that is 2 to 3 cm apart from the notch’s tip.
• Using a capillary tube, add 1 drop of the extract of the pigment in the midsection of the line.
• Let the drop dry. Repeat the same process of adding a drop and allowing it to dry for 4-5 times.
• In the chromatographic chamber, pour the ether acetone solvent.
• Make sure to folded and stapled an end side of the paper.
• Suspend the strip in the chamber.
• The loading spot remains about 1 cm above the level of the solvent.
• Let the chamber remain uninterrupted for a while.
• We can notice that the solvent passes along the paper scattering various pigments of the blend to different distances.
• Once the solvent reaches 3/4^th of the strip, carefully take the strip off.
• Allow the strip to dry.

Observation

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

Conclusion

1. The Carotene pigment is observed at the topmost as an orange-yellow band of pigments distinctively.
2. Just below this band, a yellowish band appears which indicates the pigment xanthophyll.
3. The third band appearing dark green indicates chlorophyll-a pigment.
4. The yellowish-green band present at the bottom is the chlorophyll b pigment.

Precautions

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

Viva Questions

Q.1. What Rf value or Retention factor?

A.1. The Retention factor or Rf value applies to chromatography to make the technique scientific. It is defined as the distance travelled by the compound divided by the distance travelled by the solvent.

Rf value = Distance travelled by the compound / Distance travelled by the solvent.

Q.2. What is Phycobilin?

A.2. Phycobilins are light-capturing bilins found in chloroplast organelles, cyanobacteria and in a few algae.

Q.3. What is the significance of pigment in photosynthesis?

A.3. It helps in the absorption of energy from light. The free electrons in the pigments present in their chemical structure transfer their energy to other molecules during photosynthesis when they turn into high energy electrons, thereby liberating energy they captured from light. This released energy is then used up by other molecules for the formation of sugars and related nutrients with the use of water and carbon dioxide.

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