Plant BIOTECHNOLOGY

Experiment 3

Principle

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.

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.

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.

Procedure

• In this experiment, spinach leaves and Neem leaves are used to separate different pigments.

• Pick a few fresh and green leaves of spinach and Neem and wash them.

• Cut out small pieces of leaves using scissors. Add them to the mortar.

• Accurately measure 5 ml acetone using a measuring cylinder and add it into the mortar.

• With the help of mortar and pestle, grind both the leaves into a smooth paste.

• Shift the prepared paste of both the leaves into different watch glass with the help of a spatula.

• Cut the strip of accurate size.

• Horizontally trace a line with a scale and a pencil that is 1 to 2 cm apart from the end of the strip.

• 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 petroleum ether: acetone (9:1) solvent.

• 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 3/4th of the strip, carefully take the strip off.

• Allow the strip to dry and observe for spots.

Calculation

Rf value: Distance travelled by the compound

Distance travelled by the solvent

Results

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 (Fig.- 3).

2. Just below this band, a yellowish band appears which indicates the pigment xanthophyll(Fig.- 2).

3. The third band appearing dark green indicates chlorophyll-a pigment (Fig.- 2).

4. The yellowish-green band present at the bottom is the chlorophyll b pigment (Fig.- 2).

Reference Material

1. Paper chromatography of plant pigments

2. Separation of plant pigments by Thin layer Chromatography

Questions

• What do you mean by pigment?

• What is Chromatography?

• What are pigments present in plant samples?

• Which are the different types of chromatography?