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For light energy to be used by living systems, it must first be absorbed. A pigment is any substance that absorbs light. Chlorophyll, the pigment that makes leaves green, absorbs light in the violet and blue wavelengths and also in the red because it reflects green light, it appears green. Different pigments absorb light energy at different wavelengths. The absorption pattern of a pigment is known as the absorption spectrum. The absorption spectrum of chlorophyll is between 400 nm and 700 nm. This portion of the spectrum is known as photosynthetically active region (PAR).
Electromagnetic Spectrum: Action and Absorption Spectrum of Chlorophyll a and b
The action spectrum shows how effective these pigments are in stimulating photosynthesis.In plants, chlorophyll a is the pigment directly involved in the transformation of light energy to chemical energy.
Structure of Chlorophyll
Structurally all types of chlorophyll resemble one another. All of them contain four pyrrole rings [also called porphyrin] which are linked together by methane bridges (-CH=). The skeleton of each pyrrole ring is made up of five atoms - four carbon and one nitrogen with magnesium in the centre as nucleus. One pyrrole ring is esterified with a long chain alcohol - phytol. This side chain-phytol is long and is composed of insoluble carbon and hydrogen atoms which helps to anchor the chlorophyll molecules with the thylakoids.
In plants, there are 2 types of chlorophyll - namely chlorophyll a and b. Chlorophyll molecule looks like a tadpole with porphyrin head and phytol tail. Chlorophyll a has methyl group (-CH3) at 
position and aldehyde (CHO) group in chlorophyll b.
Chlorophyll b constitutes about 1/4th of the total chlorophyll content. It acts as an accessory pigment and helps broaden the spectrum of light absorbed during photosynthesis. Chlorophyll b absorbs a different wavelength of light other than that absorbed by chlorophyll a. On absorbing light, it becomes excited and transfer its to chlorophyll a molecule.
Another group of pigments are called carotenoids. The carotenoids are red, orange or yellow pigments. In the green leaf, their colour is masked by the chlorophylls, which are more abundant.Carotenoids like chlorophyll are embedded in the thylakoid membrane of the chloroplasts. They are accessory pigments and harvest light from different regions of the spectrum. The light captured by these pigments are channelled it to the reaction centre, where light energy is converted into electrical energy.
Conversion of Light into Electrical Energy
Colour and Form of Pigments That Constitute Chlorophyll
| Name of pigment | Colour |
|---|---|
| Chlorophyll a | Yellow - Green |
| Chlorophyll b | Blue - Green |
| Carotene | Orange |
| Xanthophyll | Yellow |
The Action Spectrum
The Action Spectrum is the curve plotted on a graph paper representing the amount of oxygen evolved or the amount of carbon dioxide fixed or any other action of photosynthesis at different wavelengths of light. It has been observed that the photosynthesis occurs maximum in blue and red regions of visible light.
Action spectrum of photosynthesis determined by T.W.Englemann in 1882 using green alga. The scientist measured rate of photosynthesis as the amount of O2 released, which he detected by using bacteria that are attracted by O2
The Absorption Spectrum
The Absorption Spectrum is the curve plotted on a graph paper representing the amount of light absorbed at each wave length by that pigment.
Differences Between Chlorophyll a and b
| Chlorophyll a | Chlorophyll b |
|---|---|
| Bluish - green | Olive green |
| More soluble in petroleum ether | More soluble in 92% methanol |
| Has methyl group | Has aldehyde group |
Relation of the Visible Colour of Leaf to Absorption Spectrum
The leaf is green because wavelengths in this region of the spectrum, 550 nm are less strongly absorbed by leaf. These wavelengths are reflected.
Activity to Extract the Chloroplast Pigments and Separate them by Paper Chromatography
Materials Required
Spinach leaves, pestle and mortar, 80% acetone, calcium carbonate, Buchner funnel, beaker, measuring cylinder, glass jar with a tight cork. Whatmann No.1, filter paper, petroleum ether, acetone, hook, micropipette.
Procedure
Take 50 g of fresh spinach laves in a pestle and mortar. Crush them with 20 ml of 80% acetone. Add a pinch of calcium carbonate and again crush. Filter the extract on a Buchner filter. The deep green coloured filtrate containing chlorophylls and carotenoids is obtained. Evaporate the extract to concentrate.
Paper Chromotography of Leaf Pigments
Take a glass jar (about 45cm high) with a tight cork fitted in it. The cork should have a hole in the centre. Fit a small glass rod having a small hook, in the hole of cork. Now prepare the solvent by mixing 25ml petroleum ether and 3ml acetone. Pour the solvent into the jar and allow the jar to become saturated.Cut a strip of filter paper of the size which can easily be hung on the hook. Apply a circular spot of pigment extract about 3cm from the base of strip with the help of a micropipette. Now hang the strip inside the jar to the hook of cork and close the cork. Care should be taken that the spot is not dipped in the solvent. Make the apparatus air tight and observe.
Observation
The solvent will run on the filter paper. After few hours, the chloroplast pigments will be separated in the form of different spots on the paper. Take out the paper when the solvent reaches upto the upper level. After drying the paper, identify the different pigments with the help of their specific colours. Carotene is yellow, xanthopyhll is yellow-brown, chlorophyll-a is blue-green and chlorophyll-b is olive green in colour.
Demonstration of Fluorescence by Chlorophyll
Materials Required
Spinach leaves, pestle and morter, 80% acetone, calcium carbonate, Buchner funnel, test tube, source of light
Procedure
Take 25g of fresh spinach leaves in a pestle and morter. Crush them with 10 ml of 80% acetone. Add a pinch of calcium carbonate and crush again. Filter the extract on a Buchner funnel. The drop green coloured filtrate containing chlorophylls is obtained. Pour the filtrate in a test tube. Place the test tube before the source of light and observe.
Chlorophyll Solution as Viewed against Transmitted or Reflected Light
Observation
The solution appears green when placed between the source of light and eyes of observer is in transmitted light. The solution appears red when source of light is placed behind the observer and solution is placed in front of observer is in reflected light. The phenomenon is called fluorescence.