Methodology for Pigments

Chlorophylls

The chlorophylls are the essential components for photosynthesis, and occur in chloroplasts as green pigments in all photosynthetic plant tissues. They are bound loosely to proteins but are readily extracted in organic solvents such as acetone or ether. Chemically, each chlorophyll molecule contains a porphyrin (tetrapyrole) nucleus with a chelated magnesium atom at the centre and a long-chain hydrocarbon (phytyl) side chain attached through a carboxylic acid group. There are at least five types of chlorophylls in plants. Chlorophylls a and b occur in higher plants, ferns and mosses. Chlorophylls c, d and e are only found in algae and in certain bacteria.





Principle

Chlorophyll is extracted in 80% acetone and the absorption at 663nm and 645nm areread in a spectrophotometer. Using the absorption coefficients, the amount of chlorophyll is calculated.


Materials
» Dilute analytical grade acetone to 80% acetone (prechilled).




Procedure
1.
Weigh 1g of finely cut and well mixed representative sample of leaf or fruit tissue into a clean mortar.
2.
Grind the tissue to a fine pulp with the addition of 20mL of 80% acetone.
3.
Centrifuge (5,000rpm for 5 min) and transfer the supernatant to a 100mL volumetric flask.
4.
Grind the residue with 20mL of 80% acetone, centrifuge and transfer the supernatant to the same volumetric flask.
5.
Repeat this procedure until the residue is colorless. Wash the mortar and pestle thoroughly with 80% acetone and collect the clear washings in the volumetric flask.
6.
Make up the volume to 100mL with 80% acetone.
7.
Read the absorbance of the solution at 645, 663 and 652nm against the solvent (80% acetone) blank.


Calculation
Calculate the amount of chlorophyll present in the extract mg chlorophyll per g tissue using the following equations:
mg chlorophyll a/g tissue = 12.7 (A663) - 2.69 (A645) x

V
1000 x W

mg chlorophyll b/g tissue = 22.9 (A645) – 4.68 (A663) x

V
1000 x W

and
mg total chlorophyll/g tissue = 20.2 (A645) + 8.02 (A663) x

V
1000 x W
where  
A             = absorbance at specific wavelengths,
V             = final volume of chlorophyll extract in 80% acetone and
W            = fresh weight of tissue extracted.


Note
The amount of tissue taken for extraction may be varied. Accordingly amount of 80% acetone used may be altered so that the final extract has a volume based on 10mg plant material extracted in 1mL of acetone.




References

1. Arnon, D I (1949) Plant Physiol 24 1.
2. Witham, F H, Blaydes, D F and Devlin, R M (1971) Experiments in Plant Physiology Van Nostrand New York p 245.