ESTIMATION OF CHLOROPHYLL PIGMENTS

Aim:

To estimate the chlorophyll pigments from plant leaves.

Introduction:

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

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

Material required:

Plant leaves, Mortar and pestle, 80% acetone, centrifuge, standard flask

Procedure:

1              1.   1 g of finely cut and well mixed representative sample of leaf was                                 weighed and took into a clean mortar.

2.    The leaves  were grinded to a fine pulp with the addition of 20 ml 80%    acetone.

3.    The pulp was centrifuged (5,000 rpm for 5 min) and the supernatant was transferred to the 100 ml standard flask.

4.    The residues were mixed and grinded with 80 % of acetone, centrifuged and supernatant was transferred to the same flask.

5.    The procedure was repeated until the residue became  colorless. The mortar and pestle washed thoroughly with 80 % acetone and the clear washing were collected in the standard flask.

6.    The volume of  extract was mad to 100 ml with , 80% acetone

7.    The absorbance of the solution was read at 645,663 and 652 nm against the solvent (, 80% acetone) blank.

Calculation:  (Write Left Side using pencil) 

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)  - 20.69  (A₆₄₅) 

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

               mg   of total chlorophyll /g tissue =  20.2 (A₆₄₅)  - 8.02  (A₆₆₃) 

Result :

          mg   chlorophyll a/ g tissue = 

          mg   chlorophyll  b/g tissue = 

mg   of total chlorophyll /g tissue =