ESTIMATION OF CHLOROPHYLL (Arnon,1949 )

Estimation of chlorophyll level from leaf

For chlorophyll (a, b and total) estimation, physiologically active leaves (3 rd or 4th from the top)
were collected randomly from 5-plants of a plot to represent the sample. The method of Arnon (1949)
was followed for the chlorophyll estimation.
Materials required
1. Acetone
2. Whatmann filter paper No. 1
3. Funnel stands
4. Mortar and pestle
5. Volumetric flask (250 ml),and
6. Colorimeter
Procedure
A 100 mg of fresh leaf material excluding midribs from a mixed representative sample was
(accurately ) weighed on a electronic monopan balance and thoroughly macerated in a mortar with a
pestle in 2ml of 80% acetone . The homogenate was filtered through a sheet of Whatman filter paper
No. 1.,and the filtrate was collected in a volumetric flask. The homogenate on the filterpaper was
washed with additional 2 ml of 80% acetone 3-4 times until it became colourless. The Volume of the
filtrate was adjusted to 10 ml with 80 % acetone. The absorbance ( oA) of the chlorophyll extract
(filtrate ) was measured on a spectrophotometer at 645nm, 652 nm, and 663 nm .Absorbance of
duplicate samples were recorded. Following formulae were used for calculation of chlorophyll a, b and
total chlorophyll:
Chlorophyll-a (mg g-1 FW) = [12.7 D663 2.69 D645] V/1000 W
Chlorophyll-b (mg g-1 FW) = [22.9 D645 4.68 D663] V/1000 W
Total Chlorophyll (mg g-1 FW) = D652 1000/345 V/1000 W
where D = Optical density, V = Final volume of 80% acetone (25 ml), W = Fresh wt. Sample taken (0.1
g), FW = Fresh weight