Chlorophyll and carotenoid content estimation of the leaf

Aim of the experiment: To analyse the Chlorophyll and carotenoid content of leaf.

Objective: Estimation of chlorophyll and carotenoid content of the leaf.

Method of estimation and calculation:

The chlorophyll content of the leaf samples was measured by the 80% (v/v) acetone method. 

1. Eighty per cent acetone was prepared (80 ml of acetone + 20 ml distilled water).

 

2. Well matured leaf was taken from the plant in ziplock bag, and directly move to the next step in laboratory condition or eles could be stored in deep fridge or refrigerator  fir further use.

3. With the help of electrical weighing balance, 0.1 g of the leaf sample was weighed and cut into very thin slices using a sterilized razor blade. 

4. The leaf discs were put in 10 ml of 80% acetone solution in a previously washed test tube and covered with aluminium foil. 

5. The test tubes were kept in the dark for 24 hours. Then they were filtered by Whatman no-1 filter paper and the filtrate was used to record the absorbance in a spectrophotometer at 663 nm, 645 nm and 470 nm, keeping 80% acetone as blank. 

Calculation:

Then the amount of chlorophyll-a, chlorophyll-b, total chlorophyll and carotenoid was calculated using the following formulae and ascribed their content as mg/g of fresh weight.

Chlorophyll-a=([(12.7×A663)-(2.69×A645)]×V)/(1000×W)

Chlorophyll-b=([(22.9×A645)-(4.68×A663)]×V)/(1000×W)

Total chlorophyll=([(8.02×A663)+(20.2×A645)]×V)/(1000×W)

Carotenoid content =([(1000×A470)-(2.27×Chlorophyll-a)-(81.4 ×Chlorophyll-b)])/2270

Where, A = Absorbance at particular given wavelength.

V = Final volume of 80% acetone (ml).

W = Fresh weight of leaf sample (g).

Assignment:

Make a table for the average chlorophyll and carotenoid content of the fruit crops.