Estimation of total chlorophyll by Aron's method

Purpose:-

Estimation of total chlorophyll by Arnon's method.

Requirement:-

Spectrophotometer Buchner's funnel, filter paper test tube, Fresh leaves. measuring cylinder pestle & mortar 80% Acetone 

Formula:-

20.2 (A645) +802 A663) / 1000 x W

Principle:-

Chlorophyll is extracted in 80% acetone & the absorption at 660 nm& 645 nm are read in s spectrophotometer using the absorption co-efficient, the amount of chlorophyll is calculated

Procedure:-

1) Weigh 1 gm of finely cut & well mixed representative sample of a leaf or fruit tissue into a clean mortar

2) Grind the tissue to a fine pulp with the addition of 20mil of 80% acetone  

3) Centrifuge (5000 rpm for 5 min) & transfer the supernatant to a 100 ml volumetric flask 

4) Repeat this procedure until the residue is colorless wash the mortar and pestle

 thoroughly with 80% acetone and collect the clear washing in the volumetric flask

5) Make up the volume to 100 ml with 80% acetone 

6) Read the absorbance of the solution at 645nm & 663nm against the solvent

Tabular Column:-

Sl. No.	Wavelength	Optical Density with solution	The optical density with 80% of a solution of acetone	Actual optical density
1
2
3
4

Calculation:-

Calculate the amount of chlorophyll present in the extract mg chlorophyll per gram tissue using the following equation.

Mg total chlorophyll tissue = 20,2(A675)+8.02(A663) x V/100 x W

where:-

A=Absorbance at a specific wavelength 

V=final volume of chlorophyll extract in 80% acetone 

W=Weight of fresh tissue extracted

Result:-

Mg total chlorophyll tissue = 20,2(A675)+8.02(A663) x V/100 x W.