Method

Extraction and Determination of Chlorophyll

Take frozen plant (leaf) tissue (100 mg)

Place in 12 mL vial containing 7 mL DMSO

Incubate at 65C for 30 mm

Transfer the sample to a 25 mL graduated tube

Bring the volume to 10 mL with DMSO (dimethyl suiphoxide)

(Note: calculate the volume based on tissue wt.)

Essay immediately or transfer to a vial and store between 0-4C until analysis

Transfer 1 mL to a cuvette

Calibrate spectrophotometer against DMSO at 645 and 663 nm (Chl method)

Record absorbance at 645 and 663 nm

Calculate the chlorophyll content using Arnons equation

Arnons (1949) equations:
Chla (g l1) = 0.0127 A663 0.00269 A645;
Chlb (g l1) = 0.0229 A645 0.00468 A663;
Tot Chl (g l1) = 0.0202 A645 0.00802 A663.

References: Hiscox, J.D., and Tsraelstam, G.F. 1979. A method for the extraction of
chlorophyll from leaf
tissue without maceration. Can. J. Bot. 57: 1332-1334.