Method
Extraction and Determination of Chlorophyll
Take frozen plant (leaf) tissue (100 mg)
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Place in 12 mL vial containing 7 mL DMSO
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Incubate at 65°C for 30 mm
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Transfer the sample to a 25 mL graduated tube
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Bring the volume to 10 mL with DMSO (dimethyl suiphoxide)
(Note: calculate the volume based on tissue wt.)
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Essay immediately or transfer to a vial and store between 0-4°C until analysis
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Transfer 1 mL to a cuvette
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Calibrate spectrophotometer against DMSO at 645 and 663 nm (Chl method)
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Record absorbance at 645 and 663 nm
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Calculate the chlorophyll content using Arnon’s equation
Arnon’s (1949) equations:
Chla (g l1) = 0.0127 × A663 – 0.00269 × A645;
Chlb (g l1) = 0.0229 × A645 – 0.00468 × A663;
Tot Chl (g l1) = 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.