Ascorbic acid concentration:

Mukherjee and Choudhuri (1983) method will be followed for the determination of Ascorbic acid in plants. 0.5g of plant leaf was weighed by using a digital balance and added with 10ml of 6% trichloroacetic acid. The grinding was done on an ice bath. Care was taken while handling TCA as it caused skin burns. After the proper grinding, the extract was subjected to centrifugation at 10,000rpm for 10 minutes. Then 2ml of the extract was taken in a test tube and 0.5ml of DTC reagent (dinitro phenylhydrazine, thiourea, copper sulfate) was added to it. The test tubes were mixed well and placed at room temperature in dark for 3 hours. Then the test tubes were placed in an ice bath and the addition of 2ml of 65% ice-cold sulphuric acid was done. The chemicals were mixed well and the test tubes were again placed in the dark for another 30 minutes. The optical density was taken through a spectrophotometer at 520nm.

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