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Anthrone reagent is a 2 g/L solution of anthrone in concentrated H2SO4. It is most frequently used to measure the concentration of not only carbohydrates in a solution but also free hexoses and aldopentoses including sugar acids such as hexuronic acids.
Anthrone reagent reacts with the carbohydrates and produces blue-green color that shows maximum absorbance at 620 nm. However, you must have a calibration curve prepared with hexoses or pentoses of known concentrations treated with anthrone reagent. Then, using the calibration curve, you can measure the carbohydrate solution of unknown concentration.
Prepare glucose solutions of different known concentrations. Now, add 4 ml anthrone reagent to 1 ml of glucose solutions of known concentrations in a set of test tubes and cover the mouth with marble balls and boil the mixture in a water bath for 10 minutes and measure the absorbance at 620 nm. Prepare a standard calibration curve and do the same with glycogen and get the standard calibration curve for the glycogen.
Now, add 4 ml of anthrone reagent into 1 ml of the carbohydrate test solution and mix well. Boil the reaction mixture on the water bath for 10 minutes covering the mouth of the test tube with a marble ball. Then allow the test tube to be cool and read the absorbance at 620 nm and compare the color with standards and measure the concentration using standard curves.
However, you should be careful that the carbohydrate solution must be protein-free otherwise, you will get a false result. This is because the presence of tryptophan in proteins may react with anthrone and produce red color that will interfere with the reading at 620 nm.