*2.4.5. Estimation of carbohydrate by Anthrone method*

Carbohydrates are first hydrolyzed into simple sugars using hydrochloric acid to form furfural. This compound condenses with anthrone to form a green colored complex which can be measured by using colorimetrically at 620 nm.

**Procedure:** (1) From 30 ml of stock solution, take 0.5 ml of test sample in a test tube. (2) Make up volume of 1 ml with distilled water then add 4 ml of Anthrone reagent. (3) Heat the tubes in a boiling water bath for 10 min, the color changes from blue to green after boiling. (4) Prepare the working standards by taking 0, 0.2, 0.4, 0.6, 0.8 and 1 ml, where '0' serves as blank. (5) All the tubes including the sample tubes by adding distilled water and make up the total volume to 1 ml. (6) After adding the 4 ml of anthrone reagent. (7). Ten minutes of heat is provided in a boiling water bath. (8) After the tube is cooled rapidly and read the absorbance at 620 nm the green to dark green color. (9) The plotting concentration of the standard on the X-axis versus absorbance on the Y-axis and draw the standard graph. (10) The amount of carbohydrates present in the sample tube find out from the graph calculate.
