Estimation of starch by anthrone method
- 1. Jasmine Juliet .R Biochemistry Agricultural College & Research Institute, Madurai.
- 2. Estimation of starch by anthrone reagent Starch is an important polysaccharide. It is the storage form of carbohydrate in plants abundantly found in roots, tubers, stems, fruits and cereals. Starch, which is composed of several glucose molecules, is a mixture of two types of components namely amylose and amylopectin. Starch is hydrolysed into simple sugars by dilute acids and the quantity of simple sugars is measured colorimetrically.
- 3. Starch analysis by Anthrone method The anthrone method is one of the colorimetric methods used to determine starch concentration or total sugar content in food. Under acidic conditions, sugars react with anthrone reagents to produce a blue-greenish color. Heating needs to be conducted when reacting samples with sulfuric acid and reagents with the aim of accelerating the reaction rate, then cooled and measured its absorbance at 630 nm. Sugars reacted with sulfuric acid, it acts as a strong oxidizing agent.
- 4. Principle The sample is treated with 80% alcohol to remove sugars and then starch is extracted with perchloric acid. In hot acidic medium starch is hydrolysed to glucose and dehydrated to hydroxymethyl furfural. This compound forms a green coloured product with anthrone.
- 5. Estimation of starch by anthrone reagent
- 7. Blank colour is yellow. Standard colour intensity increases with standard concentration increases.
- 8. Materials Anthrone: Dissolve 200mg anthrone in 100mL of ice-cold 95% sulphuric acid 80% ethanol 52% perchloric acid Standard Glucose: Stock – 100mg in 100mL water. Working Standard – 10ml of stock diluted to 100mL with water.
- 9. Procedure Homogenize 0.1 to 0.5g of the sample in hot 80% ethanol to remove sugars. Centrifuge and retain the residue. Wash the residue repeatedly with hot 80% ethanol till the washing do not give color with anthrone reagent. Dry the residue well over a water bath. To the residue add 5.0mL of water and 6.5mL of 52% perchloric acid. Extract at 0°C for 20min. Centrifuge and save the supernatant. Repeat the extraction using fresh perchloric acid. Centrifuge and pool the supernatant and make up to 100mL.
- 10. Procedure Pipette out 0.1 or 0.2mL of the supernatant and make up the volume to 1mL with water. Prepare the standards by taking 0.2, 0.4, 0.6, 0.8 and 1mL in each tube with water. Add 4mL of anthrone reagent to each tube. Heat for eight minutes in a boiling water bath. Cool rapidly and read the intensity of green to dark green color at 630nm.
- 12. S.No Vol. of w.std (ml) Conc. of w.std (μg) Vol. of water (ml) Vol. of anthrone reagent (ml) Keep in boiling water bath for 8 minutes OD at 630nm Blank S1 S2 S3 S4 S5 T1 T2 Observation and Calculation
- 13. Calculation Find out the glucose content in the sample using the standard graph. Multiply the value by a factor 0.9 to arrive at the starch content. Calculate the starch percentage by using the formula, Starch content = Glucose content x 0.9.
- 14. References Hodge, J E and Hofreiter, B T (1962) In: Methods in Carbohydrate Chemistry (eds Whistler, R L and Be Miller, J N) Academic Press New York. Thayumanavan, B and Sadasivam, S (1984) Qual Plant Foods Hum Nutr 34 253.
- 15. Video link • https://www.youtube.com/watch?v=pXAf1QO BxhI • https://www.youtube.com/watch?v=C6IloM6 mQgE