Methodology for Carbohydrates

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 sugards by dilute acids and the quantity of simple sugars is measured colorimetrically.




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.



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.


Procedure

1.

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.

2.

To the residue add 5.0mL of water and 6.5mL of 52% perchloric acid.

3.

Extract at 0°C for 20min. Centrifuge and save the supernatant.

4.

Repeat the extraction using fresh perchloric acid. Centrifuge and pool the supernatant and make up to 100mL.

5.

Pipette out 0.1 or 0.2mL of the supernatant and make up the volume to 1mL with water.

6.

Prepare the standards by taking 0.2, 0.4, 0.6, 0.8 and 1mL in each tube with water.

7.

Add 4mL of anthrone reagent to each tube.

8.

Heat for eight minutes in a boiling water bath.

9.

Cool rapidly and read the intensity of green to dark green color at 630nm.





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.



Readings

1. 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.
2. Thayumanavan, B and Sadasivam, S (1984) Qual Plant Foods Hum Nutr 34 253.