Determination of total carbohydrates by anthrone method

Carbohydrates are the important components of storage and structural materials in the plants. They exist as free sugars and polysaccharides. The basic units of carbohydrates are the monosaccharides which cannot be split by hydrolysis into more simpler sugars. The carbohydrate content can be measured by hydrolyzing the polysaccharides into simples sugars by acid hydrolysis and estimating the resultant monosaccharides.

Principle
Carbohydrates are first hydrolysed into simple sugars using dilute hydrochloric acid. In hot acidic medium glucose is dehydrated to hydroxymethyl furfural. This compound forms with anthrone a gree colored product with an absorption maximum at 630nm.

Materials

⇒ 2.5 N-HCl

• Anthrone Reagent: Dissolve 200mg anthrone in 100mL of ice cold 95% H₂SO₄. Prepare fresh before use.
• Standard Glucose: Stock – Dissolve 100mg in 100mL water. Working standard – 10mL of stock diluted to 100mL with distilled water. Store refrigerated after adding a few drops of toluene.

Procedure

1. Weigh 100mg of the sample into a boiling tube.
2. Hydrolyse by keeping it in boiling water bath for 3 hours with 5mL of 2.5 N-HCl and cool to room temperature.
3. Neutralise it with solid sodium carbonate until the effervescence ceases.
4. Make up the volume to 100mL and centrifuge.
5. Collect the supernatant and take 0.5 and 1mL aliquots for analysis.
6. Prepare the standards by taking 0, 0.2, 0.4, 0.6, 0.8 and 1mL of the working standard. ‘0’ serves as blank.
7. Make up the volume to 1mL in all the tubes including the sample tubes by adding distilled water.
8. Then add 4mL of anthrone reagent.
9. Heat for eight minutes in a boiling water bath.
10. Cool rapidly and read the green to dark green color at 630nm.
11. Draw a standard graph by plotting concentration of the standard on the X-axis versus absorbance on the Y-axis.
12. From the graph calculate the amount of carbohydrate present in the sample tube.

Calculation
Note
Cool the contents of all the tubes on ice before adding ice-cold anthrone reagent.

Reading

1. Hedge, J E and Hofreiter, B T (1962) In: Carbohydrate Chemistry 17 (Eds Whistler R L and Be Miller, J N) Academic Press New York.