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Tuesday, April 26, 2011

Determination of Total Carbohydrate 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 themonosaccharides which cannot be split by hydrolysis into more simpler sugars. The carbohydrate content can be measured by hydrolysing the polysaccharides into simple sugars by acid hydrolysis and estimating the resultant monosaccharides.

Carbohydrate source


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 green coloured product with an absorption maximum at 630 nm.

MATERIALS
  •  2.5 N HCl
  • Anthrone reagent: Dissolve 200 mg anthrone in 100 mL of ice-cold 95% H2SO4. Prepare fresh before use.
  •  Standard glucose: Stock—Dissolve 100 mg in 100 mL water. Working standard—10 mLof stock diluted to 100 mL with distilled water. Store refrigerated after adding a fewdrops of toluene.
PROCEDURE

  1. Weigh 100 mg of the sample into a boiling tube.
  2. Hydrolyse by keeping it in a boiling water bath for three hours with 5 mL 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 100 mL and centrifuge.
  5. Collect the supernatant and take 0.5 and 1 mL aliquots for analysis.
  6. Prepare the standards by taking 0, 0.2, 0.4, 0.6, 0.8 and 1 mL of the working standard.'0' serves as blank.
  7. Make up the volume to 1 mL in all the tubes including the sample tubes by adding distilled water.
  8. Then add 4 mL of anthrone reagent.
  9. Heat for eight minutes in a boiling water bath.
  10. Cool rapidly and read the green to dark green colour at 630 nm.
  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

Amount of carbohydrate present in 100 mg of the sample
=   (mg of glucose / volume of test sample) X 100
NOTE:
Cool the contents of all the tubes on ice before adding ice-cold anthrone reagent.

REFERNECES

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.
2. Internet Sources