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Experiment 5-Carbohydrate Estimation by Anthrone Method

The document outlines the procedure for estimating total sugars in a sample using the Anthrone method, which involves hydrolyzing carbohydrates into monosaccharides and reacting them with anthrone reagent to produce a colorimetric response. It details the necessary equipment, reagents, and step-by-step methodology for conducting the experiment, including the preparation of standard solutions and the calculation of carbohydrate content. The final result indicates the amount of sugar present in the sample, expressed in milligrams per 100 milligrams of the sample tested.

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Sakshi Khole
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131 views3 pages

Experiment 5-Carbohydrate Estimation by Anthrone Method

The document outlines the procedure for estimating total sugars in a sample using the Anthrone method, which involves hydrolyzing carbohydrates into monosaccharides and reacting them with anthrone reagent to produce a colorimetric response. It details the necessary equipment, reagents, and step-by-step methodology for conducting the experiment, including the preparation of standard solutions and the calculation of carbohydrate content. The final result indicates the amount of sugar present in the sample, expressed in milligrams per 100 milligrams of the sample tested.

Uploaded by

Sakshi Khole
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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Experiment 5

Estimation of Total Sugars by Anthrone Method

Aim: To determine the total carbohydrate in the given sample by the Anthrone method

Theory/Principle: Carbohydrates are first hydrolyzed into simple sugars using dilute
hydrochloric acid. In a hot acidic medium, glucose is dehydrated to hydroxymethylfurfural
Active form of the reagent is anthranol, the enol tautomer of anthrone, which reacts by
condensing with the carbohydrate furfural derivative to give a green colour in dilute and a blue
colour in concentrated solutions, which isdetermined colorimetrically. The blue - green
solution shows absorption maximum at 630 nm.
Reaction:
(i) Hydrolysis to monosaccharides
Disaccharide Monosaccharide
(ii) Dehydration product is a furfural
Monosaccharide Furfural
(iii) Reaction of furfural with anthrone
Furfural + Anthrone reagent Blue green complex
Requirements:
(i) Equipments:
• UV Spectrophotometer
• Vortex mixer
• Mantle heater/Water Bath.
(ii) Chemicals/Reagents:
• 2.5 N HCl
• Ice cold water
• Sodium carbonate
• 95% H2SO4
• Anthrone Reagent
• Glucose
• Other carbohydrates as sample .
(iii) Glass wares and others:
• Test tube, Test tube stand, Pipettes, Beaker, Ice, Test tube caps, Tissue paper, Wash
bottle.
(iv) Reagents:
(i) Anthrone reagent: Dissolve 200 mg anthrone in 100 ml of ice cold 95% H2SO4. Prepare
fresh before use.
(ii) Glucose stock solution: Stock- Weigh 1 g of glucose and dissolve it carefully in to100
ml of distilled water in a standard flask.
Working standard- Dilute 10 ml of stock solution in 100 ml with distilled water. Store
refrigerated after adding a few drops of toluene.
Procedure:
Sample (unknown preparation)
1. Weigh 200 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 as unknown keep
them aside
6. Take 0, 0.2, 0.4, 0.6, 0.8 and 1 ml of working standard in a test tube. ‘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 10 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 the standard concentration on the X-axis vs.
Absorbance on the Y-axis.
12. From the graph calculate the amount of carbohydrate present in the sample tube.
Observation Table
Tubes Working Diluent Anthrone Boiling water OD at 630
standard ml (distilled reagent bath nm
µg/ml water)
200 0.2 0.8 4ml

400 0.4 0.6 4ml

600 0.6 0.4 4ml

800 0.8 0.2 4ml

Blank 0 1 4ml 10 min

Unknown 4ml

Calculation:
By graph :
Volume of unknown (sample) = ----ml
OD (Absorbance ) of the unknown sample = Amount of glucose in ---ml of unknown (sample)
= x µg = --- µg
Therefore, amount of glucose in 1 ml of sample = x µg/ Volume of unknown (sample) = y µg
Amount of glucose in 200 mg of the sample = y µg x 100 ( 100 is the dilution factor) = z µg
Amount of carbohydrate present in 100 mg of sample = (mg of sample / volume of test sample)
x 100
𝑧𝑧 µg
Amount of carbohydrate present in 100 mg of sample= �200 𝑔𝑔(𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝑜𝑜𝑜𝑜 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡)� 𝑥𝑥 100 𝑔𝑔

= a µg = ----- µg
= mg
Result :

The amount of sugar in the -------------sample is _____ mg in 100 mg sample.

Conclusion:

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