Estimation carbohydrates by colorimetric method

 Estimation carbohydrates by colorimetric method

 Principle: Carbohydrates are dehydrated by conc.H2SO4 to form furfural. The 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 is determined colorimetrically. The blue - green solution shows absorption maximum at 620 nm.

 Materials required:

 (i) Equipment:

• UV Spectrophotometer

• Vortex mixer

• Mantle heater/Water Bath.

 (ii) Chemicals/Reagents:

• Anthrone Reagent

• Glucose

• Other carbohydrates if desired

 (iii) Glass wares and others:

• Test tube, Test tube stand, Pipettes, Beaker, Ice Test tube caps, Tissue paper, Wash bottle.

 

(b) Reagents:

(i) Anthrone reagent: Dissolve 2g of Anthrone in 1 litre of concentrated H2SO4. Use freshly

prepared reagent for the assay

 (ii) Glucose stock solution: 200µg glucose per ml distilled water.

 Procedure:

1. Pipette out into a series of test tubes different volumes of glucose solution (follow up Table 1) from the supplied stock solution(200µg /ml) and make up the volume to 1 ml  with distilled water.

2. Consider tube 1 as blank and tubes 2 through 9 for the construction of a standard curve. Tubes 10-15 are for the unknown samples.

3. To each tube add 5 mL of the anthrone reagent (supplied) and mix well by vortexing.

4. Cool the tubes.

5. Cover the tubes with marbles/ Caps on top and incubate at 90 C for 17 minutes or

boiling water bath for 10 minutes.

6. Cool to room temperature and measure the optical density at 620 nm against a blank.

7. Prepare a standard curve of absorbance vs. µg glucose  

Calculation:

Determine the amount of glucose in the unknown sample by plotting a standard curve of A620 on Y-axis and µg of Glucose on X-axis.