Quantitation of Carbohydrates by Anthrone method.

Quantitation of Carbohydrates by Anthrone method.                                    
                                                                                                                  Khadga Bikram Angbuhang
                                                                                                         GoldenGate International College

Principle:

Anthrone reaction is the basis of a rapid and convenient methods for the determination of hexose, aldopentoses either in free form or in when present as polysaccharide. The reaction is not suitable if protein containing large amount of tryptophan is present, since a red color is obtained under these conditions.

In this assay, carbohydrates are dehydrated by using conc. H₂SO₄ to form furfural and its derivatives, which in turn condenses with anthrone to form a bluish green complex with an absorption maximum at 620 nm.

D- glucose + conc. H2SO4                              Hydroxymethyl furfural +3H2O

Hydroxymethyl furfural + Anthrone                              Bluish green complex

Materials and reagents:

1.  Anthrone reagents
2.  Conc. H₂SO₄ 
3. Glucose standard 50 μg/ml
4. Sample carbohydrates (Carbohydrates) 

             Procedure:

1. Take 3 test tubes and label as Test, Standard and Blank.
2.  Add 1 ml Glucose sample in test labelled test tube, 1 ml glucose standard to standard test tube and 1 ml D/W (protein free) to blank.
3. Add 4 ml of anthrone reagent to each tubes.
4. Place all tubes in a boiling water bath for 10 minutes.
5. Cool and read the absorbance at 620 nm against the blank.

Absorbance of sample X concentration of standard calculate the concentration as:

 Concentration glucose (μg/ml):              Absorbance of Standard.