Table of Contents
Anthrone test for carbohydrates post we briefly summarises about: principle, reagents requirements, procedure, result, application and limitations of anthrone test.
Anthrone Test for Carbohydrates
Anthrone test is a carbohydrate group test that provides a quick and easy method for quantifying carbohydrates that are either free or bound to any lipids or proteins. Cellulose, a primary structural polysaccharide in plants, is the most prevalent organic substance in nature, consisting of glucose units linked together in the form of the disaccharide cellobiose repeating units with multiple cross connections. It is also a significant component of many farm wastes.
If carbohydrate is present in the form of free carbohydrate (poly- or monosaccharide) or bound carbohydrate (glycoprotein or glycolipid), the concentrated acid in the anthrone test reagent first hydrolyzes it into component monosaccharide.
Similarly, the concentrated acid catalyses the monosaccharide dehydration to form furfural (from pentoses) or hydroxyl furfural (from hexoses). To form a blue-green complex, the furfural or hydroxyl furfural formed condenses with two molecules of naphthol from the anthrone test reagent. The complex can then be quantified using a spectrophotometer or a red filter colorimeter to measure the absorbance of 620 nm wavelength.
On treatment with 67 percent H2SO4, cellulose is acetolyzed with an acetic/nitric reagent, resulting in acetylated cellodextrins, which are dissolved and hydrolyzed to generate glucose molecules. This glucose molecule is dehydrated to make hydroxymethyl furfural, which reacts with anthrone test to produce a green-colored compound measured at 630 nm colour intensity.
Hydrolysis of polysaccharides to monosaccharide
Polysaccharide → Monosaccharides
Dehydration of monosaccharides to furfural
Monosaccharide → Furfural
Reaction of furfural with naphthol
Furfural + Anthrone test reagent (naphthol) → Blue-green complex
1. UV Spectrophotometer
2. Vortex mixer
3. Mantle heater/Water Bath
1. Anthrone test reagent
3. Other carbohydrates if desired
1. Test tubes
2. Test tube stand
4. Wash bottle
1. Acetic/nitric reagent: Add 150 ml of 80% glacial acetic acid and 15 ml of concentrated nitric acid.
2. Anthrone reagent: 200 mg of anthrone in 100 ml concentrated H2SO4. Prepare fresh and chill for 2 hours before use.
1. In a test tube, combine 3mL acetic/nitric reagent with a specified amount (0.5g or 1g) of the sample in a vortex mixture.
2. Place the tube in a 100°C water bath for 30 minutes. Cool for 15-20 minutes before centrifuging the contents and discarding the supernatant.
3. Allow 10mL of 67 percent sulphuric acid to sit for 1 hour after washing the residue with distilled water.
4. 1mL of the above mentioned solution is diluted to 100mL, and 10mL of anthrone test reagent is added to 1mL of this diluted solution and mixed thoroughly.
5. Heat the tubes for 10 minutes in a boiling water bath, then cool and measure the colour at 630nm. Calculate the concentration of Cellulose in the plant sample using a standard graph.
The existence of a blue-green complex in a solution suggests the presence of carbohydrates. We can deduce the concentration of unknown samples from the graph.
1. Anthrone test is used for the detection and quantification of carbohydrates in various samples like blood serum, milk, and its variation, etc.
1. Anthrone test gives a negative result in samples that are carbohydrates like D-glucose phenylosazone and D-glucose phenylosotriaole but do not form furfural or hydroxyfurfural in the dehydration step.