Molisch test is a group test for all carbohydrates, whether free or in combined form. Despite its limitations, it is routinely used to detect the presence of carbohydrates.
The reaction is based on the fact that concentrated H2SO4 catalyses the dehydration of sugars to form furfural (from pentoses) or hydroxymethyl furfural (from hexoses).These furfurals then condense with sulfonated alpha-naphthol to give a purple or violet coloured product (furfuryl-diphenyl-methane-dyes). Polysaccharides and glycoproteins also give a positive reaction. In the event of the carbohydrate being a poly- or disaccharide, the acid first hydrolyses it into component monosaccharides, which then get dehydrated to form furfural or its derivatives.
Importance of molisch’s test
Molisch test is very important because it is a general test for the presence of carbohydrates.It is used widely as compared to other tests because before every specific test for different carbohydrates a general test is important and that general test is performed via molisch test.Another important thing is that, this test is positive for all types of carbohydrates whether free or in combined form like glycoprotiens,glycolipids etc.
- i) Conc.H2SO4
- ii) Molisch’s reagent: Alpha-naphthol 5%(w/v) in 95% ethanol
- Take 1-2 mL of sample solution and add 2-3 drops of Molisch’s reagent (5% α-naphthol in 95% ethanol) and mix the contents.
- Incline the tube and carefully pour 1-2 mL of conc.H2SO4 down the side of tube so that the acid forms a layer beneath the aqueous solution.
- Observe the color at the interface between two layers and compare your result with a control test
- The formation of a purple or violet ring or zone at the junction of two layers indicates the presence of carbohydrates.
- Apply this test two different carbohydrate solutions of your own choice, preferably to one monosaccharide and one polysaccharide.
- A brown color due to charring must be ignored and the test should be repeated with a more dilute sugar solution.
- Alpha-naphthol solution is unstable and should be prepared fresh.
- The conc.H2SO4 should be added carefully along the sides of the test tube causing minimal disturbance to the contents of the tube.
- Add only few drops of molisch’s reagent. We don’t need to add much.
- Add acid with great care as we use strong acid in this test and it could burn our skin.
- Don’t shake the test tube when ring is formed. If we do so, it would destroy the ring.
In addition to carbohydrates, furfurals as such, some organic acids, aldehydes and ketones also give this test. Secondly, a concentrated sugar solution may give a red colour instead of purple owing to charring action of acid.