Molisch’s Test: Objective, Principle, Procedure, Result And Application

What Is Molisch’s Test?

Molisch’s test is a sensitive chemical test, named after Austrian botanist Hans Molisch, used for the test of presence of carbohydrates, based on the dehydration of the carbohydrate by sulfuric acid or hydrochloric acid to produce an aldehyde, which condenses with two molecules of a phenol, resulting in violet ring.

A positive reaction for Molisch’s test is given by almost all carbohydrates with exception of tetroses and trioses. It can be noted that even some glycoproteins and nucleic acids give positive results for this test (since they tend to undergo hydrolysis when exposed to strong mineral acids and form monosaccharides).

Objective of Molisch’s Test

  • To detect the presence of carbohydrate in samples.
  • To differentiate carbohydrates from other macromolecules, lipids and proteins.

Principle Of Molisch’s Test

Molisch test is a group test for all carbohydrates, whether free or in combined form. Despite its limitations, it is routinely used to differentiate carbohydrates from other macromolecules, lipids and proteins.

The reaction is based on the fact that concentrated Sulphuric acid 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.



  • Molisch reagent: Dissolve 3.75 g of α-naphthol in 25 ml of Ethanol 99%. This reagent should be prepared fresh.
  • Concentrated sulphuric acid H2SO4 (aq)
  • Test sample/solution: 5% glucose, 5% Sucrose and 5% Starch

Materials required

  • Test tubes
  • Test tube stand
  • Pipette
  • Distilled water

Molisch’s Test Procedure

  1. Take 2ml of sample in dry test tube
  2. Take 2ml of distilled water in another test tube as a control
  3. Add 2-3 drops of Molisch’s reagent to the solution
  4. Gently pipette concentrated sulphuric acid (H2SO4) drop-wise along the walls of the test tube into the test tube so as to facilitate the formation of a layer and avoid mixing.
  5. Observe color change at the junction of two layers.

Results Interpretation

  1. Positive Molisch’s Test:  The formation of a purple ring at the layer formed by the concentrated acid is a positive indicator for Molisch’s test.
  2. Negative Molisch’s Test: If no purple or reddish-purple color forms, the given analyte does not contain any carbohydrate.

What Is The Role Of Concentrated Sulphuric Acid In Molisch’s Test?

Concentrated Sulfuric acid is a strong dehydrating agent. In Molisch’s test it dehydrates sugar into hydroxy methyl furfural (from hexoses)/furfural (from pentoses) which would then condense with alpha-naphthol to give redish violet coloured ring-the response in the positive Molisch test.


  • Molisch test is a chemical test is used to detect carbohydrates (monosaccharide, disaccharide, and polysaccharide) and glycoprotein in an analyte.
  • It can be used to differentiate proteins and amino acids from carbohydrates. 

Limitation Of Molisch’s Test

The Molisch Test can give a false negative when carbohydrates are in fact present for many reasons. For example, the furfural aldehyde produced during the acid dehydration reaction step may be intereferred with in its condensation with the phenolic molecule by strong nucleophiles attacking the aldehyde. Furthermore, amino sugar residues do not dehydrate to give the proper product during acid addition. So, we generally don’t use this crude test in our laboratory as a qualitative method to verify the presence of carbohydrate in relatively complicated unpurified mixtures.