Molisch Test- Definition, Principle, Procedure, Result, Uses

Molisch test is a group test for all carbohydrates, either free or bound to proteins or lipids. It is a sensitive test that requires precision for the detection of carbohydrates.

Objectives of Molisch Test

  • To detect the presence of carbohydrates in a given sample.
  • To distinguish carbohydrates from other biomolecules.

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Principle of Molisch Test

Principle of Molisch Test

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  • The reaction is based on the fact that the concentrated acid catalyzes the dehydration of sugars to form furfural (from pentoses) or hydroxymethylfurfural (from hexoses).
  • Either of these aldehydes condenses with two molecules of naphthol to form a purple or violet colored complex at the interface of the acid and test layer.
  • If the carbohydrate is poly- or disaccharide, a glycoprotein or glycolipid, the acid first hydrolyses it into component monosaccharides, which get dehydrated to form furfural or its derivatives.
  • A green ring might be observed if any impurities are present in the reagent as they might interact with the α-naphthol and the acid.
  • A rind ring is seen if a concentrated sugar solution is used. This might be due to the charring of the sugar due to the acid.


1. Reagent

  • Molisch reagent: Dissolve 3.75 g of α-naphthol in 25 ml of Ethanol 99%. This reagent should be prepared fresh.
  • Concentrated sulphuric acid
  • Test sample

2. Materials required

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

Procedure of Molisch Test

  1. Take 2 ml of each distilled water and test sugar solutions in four test tubes separately.
  2. Add two drops of Molisch reagent to each tube.
  3. Hold the test tube in an inclined position and gently add 1 ml concentrated H2SO4 along the wall of the test tube. Do not mix the acid with the solution. A black ring may form if concentrated acid is not added slowly as the heat generated from the reaction can char the carbohydrates.
  4. Observe the test tube for the formation of a purple-colored ring at the layer between the solution and the acid.

Result and Interpretation of Molisch Test

Molisch Test
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  • The formation of the purple colored ring occurs at the interface between the sulphuric acid and the test solution.
  • The sulphuric acid remains above the test solution as the acid is denser than the test solution.
  • The absence of color indicates a negative result.

Uses of Molisch Test

  • Molisch test is used to detect the presence of carbohydrates in different samples.
  • It can be used to detect the formation of carbohydrates as a by-product in different reactions and distinguish it from other biomolecules.

Limitations of Molisch Test

  • Trioses and tetroses do not have the necessary five carbon atoms for furfural formation, so they do not give a positive result for this reaction.
  • Molisch test is not a specific test for carbohydrates. Furfurals as such or furfural yielding substance, some organic acids like citric acids, lactic acid, oxalic acid, formic acid, etc. can give a positive result.

References and Sources

  1. Tiwari A. (2015). Practical Biochemistry. LAP Lambert Academic Publishing.
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About Author

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Anupama Sapkota

Anupama Sapkota has a bachelor’s degree (B.Sc.) in Microbiology from St. Xavier's College, Kathmandu, Nepal. She is particularly interested in studies regarding antibiotic resistance with a focus on drug discovery.

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