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Date: ……/..../….
PRACTICAL NO:- 2
PERFORM MOLISCH’S TEST
THEORY
Molisch’s Test is a type of chemical test that can be used to determine carbohydrate content in a
given sample. Molisch’s test is named in honor of the Czecho-Austrianian botanist Hans
Molisch, who is recognized as the one who discovered it. Molisch’s test is based on the addition
to Molisch’s reagent (a solution of – naphthol and alcohol) in the test analyte. This is followed
by the addition of a small amount in concentrated H2SO4 (sulphuric acid) to the mix.
The appearance of a purple or a red-purplish ring at the junction of H2SO4, the analyte, and
Molisch’s reagent mix confirms the presence of carbohydrates within the analytical sample.
OBJECTIVE
To identify carbohydrate content in a particular sample.
To differentiate the carbohydrates and other biomolecules.
PRINCIPLE
Molisch’s test is a general test for all carbohydrates. In this test, carbohydrates when reacted with
conc. H2SO4 get dehydrated to form furfural and its derivatives.
When monosaccharide are treated with conc H2SO4, -OH group of sugar are removed in the form
of water and furfural is formed from pentose sugar and hydroxymethyl furfural is formed from
hexose sugar. These products reacts with sulphonated α- naphthol to give a purple (violet red)
colored complex.
Prepared By: - Ashok C. Keshari
HOD, Food & Dairy Tech.
NLPI, Bardibash
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REQUIREMENTS
Reagent
Molisch reagent: Dissolve 3.75 grams of a-naphthol within 25 milliliters of Ethanol
99.99%. This particular reagent needs to be prepared in a fresh state.
Concentrated sulphuric acid
Test sample
Materials Required
Test tubes
Test tube stand
Pipette
Distilled water
PROCEDURE
1. Take 2ml of sample in clean and dry test tube.
2. Take 2ml of distilled water in another tube as control.
3. Add 2-3 drops of Molisch’s reagent slowly to the solution.
4. Gently pipette 1ml conc. H2SO4 and slowly along the side of the tube so that two distinct
layers are formed.
5. Don’t mix the acid and the solution (A black ring can develop if concentrated acid is not
added gradually because the heat produced by the reaction could cause charring of the
carbohydrates).
6. Observe color change at the junction of two layers.
7. Check the test tube to see the appearance of a purple-colored circle in the layer between
the acid and the solution.
Result Interpretation of Molisch’s Test
Monosaccharides disaccharides, polysaccharides, and disaccharides (except trioses as well as
tetroses)are likely to cause an positive reaction. Likewise, glycoproteins and nucleic acids also
produce positive reactions, since all of these compounds are hydrolyzed into monosaccharides by
powerful mineral acids. Pentoses are then dehydrated to furfural, while hexoses are dehydrated
to 5-hydroxymethylfurfural. One or both of these aldehydes when present, will combine by
combining two molecules from a naphthol, to produce a product with a purple hue.
Positive result: If the test solution creates purple or purplish-red color layers, this indicates
carbohydrate presence. Therefore, the test sample has been found to be positive in Molisch
test. Molisch test.
Negative result: If the test solution doesn’t create purple or purplish-red color layers, this
indicates that the test solution doesn’t have carbohydrate molecules. Therefore, the test
sample does not pass this Molisch test.
Prepared By: - Ashok C. Keshari
HOD, Food & Dairy Tech.
NLPI, Bardibash
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A negative test (left) and a positive test (right)
OBSERVATIONS
A violet-colored ring forms in the intersection of two fluids i.e. the Molisch’s Reagent solution
and sulfuric acid.
RESULT AND DISCUSSION
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Prepared By: - Ashok C. Keshari
HOD, Food & Dairy Tech.
NLPI, Bardibash
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Precautions
Do not add too much Molisch’s Reagent.
Do not directly pour sulfuric acid into your solution. Otherwise, the charring of
carbohydrates could take place with a black-colored ring could develop, which will give an
inaccurate negative test.
Pour the acid into your test tube, dropping it on the inside in the tube. The acid won’t be able
to react rapidly and the whole process remains smooth and easy.
Take care when handling strong acids because it can be very harmful for the skin.
Do not disturb the violet-reddish red the ring after it has formed, as the ring is shaken and
causes it to disintegrate.
Applications of Molisch’s Test
The Molisch test can be the primary test to be performed when one wishes to find out
whether carbohydrate molecules exist or not in the sample.
Molisch is used to identify sugars like mannose, fructose, glucose and many more.
Molisch tests can be used to identify all kinds of sugars like trisaccharides, disaccharides and
monosaccharides and polysaccharides. If you wish to evaluate any product that is sugar-free
it is possible to test it using this Molisch test.
Aside from carbohydrates Apart from carbohydrates, the Molisch test can be utilized to test
the presence of various other substances like glycoproteins and nucleic acid.
Limitations of Molisch test
As the Molisch test has been described previously as being the most important test that is used to
identify any sugars in an Analyte. But, the test has an issue.
However, while all monosaccharides and trisaccharides and disaccharides polysaccharides
are positive for the Molisch test. There are few exceptions. Trise and tetrose sugars will not
react with the Molisch test. Therefore, even if the sample contains all triose and tetrose but
the test will result in negative results.
Prepared By: - Ashok C. Keshari
HOD, Food & Dairy Tech.
NLPI, Bardibash
