This is a test that is common for all carbohydrate larger than the tetroses. The test works on the basis that pentoses as well as hexoses can be dehydrated using conc. sulfuric acid, resulting in furfural or hydroxyl-methyl furfural or hydroxyl methyl furfural. These compounds condense with a-naphthol to create a purple condensation product.
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.
Objectives of Molisch Test
- To identify carbohydrate content in a particular sample.
- To differentiate the carbohydrates and other biomolecules.
Molisch’s Test Principle
In Molisch’s experiment, the carbohydrates (if present) is dehydrated upon the introduction of concentrated hydrochloric or sulfuric acid, leading to creating an aldehyde. This aldehyde experiences condensation with the -naphthol in the reagent. This results in the creation of a purple or reddish-purple-coloured complex.
Molisch’s Test Reaction
Monosaccharides disaccharides and polysaccharides (except trioses as well as tetroses)will result in positive reactions, and glycoproteins as well as nucleic acids have a positive reaction since all of these compounds are hydrolyzed into monosaccharides with strong mineral acids. Pentoses are then dehydrated to furfural, while hexoses are dehydrated to 5-hydroxymethylfurfural. Any of these aldehydes when present they will be condensed with 2 molecules of a-naphthol and produce a purple-colored compound as shown in the glucose example:
- 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
- Test tubes
- Test tube stand
- Distilled water
Molisch’s Test Procedure
- Pour 2 ml of each distilled water , and test sugar solutions within four test tubes in a row.
- Two drops of Molisch Reagent to each tube.
- Place the test tube in an incline position and slowly add 1 ml concentrated H2SO4 on the inside of the test tube.
- 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.
- Check the test tube to see the appearance of a purple-colored circle in the layer between the acid and the solution.
A violet-colored ring forms in the intersection of two fluids i.e. the Molisch’s Reagent solution and sulfuric acid.
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.
Things to Keep in Mind
- For a test to be considered a success the carbohydrate should contain at minimum five carbons. (It is this because it’s the result of furfural derivatives containing 5 carbonatoms.)
- Impurities in the reagent show a green colors, which indicate a false-negative test.
- Oligosaccharides and polysaccharides first broken down into monosaccharides using acid, which gives the Molisch’s test a positive.
- Proteins and lipids that have an attached carbohydrate may result in a positive test.
- 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 a 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.
- Tiwari A. (2015). Practical Biochemistry. LAP Lambert Academic Publishing.