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Molisch’s Test- Principle, Molisch Reagent, Procedure, Example, Limitation and Applications, Practice Problems, FAQs

You must have gone through a large number of tests from the days you started schooling to till date, sometimes in the form of a surprise test and sometime in the form of a periodic examination. But what do you think about the importance of these tests?

Let me answer this question in simple words. When a test is taken, it helps to analyse the understanding of your concept.

Similarly in chemistry, there are a lot of tests which are being performed. A chemical test is a qualitative or quantitative procedure for identifying, quantifying, or characterising a chemical compound or group.

Molisch's test is a sensitive chemical test for the presence of carbohydrates named after Austrian botanist Hans Molisch. Let us learn in this article the procedure to perform Molisch's test.


Table of Content

  • Principle of Molisch’s Test
  • Molisch Reagent
  • Molisch’s Test Procedure
  • Example of Molisch’s Test
  • Limitation of Molisch’s Test
  • Application of Molisch’s Test
  • Practice Problems
  • Frequently Asked Questions-FAQs

Principle of Molisch’s Test

Molisch's test is designed to detect the presence of carbohydrate molecules in a given test sample.

Molisch's test is based on the dehydration of carbohydrates by sulphuric acid. Pentoses on dehydration form furfural while hexoses produce hydroxy methyl furfural. The furfural reacts with aromatic phenol giving strong purple colour. When a carbohydrate-containing sample is treated with sulphuric acid, the hydroxyl group is removed from the sugar molecule. After the hydroxyl group is removed from a pentose sugar molecule, furfural is formed. The resulting furfural reacts with Molisch's reagent (sulphonated - naphthol) to produce a purplish red product.

Molisch Reagent:

  1. About 4 g of α-naphthol is dissolved in 25 ml of 99% Ethyl alcohol. This reagent has to be prepared afresh before the test.
  2. Concentrated sulphuric acid

Molisch test procedure:

Take about 2mL of water in a test tube.

Add the test sample to the test tube containing water and shake it to dissolve.

Add, 2 to 3 drops of the Molisch reagent.

Add concentrated sulphuric acid in drops along the sides of the test tube slowly.

The formation of a purple ring indicates the presence of carbohydrates.

Example of Molisch’s Test

The following example shows the Molisch test as used to detect D-glucose in a sample. D-glucose is reacted with ɑ- naphthol (Molisch reagent) and concentrated sulphuric acid.

D-Glucose loses hydroxyl groups in the form of water when concentrated sulphuric acid is added to the given solution. 5-(hydroxymethyl) furfural is formed after the loss of three water molecules. The furfural reacts with the sulfonated- naphthol present in the Molisch reagent. In the presence of the Molisch reagent, the furfural loses one more water molecule, resulting in the formation of an intermediate molecule. The intermediate molecule thus formed loses two electrons and one proton, yielding a purple or purplish-red product.

The presence of D-glucose in the given sample is confirmed by the appearance of this purple or purplish-red coloured product.

Let's look at how the product is made from an experimental standpoint. In a dry test, take 2ml of the sample solution containing D-glucose. Fill the test tube with 1ml of the Molisch reagent. Apply 1-2 drops of concentrated sulphuric acid to the test tube's walls. The formation of a purple ring indicates the presence of D-glucose in the given sample.

Limitation of Molisch’s Test

The test, however, has a limitation.

  1. Trioses and tetroses containing hydroxyl groups do not react. The Molisch test yields no results for triose and tetrose sugars. As a result, even if the given sample contains any triose or tetrose, the sample will still yield negative results.
  2. It may give positive results in the presence of any furfural forming chemicals like citric acid and lactic acid.
  3. It also gives a positive result on nucleic acids and glycoproteins.

Application of Molisch’s Test

  • The Molisch test is the first step in determining whether or not carbohydrate molecules are present in a given sample.
  • Molisch is used to detect sugars like glucose, fructose, and mannose, among others.
  • The Molisch test can detect all types of sugars, including monosaccharides, disaccharides, trisaccharides, and polysaccharides. As a result, if you want to test any sugar-free product, you can use the Molisch test.
  • However, apart from carbohydrates, the Molisch test can detect the presence of a variety of other substances such as nucleic acids and glycoproteins.

Practice Problems

Q1. Which of the following is correct for the composition of the Molisch reagent?

  1. It is typically made up of sulphonated - naphthol in ethanol solution.
  2. It is typically made up of Nitrated- naphthol in ethanol solution.
  3. It is typically made up of sulphonated or nitrated - naphthol in ethanol solution.
  4. It is typically made up of sulphonated and nitrated - naphthol in ethanol solution.

Answer:(A)

Solution: Aldehydes produce from carbohydrates by dehydration with concentrated acids like sulfuric acid or hydrochloric acid produces colour with Molisch reagent. Molisch reagent is typically made up of sulphonated - naphthol in ethanol solution. Therefore option (A) is correct.

Q2. Select the sugar which will show a negative test when the Molisch test is performed.

  1. Fructose
  2. Mannose
  3. Triose or tetrose
  4. Glucose

Answer:(C)

Solution: Molisch test is the most commonly used to detect the presence of sugars in an analyte. There are a few exceptions to the rule that all monosaccharides, disaccharides, trisaccharides, and polysaccharides pass the Molisch test.

The Molisch test yields no results for triose and tetrose sugars. Therefore option (C) is correct.

Q3. Select the correct option for the analyte which can be tested using the Molisch test.

  1. Molisch is used to detect sugars like glucose, fructose, Mannose etc.
  2. Molisch test can detect the presence of a variety of other substances such as nucleic acids and glycoproteins.
  3. Molisch test is used to determine the presence of protein in the given sample.
  4. Both A and B

Answer:(D)

Solution: Molisch test is the most commonly used to detect the presence of sugars in an analyte. There are a few exceptions to the rule that all monosaccharides, disaccharides, trisaccharides, and polysaccharides pass the Molisch test. The Molisch test yields no results for triose and tetrose sugars. Apart from carbohydrates, the Molisch test can detect the presence of a variety of other substances such as nucleic acids and glycoproteins. Therefore, option (D) is correct.

Q4. Select the correct option with respect to the Molisch test.

  1. Molisch's test is based on the principle of dehydration by sulphuric acid into furfural
  2. Molisch's test is the most commonly used to detect the presence of sugars in an analyte.
  3. Molisch test yields no results for triose and tetrose sugars
  4. All of the above

Answer: (D)

Solution: Molisch's test is based on the principle of dehydration of sulphuric acid into furfural. The Molisch test is the most commonly used to detect the presence of sugars in an analyte. There are a few exceptions to the rule that all monosaccharides, disaccharides, trisaccharides, and polysaccharides pass the Molisch test. The Molisch test yields no results for triose and tetrose sugars. Apart from carbohydrates, the Molisch test can detect the presence of a variety of other substances such as nucleic acids and glycoproteins. Therefore, option (D) is correct.

Q5. When concentrated hydrochloric or sulphuric acid is added to Molisch's test, the carbohydrate (if present) dehydrates, resulting in the formation of

  1. Ketone
  2. Sucrose
  3. Aldehyde
  4. Maltose

Answer: C

Solution: Aldehydes condenses with phenols and naphthols to form colours. Carbohydrates are dehydrated to aldehydes by concentrated acids like sulphuric and hydrochloric acids.

Frequently Asked Questions-FAQs:

Q1. How positive result of the Molisch test indicated?

Answer: Two drops of the Molisch reagent (ɑ-naphthol solution in 99 per cent by volume ethanol) are added. The solution is then slowly poured into a tube containing two millilitres of concentrated sulfuric acid, forming two layers. A positive test is indicated by the formation of a purple product at the interface of the two layers.

Q2. What are the precautions to be taken in performing Molisch’s test?

Answer: Few precautions that should be taken in Molisch’s test are:

  • Drop the acid into the test tube through the test tube's wall. This will prevent the acid from reacting quickly, and your entire procedure will remain smooth and simple.
  • Handle the strong acid with caution because it is extremely harmful to the skin.
  • Shaking causes the reddish violet ring to disappear, so never disturb it once it has formed.

Q3. How does Molisch’s test give a positive test for polysaccharides, glycoprotein and nucleic acid?

Answer: All carbohydrates, barring trioses and tetroses, gives a positive test. Nucleic acids and glycoproteins also give a positive test under severe conditions of dehydration..

Q4. What is the other test that can be to determine the sugar present in the sample?

Answer: Few other tests that can determine the sugar present in the sample are:

  • Fehling’s test: This test is performed by reducing sugar levels. Fehling's solution is added to the aqueous carbohydrate solution and heated in a water bath. The presence of reducing sugars is confirmed by the formation of a red precipitate.
  • Benedict’s test: This test is performed by reducing sugar levels. Glucose is converted into an enediol in an alkaline medium. The diol reduces cupric to cuprous ions, forming a red cuprous hydroxide precipitate.
  • Tollen’s test: This test is performed by reducing sugar carbohydrates. Tollens reagent forms form a silver mirror on the test tube's inner walls. This affirms the presence of reducing sugars. Silver ions are transformed into metallic silver.

Q5. Is the Molisch test positive for starch?

Answer: Molisch test results are positive for all carbohydrates (monosaccharides, disaccharides, and polysaccharides). It works by dehydrating the carbohydrate with Sulphuric acid to make an aldehyde, which then condenses with two molecules of ɑ-naphthol to form a purple ring at the interface.

Q6. What are the indications of a positive Molisch test result?

Answer: Two drops of Molisch reagent (a 99 per cent ethanol solution of ɑ-naphthol) are added. After that, the solution is slowly poured into a tube holding two ml of pure sulfuric acid, forming two layers. The creation of a purple product at the interface of the two layers indicates a positive test.

Q7. What is the purpose of Molisch's test?

Answer: Molisch's test determines the presence of carbohydrates in an analyte using a chemical reaction. Hans Molisch, a Czech-Austrian botanist, is the inventor of this test.

Q8. With Molisch's test, which carbohydrate yielded a positive result?

Answer: The Molisch test is passed by all carbohydrates (monosaccharides, disaccharides, and polysaccharides). It works by dehydrating the carbohydrate to produce an aldehyde, which then condenses with two molecules of -naphthol to form a purple ring at the interface.

Related Topics

Disaccharides

Glucose-osazone formation

Polysaccharides-Glycogen

Monosaccharides-Cyclic structure of glucose and fructose

Biomolecules-Classification

Classification of Protein

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