Ninhydrin Test

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What is the Ninhydrin Reaction?

Organic chemistry consists of several tests for qualitative analysis of the compounds. One such test is the ninhydrin test reaction. This test consists of a chemical reaction that determines whether a sample compound contains amines or alpha-amino acids. The main reactant in this process is ninhydrin, which is a hydrocarbon with the formula C9H6O4. By IUPAC nomenclature standards, ninhydrin is also called 2,2-di-hydroxyindane-1,3-dione. This chemical is added to a solution of the sample compound. The marker for a positive ninhydrin test is a deep blue colouration obtained in the solution. This reaction indicates the presence of amino acids, other amines and ammonia in the test sample. In this article, we will discuss the ninhydrin test reaction in detail. 

Ninhydrin Test Reaction 

The chemical reaction involved in the ninhydrin reaction mechanism is as shown below. The illustration portrays a positive ninhydrin test. 

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The reaction takes place between the amino group in the sample compound and the ninhydrin reagent. The end product formed is similar to di-ketohydrin. Di-ketohydrin has a characteristic deep blue pigmentation which we often name as Ruhemann's purple. 

Ninhydrin Test Principle

The ninhydrin reaction is essentially a redox reaction. Here ninhydrin acts as an oxidizing agent, and itself gets reduced. Ninhydrin reacts with the amino group of the free amino acid in the test sample and oxidizes the compound, leading to deamination. In this reaction, two gases get released. These are ammonia (NH3) and carbon dioxide (CO2). Besides the gases, we obtain an aldehyde and hydrindantin, which is formed by the reduction of ninhydrin. Now, the released ammonia further reacts with the ninhydrin giving rise to di-ketohydrin, which forms a coloured complex. This coloured complex is what we call Ruhemann's purple, and this gives our solution the deep blue colouration. This process is the ninhydrin test mechanism.

There are other variations of the ninhydrin test reaction, such as:

  • If the test sample contains proteinogenic amino acid such as proline, the colouration obtained is yellow.

  • If we use the reagent asparagine instead of ninhydrin, we obtain a brown coloured complex. 

Ninhydrin Test Procedure 

The following are the steps we carry out to run the ninhydrin test:

  • We begin with a 2% solution of ninhydrin which we prepare by dissolving 0.2g of ninhydrin per 10ml of a carrier solvent such as acetone or ethanol. 

  • Next, we prepare a 1% solution of the test compound using distilled water. To this, we add a few drops of our ninhydrin solution. 

  • Next, we place our test tube in a warm water bath for a few minutes. 

  • If the solution develops a deep blue or purplish colour, we have a positive ninhydrin test. 

Interpretation of Ninhydrin Test Mechanism and Reaction 

  • If our test sample contains ammonia, a primary or secondary amine or any amino acid heteroatom, then the ninhydrin test reaction will yield a Ruhemann's purple colouration. 

  • For compounds such as the likes of hydroxyproline or proline, the colouration we obtain is yellow. 

  • We obtain a brown colour when we use asparagine as a reactant. 

  • If carrying out the above procedure does not lead to colour change, this means that the test sample does not contain an amino group. 

Solved Examples 

Q. Explain What You Mean By Ninhydrin Test.

Answer: The ninhydrin reaction is a qualitative analysis test of hydrocarbons. In this test, we use the reagent known as ninhydrin which is a compound with the formula C9H6O4. This reagent acts as an oxidizing agent for compounds containing an amino group. This test is essentially meant to verify the presence of an amino group in the given test sample. On obtaining a positive ninhydrin test result, we get a blue colouration which we call Ruhemann's purple. 

FAQ (Frequently Asked Questions)

Q1. Explain the Procedure of Carrying Out the Ninhydrin Reaction.

Answer: The objective of carrying out a ninhydrin test is to verify the presence of an amino group in the given compound. To carry out the ninhydrin reaction, we will require ninhydrin reagent, test tubes, a carrier solvent such as ethanol, distilled water, spatula, a warm water bath and the test sample. The steps to carry out the reaction are as follows:

  • We prepare a 2% solution of ninhydrin using a carrier solvent such as acetone or ethanol. We do this by taking 0.2g of ninhydrin and dissolve it in 10ml of the solvent. 

  • Next, we prepare a solution of the given test compound using distilled water. 

  • We transfer the test solution to a test tube, and to this, we add a few drops of the ninhydrin solution. 

  • We let the test tube rest for a few minutes in a water bath at a slightly higher temperature. 

  • If the colour of the solution changes to a deep blue colouration, we have a positive ninhydrin test result. 

Q2. Discuss the Mechanism of the Ninhydrin Reaction.

Answer: The ninhydrin reaction mechanism is essentially an oxidation and reduction reaction process. When we add drops of the ninhydrin solution to the given test sample, the ninhydrin acts as an oxidizing agent. It reacts with the amino group of the compound, leading to delamination. This process results in the release of two gaseous products, namely, ammonia and carbon dioxide. This redox reaction reduces the ninhydrin and forms a reduced product known as hydrindantin, besides forming an aldehyde. Now the released ammonia reacts with another molecule of ninhydrin, forming a di-ketohydrin complex which has a deep blue colouration. This colour verifies the presence of amines and gives us a positive ninhydrin test.