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.
The reaction takes place between the amino group in the sample compound and the ninhydrin reagent. The end product formed is similar to di-dehydrin. Di-ketohydrin has a characteristic deep blue pigmentation which we often name 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 delamination. In this reaction, two gasses get released. These are ammonia (NH3) and carbon dioxide (CO2). Besides the gasses, 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 acids 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.
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.
FAQs on Ninhydrin Test
1. Explain the Procedure of Carrying Out the Ninhydrin Reaction.
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 color of the solution changes to a deep blue colouration, we have a positive ninhydrin test result.
2. Discuss the Mechanism of the Ninhydrin Reaction.
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 color verifies the presence of amines and gives us a positive ninhydrin test.
3. What is Ninhydrin?
The chemical ninhydrin (2,2-dihydroxybenzene-1,3-dione) is used to find ammonia with other primary and secondary amines. Ruhemann's purple is a deep blue or purple colour that is a consequence due to the interaction between interacting with these free amines. As the terminal amines of lysine residues in peptides and proteins sloughed off in fingerprints react with ninhydrin, it is most typically employed to identify fingerprints. At room temperature, it is a white solid that is soluble in ethanol and acetone. Indane-1,2,3-trione hydrate can be considered to be ninhydrin. Visit Vedantu’s website to learn more.
4. What are the applications of Ninhydrin?
In solid-phase peptide synthesis, ninhydrin can also be employed to monitor deprotection (Kaiser test). The C-terminus of the chain is connected to the solid support, whereas the N-terminus extends away from it. A ninhydrin test results in blue when that nitrogen is deprotected. Because amino-acid residues are connected with their N-terminus protected, the test yields a colourless or yellow result if the next residue is correctly coupled onto the chain.
A solution suspected of containing the ammonium ion can be examined with ninhydrin by dotting it onto a solid support (such as silica gel); if the solution contains this species, treatment with ninhydrin should result in vivid purple colour. The reagent can also be employed in thin layer chromatography (TLC) to analyse a chemical reaction (usually 0.2 per cent solution in either n-butanol or ethanol). It can identify practically all amines, carbamates, and amides on a TLC plate after rigorous heating.
5. How is a ninhydrin solution used during forensics?
Forensic investigators frequently employ a ninhydrin solution to examine latent fingerprints on porous materials like paper. The amino acids in the sweat secretions that collect on the distinctive ridges of the finger transmit to surfaces that are touched. When the surface is exposed to ninhydrin, the amino acids are converted into visibly coloured products, revealing the print. The long-term stability of the test solutions is poor, especially if they are not kept cool.
Before ninhydrin, a solution of 1,2-indandione and zinc chloride (IND-Zn) can be used to improve the ability of ninhydrin. This sequence causes more overall amino acid reactions, probably because IND-Zn aids in their release from the surface for the succeeding ninhydrin reaction.
6. How is the reactivity to ninhydrin?
The partial positive charge on a carbonyl's carbon atom is amplified by surrounding electron-withdrawing groups like carbonyl itself. As a result, the central carbon of a 1,2,3-tricarbonyl molecule is substantially more electrophilic than a ketone's central carbon. As a result, indane-1,2,3-trione rapidly interacts with nucleophiles, such as water. Because of the destabilising action of the neighbouring carbonyl groups, ninhydrin generates a stable hydrate of the central carbon, whereas most carbonyl compounds have a carbonyl form that is more solid and reliable than a product of water addition (hydrate).
The amine is condensed with a molecule of ninhydrin to give a Schiff base, which is then used to make the ninhydrin chromophore (2-(1,3-dioxolan-2-yl amino in an-1,3-dione). As a result, only ammonia and primary amines can pass through this stage. The Vedantu app and website contain free study materials.
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