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. What is the primary purpose of the Ninhydrin test in Chemistry?
The primary purpose of the Ninhydrin test is to detect the presence of ammonia, primary amines, and secondary amines. It is most famously used in biochemistry as a qualitative and quantitative test for amino acids, which are the building blocks of proteins. A positive test indicates the presence of these functional groups in a given sample.
2. What is the fundamental principle behind the Ninhydrin test?
The principle of the Ninhydrin test involves a sequence of chemical reactions. Ninhydrin, a powerful oxidising agent, reacts with the alpha-amino group of an amino acid in a process called oxidative deamination. This reaction releases ammonia, carbon dioxide, an aldehyde, and a reduced form of ninhydrin called hydrindantin. The liberated ammonia then reacts with another ninhydrin molecule and the hydrindantin to form a deep blue-purple complex known as Ruhemann's purple, which is the visible indicator of a positive test.
3. How is the Ninhydrin test performed in a laboratory?
The standard procedure for performing a Ninhydrin test is as follows:
- First, a small amount of the test sample is dissolved in a solvent like distilled water to create an aqueous solution.
- A few drops of Ninhydrin reagent (typically a 2% solution in acetone or ethanol) are added to this test solution in a test tube.
- The mixture is then gently heated in a water bath for about 2-5 minutes to facilitate the reaction.
- Finally, the test tube is observed for a characteristic colour change. The appearance of a deep purple or blue colour indicates a positive result.
4. Which compounds will give a positive result for the Ninhydrin test?
The Ninhydrin test gives a positive result for a wide range of compounds that possess a free amino group. This includes all alpha-amino acids (except for a few cases), peptides, and proteins. Additionally, it reacts with ammonia and other primary and secondary amines, making it a general test for these functional groups.
5. Why do proline and hydroxyproline give a yellow colour instead of purple with ninhydrin?
Proline and hydroxyproline are unique because they are technically imino acids, not amino acids. Their amino group is a secondary amine that is part of a rigid ring structure. This structural difference prevents the typical reaction pathway that forms the purple Ruhemann's complex. Instead, they form a different, unstable yellow-orange coloured product. This distinct result allows the Ninhydrin test to be used to differentiate proline and hydroxyproline from other primary amino acids.
6. Can the Ninhydrin test be used to detect proteins?
Yes, the Ninhydrin test can be used to detect proteins. Proteins are long chains of amino acids linked by peptide bonds. The test reacts with the free amino group at the N-terminus of the protein chain, as well as with the free amino groups in the side chains of certain amino acids like lysine. Therefore, a solution containing protein will yield a positive purple colour, confirming the presence of these accessible amino groups.
7. Why is heat required during the Ninhydrin test?
Heat acts as a catalyst and is essential for the Ninhydrin test to proceed at a practical rate. The reaction involves multiple steps, including oxidation, deamination, and condensation, which are kinetically slow at room temperature. Heating the mixture in a water bath provides the necessary activation energy to speed up these reactions, allowing the formation of the coloured Ruhemann's purple complex within a few minutes.
8. What are some important real-world applications of the Ninhydrin test?
The Ninhydrin test has several important applications across different fields:
- Forensic Science: It is widely used to develop latent fingerprints on porous surfaces like paper and cardboard. The ninhydrin reacts with the amino acids present in sweat residues left behind.
- Biochemistry: It is used for the quantitative analysis of amino acids after they have been separated using techniques like chromatography.
- Peptide Chemistry: In solid-phase peptide synthesis, it is used as the Kaiser test to confirm whether the deprotection of an amino group has been successfully completed.
9. What are the main limitations of the Ninhydrin test?
While useful, the Ninhydrin test has some limitations. Firstly, it is not entirely specific to alpha-amino acids, as it also reacts with ammonia and any primary or secondary amine. Secondly, the test is destructive; the amino acid being tested is consumed in the reaction and cannot be recovered for further analysis. Lastly, the colour intensity can vary between different amino acids, and some, like proline, give a different colour altogether, which requires careful interpretation.
