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Ninhydrin test reaction is a test to determine whether a sample compound contains alpha-amino acid or amines. In the Ninhydrin test reaction, reactant is ninhydrin, which is a hydrocarbon with a formula C9H6O4.
According to IUPAC nomenclature, ninhydrin is also called as 2, 2-di-hydroxyindane-1, 3,-dione. The ninhydrin test gives deep blue color which indicates the presence of ammonia, primary or secondary amines, or amino acids in the given compound.
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Key Terms: Ninhydrin (2, 2-di-hydroxyindane-1,3,-dione), Redox reaction, amines, di- ketohydrin, Aldehyde
Ninhydrin Test
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The Ninhydrin test is a chemical test used to identify primary or secondary amines, ammonia and amino acids. In the Ninhydrin Test, ninhydrin reagent is added to the testing material, which results in production of deep blue color. This deep blue color is also called Ruhemann's purple in the presence of amino group.
Ninhydrin is most commonly used for detecting latent finger marks on porous materials like cardboard and paper. The chemical process involved in Ninhydrin test is very complex. The blue colour develops based on reactant temperature, acidity (pH), humidity.
Ninhydrin Test
Ninhydrin Test Objective
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The test is performed to distinguish between carbohydrates and amino acids.
- It is also performed to measure the number of amino acids present in given sample.
- This test is performed for identification of amino group and amines.
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Ninhydrin Test Principle
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The principle behind Ninhydrin test is redox reaction which is combination of both oxidation and reduction reaction. In the test, ninhydrin acts as an oxidizing agent and itself get reduced.
- In the test sample, ninhydrin reacts with the amino group of free amino acid and oxidizes the compound, leading to delamination.
- In the performed reaction, two gases i.e., NH3 (Ammonia) and CO2 (Carbon dioxide) is released.
- Along with NH3 and CO2, the other two by-products are aldehyde and hydrindantin. The latter two are formed by the reduction of ninhydrin.
- The released by- product, ammonia further reacts with ninhydrin and give another by product di- ketohydrin, which forms a coloured complex.
- This coloured complex so formed is called as Ruhemann’s purple, and this give solution deep blue colouration. This whole process is termed as Ninhydrin Test Mechanism.
Variations of Ninhydrin Test ReactionIf the amino acid contains protein substrate like proline then the test sample will give yellow colouration. If the Ninhydrin reagent is replaced by asparagines then, the resultant obtained will be brown colour complex. |
Ninhydrin Test Procedure
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The procedure of performing Ninhydrin test is as follows:
- Firstly, take 2% solution of Ninhydrin. Ninhydrin solution is prepared by dissolving 0.2 g of ninhydrin per 10 ml of a carrier solvent such as alcohol or acetone.
- Now, prepare 1% solution of test compound using distilled water. In this prepared solution add few drops of ninhydrin solution which was prepared in step above.
- In this step, place test tube of solution contained in a warm bath for few minutes.
- If the solution turns into deep blue or purplish colour, then ninhydrin test is positive.
Ninhydrin Test Result Interpretation
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If the above sample contains ammonia, any amino acid or primary/ secondary amine, then the reaction will show Ruhemann’s purple colouration.
- For other compounds like proline or hydroxyproline, the colouration obtained will be yellow.
- If the reaction shows brown colour then asparagines would have been used a reactant.
- If the Ninhydrin test is negative then the resultant solution will give no colour at all and it shows that the given solution does not contain any amino group.
Uses of Ninhydrin Test
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- This test is used to identify whether the given solution contain amino acids or not.
- This test is also used in solid-phase peptide synthesis so as to ensure that proteins are properly stored for amino acid analysis.
- Ninhydrin test also finds its use in detecting fingerprints due to its high sensitivity.
Things to Remember
- The Ninhydrin test is a chemical test used to identify primary or secondary amines, ammonia and amino acids.
- The principle behind Ninhydrin test is redox reaction.
- If the ninhydrin test is positive then, it gives blue colouration which is also called as Ruhemann’s purple complex.
- If the ninhydrin test is negative, then the reaction result will be colourless.
- Other variation of ninhydrin test is asparagines which gives brown colour and proline which gives yellow colouration.
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Sample Questions
Ques 1. The positive ninhydrin test will give: (1 Mark)
(a) Yellow colour
(b) Purple colour
(c) Blue colour
(d) Colourless
Ans. c. Blue colour
Explanation: If the sample to be tested contains primary or secondary amines, ammonia and amino acids, then Ninhydrin Test will be positive as it forms Ruhemann’s purple complex which gives purplish blue or blue colour. This ninhydrin test is based on the principle of redox reaction.
Ques 2. The main principle behind Ninhydrin test is: (1 Mark)
(a) Redox reaction
(b) Oxidation reaction
(c) Decomposition reaction
(d) Reduction reaction
Ans. a. Redox reaction
Explanation: The main principle behind Ninhydrin Test is redox reaction where ninhydrin act as oxidizing agent and itself get reduced. The bi- product of Ninhydrin reaction is NH3 and CO2, the other two by-products are aldehyde and hydrindantin. The latter two are formed by the reduction of ninhydrin.
Ques 3. What is Ninhydrin test? (2 Marks)
Ans. The Ninhydrin test is a chemical test used to identify primary or secondary amines, ammonia and amino acids. In the Ninhydrin Test, ninhydrin reagent is added to the testing material, which is a compound having formula C9H6O4, which results in production of deep blue color or Ruhemann’s purple in the presence of amino group.
Ques.4. Which amino acid does not give positive result of ninhydrin test? (2 Marks)
Ans. Ninhydrin test is useful in identification of amino acids but not all amino acid give positive ninhydrin test. One such amino acid is Proline as it does not get hydrolyzed like other amino acid and therefore doesn’t react with ninhydrin.
Ques 5. List the limitations of Ninhydrin test. (3 Marks)
Ans. The major limitation of ninhydrin test is that, it reacts with ammonia nitrogen and other free amines in addition to (α + ε) amino group.
Additionally, historic or archeological protein fibres are sometime contaminated with soil residues and hence, it is difficult to extract them which ultimately affect the result given by Ninhydrin test.
Ques 6. Why does Ninhydrin test causes blue stain on skin? (2 Marks)
Ans. As human body is made up of protein, of which amino acid is a unit and hence if ninhydrin reacts with skin it gives positive blur colour due to presence of amino acids in the skin. Ninhydrin is capable of detecting NH3 and amines.
Ques 7. Explain the procedure of Ninhydrin test. (4 Marks)
Ans. The procedure to carry out Ninhydrin test is as follows:
- Take 2% solution of Ninhydrin. Ninhydrin solution is prepared by dissolving 0.2 g of ninhydrin per 10 ml of a carrier solvent such as alcohol or acetone.
- Now, prepare 1% solution of test compound using distilled water. In this prepared solution add few drops of ninhydrin solution which was prepared in step above.
- Place test tube of solution contained in a warm bath for few minutes.
- If the solution turns into deep blue or purplish colour, then ninhydrin test is positive.
- If the solution remains colourless then, the ninhydrin test is negative.
Ques 8. what are the uses of Ninhydrin test? (3 Marks)
Ans. The uses of Ninhydrin test are as follows:
- This test is use to identify whether the given solution contain amino acids pr not.
- This test is also used in solid-phase peptide synthesis so as to ensure that proteins are properly stored for amino acid analysis.
- Ninhydrin test also finds its use in detecting fingerprints due to its high sensitivity. In this, ninhydrin solution is sprayed over fingerprints then the ninhydrin reacts with amino acid in the residue of fingerprints.
Ques 9. What is the main objective of Ninhydrin Test. (3 Marks)
Ans. The main objectives of Ninhydrin Test are:
- The test is performed to distinguish between carbohydrates and amino acids as it give distinguished Ruhemann’s blue colour.
- It is also performed to measure the number of amino acids present in given sample.
- This test is performed for identification of amino group and amines.
Ques 10. Explain the Ninhydrin Test reaction mechanism. (5 Marks)
Ans.
- The mechanism behind ninhydrin test reaction is redox reaction i.e., oxidation and reduction reaction.
- Add droplets of ninhydrin solution to a sample to be tested , here ninhydrin will act as an oxidizing agent. Ninhydrin will interact with amino acid present in the sample and further causing delamination.
- In this procedure two gaseous by- products are also produced and they are NH3 and CO2. This reaction also produces an aldehyde, and further reduces the ninhydrin to produce a product known as hydrindantin.
- The NH3 that has been released as by product, now, interact with another molecule of ninhydrin to generate a di- ketohydrin complex which give deep blue colouration.
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