Ninhydrin Test - Reaction, Procedure, Result and Inferences

Definition of Ninhydrin Test

The ninhydrin test is a chemical test to detect the presence of amino acids. Ninhydrin test is a confirmatory test for protein after performing burnt test. It is optional test for biuret test. If this ninhydrin test observed positive then only you have to perform further tests for amino acids. This test involves the addition of ninhydrin reagent to the sample solution that results in the formation of deep blue color gives +ve test.

IUPAC name of Ninhydrin reagent is 2,2-dihydroxyindane-1,3-dione.

Ninhydrin test is generally used to detect the fingerprints over any surface because ninhydrin reagent reacts with dried fingerprints.

Why Ninhydrin Test?

As this test is confirmatory test so, ninhydrin test is used to detect the presence of amino acids and carbohydrates in the given sample.
To detect the presence of amines and specific amino groups in the sample solution.
To detect the exact quantity the amino acids present in the given sample.

Principle of Ninhydrin Test

This test is positive test for amino acids and proteins. The formation of a complex called Rheumann’s purple due to the condensation of two molecules of ninhydrin with one molecule of ammonia from amino acid is responsible for violet color. All amino acids give blue or purple color except proline and hydroxyproline, which give a yellow color under the same conditions. In amino acids like proline and hydroxyproline, this test yields turns yellow-orange in color.

Reaction of Ninhydrin Test

Requirements of Ninhydrin Test

Instruments and Materials

Test tubes
Test tube stand
Pipettes
Dropper
Hot Water Bath
Spectrophotometer

Chemicals and Reagents

1 ml of test solution
5 drops of Ninhydrin Reagent: 0.35g of ninhydrin in 100 ml ethanol

Procedure of Ninhydrin Test

Take a dry and sterile test tube.
Add 1 ml of the given sample solution into the test tube with the help of dropper.
Add 5 drops of ninhydrin solution to test tube side by side.
Boil the mixture for 2 minutes and cool.
Note the change in color.
If there is a deep blue or purple appearance then the presence of protein is confirmed.

Observation and Inferences of Ninhydrin Test

If there is appearance of a deep blue or purple then the presence of protein is confirmed. This test is confirmatory test only.
If there is appearance of yellow-orange color then it will be specific test for proline or hydroxyproline.

Results and Interpretations of Ninhydrin Test

The presence of a purple-colored complex in the test tube gives +ve test and confirms the presence of protein and amino acid in the given sample solution.
But the absence of the purple color in the test tube gives -ve test.
The presence of yellow color indicates the presence of proline or hydroxyproline amino acid.

FAQs – Ninhydrin Test

1. What is the ninhydrin test for?

Ans – Ninhydrin test is used to detect the presence of protein and amino acid. This test is specific for amino acid proline and Hydroxyproline.

2. Which is the confirmatory test for protein?

Ans – Ninhydrin test is performed to detect the presence of protein. This test is confirmatory test for qualitative test protein.

3. What is the positive result of ninhydrin test?

Ans – There are 2 positive results in ninhydrin test: (1) All amino acids give deep blue or purple color as a positive result except Proline and Hydroxyproline. (2) Proline and Hydroxyproline give yellow color as a positive result.

4. Is ninhydrin test positive for all proteins?

Ans – No. Ninhydrin test is positive for all the amino except two amino acids. i.e. Proline and Hydroxyproline (because these are having free amino group).

5. Which is a better test for proteins biuret and ninhydrin?

Ans – Both the ninhydrin test and biuret test are confirmatory test for protein. But ninhydrin test is most accurate and easy test as compared to biuret test.

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