The Ninhydrin test is a simple chemical test for detecting amino acids, primary and secondary amines and ammonia. In this test, ninhydrin reagent (C9H6O4; IUPAC name: 2,2-dihydroxyindane-1,3-dione) is added to the test solution. The development of a deep blue color also called Ruhemann's purple indicates the presence of amino acids, primary and secondary amines and ammonia.
The ninhydrin test is based on the reaction of two molecules of ninhydrin with free α - amino acid, resulting in the formation of a deep purple or blue colored complex.
In this reaction, ninhydrin reagent acts like an oxidizing agent and causes the deamination (lose its amino group -NH2) and decarboxylation (lose its carboxyl group -COOH)of the amino acid upon heating. This produces ammonia (NH3) and a reduced form of ninhydrin. The released ammonia reacts with the reduced ninhydrin and another molecule of ninhydrin through condensation reaction, finally forming the purple colored complex.
The intensity of the purple color complex is directly proportional to the amount of amino acids present in the given sample or test sample.
Primary amines and amino acids reaction with ninhydrin give this purple color. Secondary amines like (proline or hydroxyproline) produce a yellow or orange color due to formation of an iminium salt during reaction.
Ninhydrin reagent: Dissolve 0.35 gram of ninhydrin in 100 ml of ethanol. Acetone can also be used instead of ethanol.
Diluent solvent: Diluent solvent is used for qualitative analysis. Mix equal volume of water and n-propanol.
1. Take 1 ml of test solution in a clean dry test tube. Add a few drops (most commonly 0.5ml to 1 ml) to the test tube.
2. Place the test tube in a boiling water bath for 5 minutes and allow them to cool to room temperature.
3. Observe the color change. If amino acids or primary amines are present in the solution, a deep blue color develops.
1. Tube 1 should be blank, containing only 1 ml of distilled water. Label test tube 2 to 8 for standard solution. Tubes 9 and 10 for unknown samples.
2. Pipette out different volumes of standard amino acid solution into labelled tubes. Make the final volume of each tube upto 2 ml with distilled water.
3. Add 1 ml of ninhydrin reagent to each tube and mix it well. Add 5 ml of diluent solvent to each tube and mix it well again.
4. Incubate all the test tubes in a boiling water bath for 15 minutes and allow them to cool down to room temperature.
5. Measure the optical density (absorbance) at 570 nm using a colorimeter or spectrophotometer, using the blank as the reference. For proline and hydroxyproline, measure at 440 nm if specified by the method.
6. After the test tubes cooled down, add 1 ml of ethanol to each test tube and mix well. Record the absorbance of all tubes against the blank at 570 nm using a colorimeter or spectrophotometer.
The presence of a deep purple or blue color complex (Ruhemann’s Purple) indicates the presence of amines or free amino acids in the sample.
No color change in the test tube means absence of detectable amino acids or amines in the sample. It is a negative result.
Yellow or orange color indicates the presence of secondary amines and brown color development seen with asparagine and some proteins with free amide groups.
Ninhydrin test is frequently employed in biochemistry labs to test the presence of alpha-amino acids, peptides and proteins containing free amino groups.
Used in colorimetric or spectrophotometric analysis to measure the concentration of amino acids.
In addition it is widely used by forensic investigators to develop latent fingerprints on porous surfaces like paper, wood. Ninhydrin reacts with amino acids present in the eccrine sweat residue left behind by a fingerprint.
Ninhydrin reacts with all alpha-amino acids, it cannot be used to identify a specific amino acid in a complex.
Proline and hydroxyproline do not produce a purple color complex, instead they produce yellow or orange color, which can cause false negatives.
If the test solution has other substances that contain free amines such as ammonia, drugs or contaminants, it can also react with ninhydrin and create false positive results.
Principle of Ninhydrin Test
The ninhydrin test is based on the reaction of two molecules of ninhydrin with free α - amino acid, resulting in the formation of a deep purple or blue colored complex.
In this reaction, ninhydrin reagent acts like an oxidizing agent and causes the deamination (lose its amino group -NH2) and decarboxylation (lose its carboxyl group -COOH)of the amino acid upon heating. This produces ammonia (NH3) and a reduced form of ninhydrin. The released ammonia reacts with the reduced ninhydrin and another molecule of ninhydrin through condensation reaction, finally forming the purple colored complex.
The intensity of the purple color complex is directly proportional to the amount of amino acids present in the given sample or test sample.
Primary amines and amino acids reaction with ninhydrin give this purple color. Secondary amines like (proline or hydroxyproline) produce a yellow or orange color due to formation of an iminium salt during reaction.
Ninhydrin Reagents Preparation
Ninhydrin reagent: Dissolve 0.35 gram of ninhydrin in 100 ml of ethanol. Acetone can also be used instead of ethanol.
Diluent solvent: Diluent solvent is used for qualitative analysis. Mix equal volume of water and n-propanol.
Procedure of Ninhydrin Test
Qualitative analysis
1. Take 1 ml of test solution in a clean dry test tube. Add a few drops (most commonly 0.5ml to 1 ml) to the test tube.
2. Place the test tube in a boiling water bath for 5 minutes and allow them to cool to room temperature.
3. Observe the color change. If amino acids or primary amines are present in the solution, a deep blue color develops.
Quantitative Analysis
1. Tube 1 should be blank, containing only 1 ml of distilled water. Label test tube 2 to 8 for standard solution. Tubes 9 and 10 for unknown samples.
2. Pipette out different volumes of standard amino acid solution into labelled tubes. Make the final volume of each tube upto 2 ml with distilled water.
3. Add 1 ml of ninhydrin reagent to each tube and mix it well. Add 5 ml of diluent solvent to each tube and mix it well again.
4. Incubate all the test tubes in a boiling water bath for 15 minutes and allow them to cool down to room temperature.
5. Measure the optical density (absorbance) at 570 nm using a colorimeter or spectrophotometer, using the blank as the reference. For proline and hydroxyproline, measure at 440 nm if specified by the method.
6. After the test tubes cooled down, add 1 ml of ethanol to each test tube and mix well. Record the absorbance of all tubes against the blank at 570 nm using a colorimeter or spectrophotometer.
Interpreting Ninhydrin Test Results
The presence of a deep purple or blue color complex (Ruhemann’s Purple) indicates the presence of amines or free amino acids in the sample.
No color change in the test tube means absence of detectable amino acids or amines in the sample. It is a negative result.
Yellow or orange color indicates the presence of secondary amines and brown color development seen with asparagine and some proteins with free amide groups.
Applications of Ninhydrin Test
Ninhydrin test is frequently employed in biochemistry labs to test the presence of alpha-amino acids, peptides and proteins containing free amino groups.
Used in colorimetric or spectrophotometric analysis to measure the concentration of amino acids.
In addition it is widely used by forensic investigators to develop latent fingerprints on porous surfaces like paper, wood. Ninhydrin reacts with amino acids present in the eccrine sweat residue left behind by a fingerprint.
Limitations of Ninhydrin Test
Ninhydrin reacts with all alpha-amino acids, it cannot be used to identify a specific amino acid in a complex.
Proline and hydroxyproline do not produce a purple color complex, instead they produce yellow or orange color, which can cause false negatives.
If the test solution has other substances that contain free amines such as ammonia, drugs or contaminants, it can also react with ninhydrin and create false positive results.
