Ninhydrin Test Definition

The ninhydrin test is performed to determine the presence of ammonia, primary or secondary amines, or amino acids. During this test, the sample is combined with ninhydrin reagent, which produces a deep blue colour while an amino group is found, also known as Ruhemann’s purple.

Objectives of Ninhydrin Test

• To determine whether the test fluid contains amines or amino groups.
• To calculate the amount of amino acids in the sample.
• To differentiate amino acids from carbohydrates.

Principle of Ninhydrin Test

• The Ruhemann’s purple or blue colour that results from the reaction of two molecules of ninhydrin (2,2-dihydroxyindane-1,3-dione) with a free alpha-amino acid serves as the basis for this assay.
• The amino acids are deaminated and decarboxylated at a high temperature in this reaction, which ninhydrin catalyses as an oxidising agent.
• Condensation happens between the second ninhydrin molecule, the released ammonia, and the reduced ninhydrin molecules after this reaction.
• A deep purple diketohydrin complex is created at the end of the reaction.
• This assay produces a yellow-orange iminium salt in amino acids such as proline and hydroxyproline.
• A black product is produced, similar to how the amino group-free protein asparagine reacts with the ninhydrin reagent.
• The concentration of amino acids in the solution directly relates to the intensity of the complex that forms.
• The kind of amino acid present in turn affects the colour intensity.

Reaction

Requirements

Reagents

• Ninhydrin reagent:You can also use iso-propanol or a 1:1 combination of acetone and butanol in place of ethanol. Dissolve 0.35g of ninhydrin in 100 ml of ethanol.
• (For the quantitative test) Diluent solvent, Equilibrate the amounts of water and n-propanol.
• Standard solution (1% protein solution)
• Sample solution

Materials required

A test tube

Test tube holder

Pipette

Equipment

• Water bath
• Spectrophotometer

Procedure of Ninhydrin Test

For qualitative analysis,

• Place 1 ml of the test sample and 1 ml of the reference protein solution in separate dry test tubes.
• Drops of the ninhydrin reagent should be added to both test tubes.
• After 5 minutes in the water bath, remove the test tubes and let them cool to room temperature.
• Observe the formation of colour and note down the result.

For quantitative analysis,

• Pipette various amounts of the protein solution—10 l, 20 l, and so forth—from the provided stock solution into a string of test tubes, then top off the volume to 1 mL with distilled water.
• Take a tube marked “one” and fill it with 1 mL of just distilled water. Use the other tubes, numbered “2” through “9,” to build a standard curve. Tubes 10–15 contain the unidentified samples.
• Each tube should contain 1 ml of the ninhydrin reagent and 5 ml of the diluent solvent. Mix thoroughly by vortexing.
• Refresh the tubes.
• Top the tubes with caps and incubate for 17 minutes at 90 °C or 20 minutes in a boiling water bath.
• When the tubes are at room temperature, measure the solutions’ optical densities at 570 nm (or 440 nm for proline and hydroxyproline) in comparison to a reference sample.
• Make an absorbance standard curve against an amino acid concentration.
• Plotting the concentration of the amino acid on the X-axis and the A570 standard curve on the Y-axis will allow you to estimate the quantity of amino acid in an unidentified sample.

Results and Interpretation of the Ninhydrin Test

• A positive result and an indication that an amino acid was present in the sample is the existence of a compound with a purple hue in the test tube.
• Negative results and a paucity of amino acids in the sample are indicated by the lack of complex in the tube.
• The graph allows us to estimate the percentage of unidentified samples.

Test for Ninhydrin: Use

• The ninhydrin test is used to determine whether amino acids are present in unidentified materials.
• This assay is also employed in solid-phase peptide production to check the level of protein protection from amino acid analysis.
• Due to its sensitivity, the ninhydrin test is commonly utilised to identify fingerprints. Because ninhydrin combines with the terminal amines of lysine residues in peptides as well as proteins that leave fingerprints, it is possible.

Limitations of Ninhydrin Test

• Besides -amino groups, ninhydrin interacts with nitrogen in ammonia as well as other free amines.
• Steric obstruction inhibits ninhydrin from entering the amino groups, rendering the test inefficient for detecting proteins with a high molecular weight.

References and Sources

• Tiwari A. (2015). Practical Biochemistry. LAP Lambert Academic Publishing.
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