Ninhydrin Test

At room temperature, ninhydrin is a chemical powder that is soluble in ethanol or acetone. When a ninhydrin solution is put on fingerprints (typically with a simple spray bottle), the ninhydrin reacts with the amino acids found in fingerprint residue.

Ninhydrin Test Definition

The ninhydrin test is a chemical test useful to identify ammonia, primary/secondary amines, or amino acids. In this test, ninhydrin reagent is added to the test material, resulting in the production of deep blue colour, also known as Ruhemann’s purple, in the presence of an amino group.

Ninhydrin is the most commonly in use chemical reagent for detecting latent fingermarks on porous materials like paper and cardboard. The chemical reacts with the amino acid (eccrine) component of the fingerprint deposit to form Ruhemann’s purple, which has a dark purple colour. The chemical processes involved are quite complex, and development parameters such as temperature, acidity (pH), and humidity must be controlled to produce optimal results.

Objectives of Ninhydrin Test

• The test solution needs to be tested for the presence of amines and amino groups.
• To distinguish between carbohydrates and amino acids.
• To measure the number of amino acids present in a sample.

Principle of Ninhydrin Test

When subject to ninhydrin, the amino acid undergoes oxidative deamination, releasing carbon dioxide, ammonia, and an aldehyde to produce hydrindantin. The ammonia then interacts with another molecule of ninhydrin to generate diketo hydrin (also known as Ruhemann’s complex).

After then, the ammonia combines with another ninhydrin molecule to generate diketo hydrin (also known as Ruhemann’s complex). This compound is responsible for the strong blue colour. A yellow complex forms when amino acids such as proline are present in the analyte. When asparagine is used, the resulting complex has a brown colour.

Reaction Requirements

Reagent for Ninhydrin: In 100 mL of ethanol, dissolve 0.35g of ninhydrin. Solvent for dilution (for quantitative testing): Combine equal parts of water and n-propanol, Standard solution (1 per cent protein solution), and Sample solution.

The procedure of the Ninhydrin Test

For qualitative analysis

• Combine 1 ml of the test sample and 1 ml of the standard protein solution in a single dry test tube.
• Add a few drops of the ninhydrin reagent to each test tube.
• After five minutes in the water bath, let the test tubes cool to room temperature.
• Pay attention to how colours are created and how they turn out.

For quantitative analysis

• Pipette increasing volumes of the protein solution (10 l, 20 l, etc.) from the given stock solution into a series of test tubes, then distil the liquid to 1 mL.
• Take a blank tube, 1ml of distilled water, and the remaining tubes labelled 2 to 9 for the construction of a standard curve. Unidentified samples are stored in tubes 10-15.
• Add 1 ml of the ninhydrin reagent and 5 mL of the diluent solvent to each tube and fully mix by vertexing.
• Allow time for the tubes to cool.
• Cover the tubes and incubate at 90°C for 17 minutes or 20 minutes in a boiling water bath.
• Cool the tubes to room temperature before measuring the optical density of the solutions against a blank at 570 nm (440 nm for proline and hydroxyproline).
• To quantify the amount of amino acid in the unknown substance, draw a standard curve of A570 on the Y-axis and the concentration of amino acid on the X-axis.

Result and Interpretation of Ninhydrin Test

A positive result shows by the appearance of a complex with a purple colour in the test tube. This colouration denotes the presence of an amino acid in the sample. The absence of the complex in the test tube constitutes a negative result and demonstrates that the sample does not contain any amino acids.

Uses of Ninhydrin Test

The ninhydrin test detects amino acid content in unknown compounds. It is also used in solid-phase peptide synthesis to ensure that proteins are adequately stored for amino acid analysis. Because of its high sensitivity, the ninhydrin test is frequently in use to detect fingerprints. Because ninhydrin combines with the terminal amines of lysine residues in peptides and proteins shed off in fingerprints, it is possible.

Limitations of Ninhydrin Test

One drawback of the test is that ninhydrin reacts with ammonia nitrogen and other free amines in addition to (α + ɛ ) amino groups. Furthermore, archaeological and historic protein fibres are frequently contaminated with soil residues. These are difficult to extract and can thus affect test results.

FAQs on Ninhydrin Test

Question 1. Which amino acid is ninhydrin-resistant?

Answer. Ninhydrin analyses protein amino acids. Most amino acids, excluding proline, react with ninhydrin.

Question 2. Is ninhydrin cancerous?

Answer. Mammalian skin irritates. No long-term in vivo bioassay has been undertaken to test ninhydrin’s carcinogenic and co-carcinogenic potential in lab animals.