Introduction

The ninhydrin assay is a chemical method utilized to determine if a sample contains amines or α-amino acids. In this procedure, ninhydrin (a chemical compound with the molecular formula C9H6O4, formally known as 2,2-dihydroxyindane-1,3-dione) is introduced to a solution containing the analyte. The formation of a deep blue color signifies the presence of ammonia, primary or secondary amines, or amino acids within the sample.

Ninhydrin Test Theory

The chemical reaction typically observed in a positive ninhydrin test is depicted below.

The amino group of the analyte reacts to form a compound resembling diketohydrin. This compound exhibits a deep blue color, commonly known as Ruhemann’s purple.

The amino group of a free amino acid reacts chemically with ninhydrin, acting as an oxidizing agent. In the presence of ninhydrin, the amino acid undergoes oxidative deamination, leading to the release of CO2, NH3, and an aldehyde, along with the formation of hydrindantin (the reduced form of ninhydrin).

The released ammonia then reacts with another ninhydrin molecule, producing diketohydrin (also known as Ruhemann’s complex), which is responsible for the characteristic deep blue color. If the analyte contains imino acids like proline, a yellow complex is formed. In the case of asparagine, the resulting complex is brown.

Ninhydrin Test: A Detailed Guide to The Amines and Amino Acids Determination

Procedure

Test A

To carry out the ninhydrin test, a TLC plate is needed (you can use thick filter paper), a 10% ninhydrin ethanol solution, and samples aqueous solutions.

• Several drops of the studied samples are spoted on the TLC plate.
• Sample 1 is an aqueous methamphetamine solution.
• Sample 2 is an aqueous amphetamine solution.
• Sample 3 is an aqueous ephedrine solution.
• Next, few drops of 10% ninhydrin solution in ethanol are added to each sample.
• Sample 4 – only ninhydrin is spotted.
• This is a control test to ensure that the reagent itself doesn’t give a coloring. If it gives a coloring, then the reagent or plates are unsuitable for analysis, have impurities.
• Then the plate has to be heated in an oven or on an electric stove at least at 120 °C for 15 min.

Primary amines such as amphetamine and secondary amines such as methamphetamine form pink or purple spots. Ephedrine also forms a purple spot. It can be seen that the reactions are positive on samples 1, 2, and 3. Samples number 1 and 2 have pink spots. Sample 3 have a purple color. Sample 4 did not give a characteristic color and remained clean.

Ninhydrin Test B

To carry out the ninhydrin test again, it takes: a TLC plate with silica gel (TLC Silica gel 60 F254), a 10% Ninhydrin in ethanol solution, the test sample aqueous solution, solvent (ethanol), UV lamp with a wavelength of 254 nm or/and 366 nm.

• Beakers contain aqueous solutions of ephedrine. Three different ephedrine samples from different syntheses, which can have different purity levels. In this case, a test is carried out in order to determine an amount of substances (impurities) in these ephedrine samples.
• A few drops of ephedrine aqueous solutions are spotted to the TLC plate.
• TLC plate is immerced in ethanol. The solvent is soaked with the plate. Ethanol is fill up the silica gel plate’s surface and pulled substances that aren’t bound well to silica gel on spots.
• The plate is turned out of the solvent.
• The plate is air dried (with warm but not hot air).
• Next, a developings with a UV lamp and ninhydrin is carried out.
• TLC plate is developed under UV lamp, pink spots can be seen on the picture.
• Next, the TLC plate is sprayed with a solution of ninhydrin in ethanol.
• Then the plate has to be heated in an oven or on an electric stove at least at 120°C 15 min for developing.
• The test results are evaluated.

Ephedrine gave a characteristic purple color. At the top, a small amount of impurities can bee seen. According to the test results, the studied ephedrine samples are relatively pure.

Conclusion

Ninhydrin is used as a qualitative and quantitative reagent in the determination of primary amines and amino acids. The ninhydrin test is used in criminology.

Reacting with proteins contained in human sweat, it turns their traces purple. This allows it to be used in fingerprinting studies. A 2% solution of ninhydrin in ethyl alcohol or acetone is used in forensics to identify papillary pattern prints on porous surfaces, such as paper.

Bibliography

• Dhuner, K. G., S. Edshage, and A. Wilhelm. “Ninhydrin test—an objective method for testing local anaesthetic drugs.” Acta Anaesthesiologica Scandinavica 4.4 (1960): 189-198. https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1399-6576.1960.tb00074.x
• https://en.wikipedia.org/wiki/Ninhydrin