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March 24, 2022 - BY Ruchika Dwivedi

Ninhydrin; A Novel Method for Fingerprint Development

Siegfried Ruhemann accidentally created ninhydrin in 1910, which led to its discovery. Ruhemann noticed that this new molecule produced a purple byproduct when it reacted with amino acids present in the skin surface, and he published a series of papers describing this reaction. He hypothesized a structure for this deep purple-colored byproduct, which is now known as Ruhemann's purple, and discussed how the reaction could be used to identify minute levels of amino acids and protein products in biological materials.

Ninhydrin has been widely used in analytical chemistry and biochemistry since its discovery by Ruhemann. The amino acid reaction has been used as a diagnostic test for the presence of protein and amine molecules in biological materials since 1913. The reaction became much more valuable with the advent of chromatography for the localization of amino acids on paper chromatograms or in fractions produced by liquid chromatography.

The amino acid content of samples was also quantified using Ruhemann's purple and other by-products of the ninhydrin and amino-acid reaction. Some writers claimed that the reagent was the most extensively used reaction in analytic laboratories because it was so potent and versatile.

The use of ninhydrin was commonly accompanied by warnings to avoid coming into touch with the reagent on exposed skin or other surfaces. This was owing to the strong reactivity of ninhydrin with sweat, which caused fingerprints to emerge on chromatograms. Despite these indications, which clearly demonstrated ninhydrin's capacity to generate fingerprints, the reagent was not used in forensics until 1954.

Ninhydrin quickly became an indispensable tool in the detection of latent fingerprints after this original discovery, with widespread use noted as early as 1959 among jurisdictions. The approach is now one of the most widely used methods for detecting fingerprints on porous surfaces such as paper. However, this approach has disadvantages, which scientists have addressed by synthesizing analogues (compounds structurally similar to ninhydrin that have comparable reactions with amino acids) to improve the clarity of the developed fingerprint. Several of these analogues have proven to be quite effective (e.g., 1,8-diazafluoren-9-one [DFO], 1,2-indanedione, and 5-methylthioninhydrin), but none has been able to totally replace ninhydrin as the most commonly employed method.

Amino Acid Reagents for Fingerprint Detection 

When the fingers come into contact with a surface, they deposit sweat, which leaves certain fingerprints. Sweat is mostly made up of watery components, which make up 98 percent of a fingerprint's volume. A modest but detectable amount of amino acids can be found in these aqueous deposits, averaging about 250 ng per fingerprint. The amino acids remain solid after the water has evaporated from the surface.

For porous surfaces like paper, amino acids are desirable targets for fingerprint development reagents. Although uncontrollable variables (such because the total amount of sweat deposited by the finger, the aminoalkanoic acid concentration of the individual’s excretions, and therefore the age of the fingerprint) influence the quantity of amino acids transferred to the paper, amino acids are always present in perspiration in some amount. On contact with the paper, these amino acids impregnate the surface of the paper, where they're retained by their high affinity for cellulose.

Because of this affinity, amino acids don't migrate significantly from their initial deposition sites; however, the number of amino acids retained within the fingerprint decreases gradually over time. Furthermore, amino acids react with a wide variety of chemicals to supply colored compounds. These qualities are exploited to produce clear, sharp images of fingerprints that were up to 40 years old.

Fingerprint residues may have at least 14 amino acids. The optimum reagent must be nonspecific to a certain aminoalkanoic acid in order to yield the best-developed fingerprint (i.e., reacts well with all).

Ninhydrin is one of many compounds that functions as a nonselective aminoalkanoic acid reagent, making it ideal for fingerprint formation.

Properties of Ninhydrin

Ninhydrin is a colorless to light yellow crystalline solid that is readily soluble in polar solvents like water and methanol. When heated to 125 degrees Celsius, the solid becomes pink to crimson, melts around 130–140 degrees Celsius, and decomposes at 241 degrees Celsius. In the presence of any water, the chemical exists as a stable hydrate, but in anhydrous circumstances, it takes on a triketone structure.

The reaction of Ninhydrin with Amino Acids. In 1910, the reaction of ninhydrin with skin to generate a deep purple compound was recorded for the first time. The purple color was discovered to be the consequence of a reaction between ninhydrin and amino acids, and the product was named diketohydrindylidene–diketohydrindamine, which is now known as Ruhemann's purple. An aldehyde derivative of the amino acid and carbon dioxide are produced as by-products of this reaction. Several attempts have been made to figure out how this response works. Other reagents required for this reaction include acid and water. Ruhemann's initial product structure was right, and the reaction with amino acids creates the ammonium salt of Ruhemann's purple, according to structural investigations of the reaction product.

To proceed at an acceptable rate, this reaction is complex and necessitates a precisely adjusted set of circumstances. The pH of the reaction should be higher than 4, ideally between 4.5 and 5.2. Because water is an essential reactant, development in a high-humidity environment is critical. Finally, because Ruhemann's purple is known to fade when exposed to light and oxygen, the fingerprint should be kept in a dark, cool location. Fingerprints that have been treated with ninhydrin are purple in color and have outstanding contrast and clarity of detail.

Optical Enhancement of Ninhydrin-Developed Fingerprints. Under ideal conditions, the ninhydrin treatment produces good contrast (e.g., fresh fingerprints on white paper). However, on colorful paper or with old fingerprints, the results can be less than ideal.

To improve the contrast between ninhydrin-produced fingerprints and a colored substrate or to enhance weakly developed fingerprints, several approaches have been developed. The absorption maxima—wavelengths of light that are significantly absorbed by the compound—can be seen in the UV-to-visible light spectrum of Ruhemann's purple. These peaks, located at λ  = 407 nm and λ = 582 nm, can be employed to boost the contrast between a developed fingerprint and a nonabsorbing background. When lasers became available to the forensic community in the late 1970s to early 1980s, treatment with zinc chloride was described for enhancing weak ninhydrin prints by using the light of an argon-ion laser. This method was capable of drastically increasing the number of identifiable latent fingerprints developed by the ninhydrin process. With the current ubiquity of forensic light sources, both absorption bands of Ruhemann’s purple can be exploited to produce high-contrast fingerprints. 

Post-Treatment with Metal Salts

The reaction between Ruhemann’s purple and metal salts such as zinc, cadmium, cobalt, and copper was used in a biochemical context to preserve ninhydrin spots on chromatograms. Formation of a metal-salt complex alters the color of Ruhemann’s purple from deep violet to red or orange, depending upon the salt used. The lighter hue may provide a greater contrast against a dark-colored back¬ground, especially when observed at 490–510 nm, where the metal–Ruhemann’s purple complex has an absorption maximum. 

It has been reported that viewing zinc-complexed ninhydrin-treated fingerprints under an argon ion laser could induce fluorescence of even weakly developed prints (Herod and Menzel, 1982b, pp 513–518). This discovery had a profound impact on fingerprint development because fluorescent reagents are more sensitive than chromogenic ones and can be viewed more clearly against colored backgrounds. Subsequent studies revealed that intense laser light was not necessary if the zinc-treated samples were cooled to the temperature of liquid nitrogen (-196 °C or 77 K); the fluorescence could be observed under a xenon arc lamp. This technique required submersion of the document in liquid nitrogen, a glass plate being placed between the sample and the light source and camera, and a heat source to prevent condensation on the glass . Later research showed that cadmium complexes provided an improved luminescence under these conditions. 


Ninhydrin Formulations. Several ninhydrin formu¬lations have been reported in the literature. Ninhydrin solutions are typically prepared in two steps: first, a stock solution is prepared that has a high proportion of polar solvent to facilitate the stability of the mixture; second, a portion of the stock solution is diluted with a nonpolar carrier solvent to produce a reagent suitable for application to evidential items. 

Application of ninhydrin working solutions can be per¬formed by dipping, spraying, or brushing, with the dipping method preferred in most instances. The item to be examined is briefly submerged in the working solution and allowed to air-dry to evaporate the solvent. 

Following treatment with ninhydrin solution, development should ideally proceed at room temperature, in a dark and humid environment (50–80% humidity), for a period of 1–2 days. If ambient humid¬ity is low, development in a specialized, humidity-controlled fingerprint development chamber may be necessary. The development may be accelerated by the application of steam or heat, but this may result in a greater degree of background development, reducing the clarity and contrast of the resulting fingerprints. Steaming can be achieved by holding a steam iron above the exhibit; heat can be delivered in a press, oven, fingerprint development cabinet, or by a mi¬crowave oven and should not exceed 80 °C. 

Ninhydrin crystals may be ground in a mortar and pestle to form a fine powder and applied directly to the fingerprints with a fingerprint brush. This method is slow and produces only faint prints but may be suitable for some types of heat- or solvent-sensitive paper. Ninhydrin may also be applied by a fuming method; a forensic fuming cabinet is used to heat the ninhydrin until it sublimes, allowing gaseous ninhydrin to deposit on the fingerprint residues. The reagent is most suited to paper, although any porous substrate may give visible results, and some nonporous substrates have been reported to produce visible fingerprints. 

Metal Salt Post-Treatment. The application of zinc or cadmium salts to ninhydrin-developed fingerprints will result in an immediate color change from purple to orange or red, respectively. Note that the use of zinc is preferred to cadmium because of cadmium’s toxicity. Dipping the exhibit into the solution is preferred over spraying because of the toxicity of some of the reagents. If humidity is low, a short blast of steam may be required to produce development. However, the humidity must be carefully controlled if zinc salts are used because high moisture levels cause the formation of an unstable, nonfluorescent, red complex that will reduce the contrast of the resulting fingerprint. 

Post-treated fingerprints may be further enhanced by viewing under 490 nm light (for zinc-treated residues) or 510 nm light (for cadmium-treated residues). Fluorescence may be induced by submerging the article in liquid nitro¬gen and exciting the treated fingerprint with the above-mentioned wavelengths of light. The fluorescent emissions should be viewed using a 550–570 nm band-pass filter or a 550 nm long-pass filter.