In researching substances in organic chemistry, more than a dozen different qualitative reactions are used to determine the content of certain compounds. Such a visual analysis allows you to immediately understand whether the necessary substances are present, and if they are not, significantly reduce further experiments to identify them. The ninhydrin reaction, which is the main one in the visual determination of amino compounds, also belongs to such reactions.
What it is?
Ninhydrin is a dicarbonyl compound containing one aromatic ring with a heterocycle attached to it, at the second atom of which there are 2 hydroxyl groups (OH - ). This substance is obtained by direct oxidation of inanedione - 1.3, and therefore, according to international nomenclature, it has the following name: 2.2 - dihydroxyinandion -1.3 (Fig. 1).
Pure ninhydrin is a yellow or white crystals, which, when heated, dissolve well in water and other polar organic solvents, for example, in acetone. This is a rather harmful substance, in contact with the skin in large quantities or on mucous membranes, it causes irritation, including by inhalation. Work with this compound should be careful and only with gloves, since when it comes into contact with the skin it gives a reaction with the skin cells proteins and turns it purple.
Reactive Substances
As indicated above, the ninhydrin reaction is primarily used to visually determine the content of amino compounds:
- α-amino acids (including proteins);
- amino sugars;
- alkaloids containing –NH 2 and –NH groups;
- various amines.
It should be noted that secondary and tertiary amines sometimes react very weakly; therefore, additional studies are necessary to confirm their presence.
For quantitative determination, various chromatography methods are used, for example, paper (BH), thin-layer (TLC) or washing of solid media with a solution of ninhydrin in various media.
This reaction is not specific with respect to amino compounds, since a reagent can enter into it with everyone at once. However, on the part of the reaction products, it has a peculiarity in the form of emission of carbon dioxide bubbles (CO 2 ), and this is characteristic only when interacting with α-amino acids.
Mechanism features
In the literature, there are different interpretations of the ninhydrin reaction equation. Some researchers omit the formation of hydrindindin from 2-aminoinanedione, which, with the participation of ammonia and ninhydrin, also forms a coloring substance called "purple Rueman" (or "Blue Rueman"), while others, on the contrary, suggest only its participation without the presence of intermediate amino products. There are also some interesting points in the record of the reaction itself, in particular, with regard to the methods of coupling the amino derivative of ninhydrin to its main molecule with the formation of a dye. The question of the location of “walking hydrogen” obtained by the intermediate amine from the aqueous medium also remains in doubt: it can be either in the ketone group or next to –NH 2 .
In fact, the nuance with the H atom is insignificant, since its position in the compound does not play a special role in the course of the reaction; therefore, one should not pay attention to it. As for the omission of one of the possible stages, the reason lies in the theoretical aspect: the exact mechanism of the formation of purple Rueman has not yet been precisely determined, therefore, quite different schemes of the ninhydrin reaction can be found.
Below will be proposed the most complete possible course of the interaction of the reagent with amino compounds.
Reaction mechanism
Initially, ninhydrin interacts with the α-amino acid, attaching it at the site of cleavage of hydroxy groups and forming a condensation product (Fig. 2a). Then the latter is destroyed, releasing an intermediate amine, aldehyde and carbon dioxide (Fig. 2b). Upon addition of ninhydrin, the structure of purple Rueman is synthesized from the final product (diketohydrindenketohydrinamine, Fig. 2c). Also indicated is the possible formation of hydrindindin (reduced ninhydrin) from an intermediate amine, which in ammonia (more precisely, ammonium hydroxide) with an excess of the reagent itself also turns into a coloring compound (Fig. 2d).
The formation of hydrindantine was proved by Ruehman himself under the action of hydrogen sulfide on the ninhydrin molecule. This compound is able to dissolve in sodium carbonate Na 2 CO 3 , staining the solution in dark red. And when diluted hydrochloric acid is added, hydrindantine precipitates.
Most likely, the intermediate amine, hydridantine, ninhydrin, and the dye structure, due to their instability upon heating, are in some equilibrium, which allows the presence of several additional stages.
This mechanism is also suitable for explaining the ninhydrin reaction with other amino compounds, with the exception of by-products resulting from the cleavage of the rest of the structure from –NH 2 , -NH or -N.
Biuret test and other reactions to proteins
Qualitative analysis of peptide bonds even of non-protein structures can take place not only with the participation of the above reagent. However, in the case of the ninhydrin reaction to proteins, the interaction proceeds not on –CO-NH‒ groups, but on amine ones. There is a so-called “biuret reaction”, which is characterized by the addition of divalent copper ions from CuSO 4 or Cu (OH) 2 to the solution with amino compounds in an alkaline medium (Fig. 3).
During the analysis, if the necessary structures are present, the solution turns dark blue due to the binding of peptide bonds to a color complex, which distinguishes one reagent from another. That is why biuret and ninhydrin reactions are universal with respect to protein and non-protein structures with the –CO-NH‒ group.
When determining cyclic amino acids, a xanthoprotein reaction is used with a concentrated solution of nitric acid HNO 3 , which, when nitrated, gives a yellow color. A drop of reagent that gets on the skin also shows a yellow color, reacting with amino acids in the skin cells. Nitric acid can leave burns, so gloves should also be used with it.
Examples of interaction with amino compounds
The ninhydrin reaction to α-amino acids gives a good visual result, except for the color proline and hydroxyproline structures, which react with the formation of a yellow color. A possible explanation for this effect was found in other environmental conditions of the interaction of ninhydrin with these structures.
Amino group reaction
Since the sample is not specific, visual detection of alanine using the ninhydrin reaction in the mixture is not possible. However, by paper chromatography, when applying samples of various α-amino acids, spraying them with an aqueous solution of ninhydrin and developing in a special medium, it is possible to calculate the quantitative composition of not only the claimed compound, but also many others.
Schematically, the interaction of alanine with ninhydrin follows the same principle. It joins the reagent at the amine group, and under the influence of active ions, hydroxonium (H 3 O + ) is cleaved at the carbon-nitrogen bond site, decomposing into acetic aldehyde (CH 3 COH) and carbon dioxide (CO 2 ). Another ninhydrin molecule attaches to nitrogen, displacing water molecules, and a coloring structure forms (Fig. 4).
Heterocyclic amino compound reaction
The ninhydrin reaction with proline is specific, especially in chromatographic analyzes, since such structures in an acidic medium are first colored yellow and then become purple in a neutral color. The researchers explain this by the peculiarity of the rearrangement of the cycle in the intermediate compound, which is affected precisely by the presence of a large number of hydrogen protons, supplementing the external energy level of nitrogen.
The destruction of the heterocycle does not occur, and another ninhydrin molecule joins in it at the 4th carbon atom. With further heating, the resulting structure in a neutral medium transforms into purple Rueman (Fig. 5).
Preparation of the main reagent
The ninhydrin test is carried out in different solutions, depending on the dissolution of the amino structures in various organic and inorganic compounds.
The main reagent is the preparation of a 0.2% solution in water. This is a universal mixture, since most of the compounds dissolve precisely in H 2 O. To obtain a freshly prepared reagent, a 0.2 g sample of chemically pure ninhydrin is diluted in 100 ml of water.
It should be noted that for some of the analyzed solutions this concentration is insufficient, therefore, 1% or 2% solutions can be prepared. This is characteristic of extracts from medicinal raw materials, since they contain various classes of amino compounds.
When conducting chromatographic studies, solutions, for example, when washing a mixture on a solid support through a column, can be prepared with alcohol, dimethyl sulfoxide, acetone and other polar solvents - everything will depend on the solvent of certain amino structures.
Application
The ninhydrin reaction allows one to detect many amino compounds in a solution, which determined it as one of the first to be used in a qualitative analysis of organic substances. Visual determination significantly reduces the number of experiments, especially when analyzing poorly studied plants, drugs and dosage forms, as well as unknown solutions and mixtures.
In forensics, this method is widely used to determine the presence of traces of sweat on any surface.
Even despite the non-specific nature of the reaction, the withdrawal of the ninhydrin reaction from chemical practice is impossible, since the replacement of this substance with less toxic analogues (for example, oxolin) proved that they have worse sensitivity to amino groups and do not give good results in photometric analyzes.