When studying 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 present, you can significantly reduce further experiments to identify them. These reactions include ninhydrin, which is the main one in the visual determination of amino compounds.
What is this?
Ninhydrin is a dicarbonyl compound containing one aromatic ring with a heterocycle attached to it, the second atom of which has 2 hydroxyl groups (OH-). This substance is obtained by direct oxidation of inandione - 1, 3, and, therefore, according to the international nomenclature, it has the following name: 2, 2 - dihydroxyinandione -1, 3 (Fig. 1).
Pure ninhydrin is a yellow or white crystalcolors that, when heated, dissolve well in water and other polar organic solvents, such as acetone. This is a rather harmful substance, if it comes into contact with the skin in large quantities or mucous membranes, it causes irritation, including when inhaled. Work with this compound should be careful and only with gloves, as when it comes into contact with the skin, it reacts with skin cell proteins and stains it purple.
Reactive Substances
As mentioned above, the ninhydrin reaction is used primarily for the visual determination of the content of amino compounds:
- α-amino acids (including in proteins);
- amino sugars;
- alkaloids containing –NH2 and -NH groups;
- various amines.
It should be noted that secondary and tertiary amines sometimes react very weakly, so more research is needed to confirm their presence.
Various methods of chromatography are used for quantitative determination, for example, paper chromatography (BC), thin layer chromatography (TLC) or with washing of solid carriers with a solution of ninhydrin in various media.
This reaction is not specific for amino compounds, since the reagent can enter into it with all of them at once. However, on the part of the reaction products, it has a peculiarity in the form of the release of carbon dioxide bubbles (CO2), and this is typical only when interacting with α-amino acids.
Mechanism features
BThere are different interpretations of the ninhydrin reaction equation in the literature. Some researchers omit the formation of hydrindanthine from 2-aminoinandione, which, with the participation of ammonia and ninhydrin, also forms a coloring substance called "Rueman's purple" (or "Rueman's blue"), while others, on the contrary, assume 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, this concerns the methods of attaching the amino derivative of ninhydrin to its main molecule to form a dye. The indication of the place of the “walking hydrogen” obtained by the intermediate amine from the aqueous medium also remains questionable: it can be either in the ketone group or next to –NH2.
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, so it should not be paid attention to. As for the omission of one of the possible stages, the reason here lies in the theoretical aspect: until now, the exact mechanism for the formation of Rueman's purple has not been precisely determined, so quite different schemes of the ninhydrin reaction can be found.
The most complete possible course of interaction of the reagent with amino compounds will be proposed below.
Reaction mechanism
First, ninhydrin interacts with 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 the intermediate amine, aldehyde, and carbon dioxide (Fig. 2b). From the final product when joiningninhydrin, the Rueman purple structure (diketonhydrindenketohydrinamine, Fig. 2c) is synthesized. Also indicated is the possible formation of hydrindanthine (reduced ninhydrin) from the intermediate amine, which also turns into a coloring compound in the presence of ammonia (more precisely, ammonium hydroxide) with an excess of the reagent itself (Fig. 2d).
The formation of hydrindantine was proved by Rueman himself when hydrogen sulfide acts on the ninhydrin molecule. This compound is able to dissolve in sodium carbonate Na2CO3, coloring the solution dark red. And when dilute hydrochloric acid is added, hydrindanthine precipitates.
Most likely, the intermediate amine, hydridanthin, ninhydrin and the structure of the dye, due to their instability when heated, are in some equilibrium, which allows for the presence of several additional stages.
This mechanism is suitable for explaining the ninhydrin reaction with other amino compounds, with the exception of by-products resulting from the elimination of the rest of the structure from –NH2, -NH or -N.
Biuret test and other reactions to proteins
Qualitative analysis for 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 does not take place along the –CO-NH‒ groups, but along the amine ones. There is a so-called "biuret reaction", which is characterized by the addition of ions to the solution with amino compoundsbivalent copper from CuSO4 or Cu(OH)2 in an alkaline medium (Fig. 3).
During the analysis, in the presence of the necessary structures, the solution turns dark blue due to the binding of peptide bonds into a color complex, which distinguishes one reagent from another. That is why the biuret and ninhydrin reactions are universal in relation to protein and non-protein structures with the –CO-NH‒ group.
When determining cyclic amino acids, a xantoprotein reaction with a concentrated solution of nitric acid HNO3 is used, which gives a yellow color when nitrated. A drop of reagent on the skin also exhibits a yellow color by reacting with the amino acids in the skin cells. Nitric acid can cause burns and should also be handled with gloves.
Examples of interaction with amino compounds
Ninhydrin reaction for 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.
Reaction with amino group
Since the test 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, one cancalculate 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 attaches to the reagent at the amine group, and under the action of active hydronium ions (H3O+) is split off at the carbon-nitrogen bond, decomposing into acetaldehyde (CH3COH) and carbon dioxide (CO2). Another ninhydrin molecule attaches to nitrogen, displacing water molecules, and a coloring structure is formed (Fig. 4).
Reaction with a heterocyclic amino compound
The ninhydrin reaction with proline is specific, especially in chromatographic analyses, since such structures in an acid medium first turn yellow, and then turn purple in a neutral one. The researchers explain this by a feature of the cycle rearrangement in the intermediate compound, which is affected precisely by the presence of a large number of hydrogen protons that complement the external energy level of nitrogen.
The destruction of the heterocycle does not occur, and another ninhydrin molecule is attached to it at the 4th carbon atom. Upon further heating, the resulting structure in a neutral medium turns into Rueman purple (Fig. 5).
Preparation of the main reagent
Ninhydrin test is carried out with different solutions, depending on the dissolution of amino structures in certain organic andinorganic compounds.
The main reagent is the preparation of a 0.2% solution in water. This is a versatile mixture, since most compounds dissolve well in H2O. To obtain a freshly prepared reagent, a sample of 0.2 g of chemically pure ninhydrin is diluted in 100 ml of water.
It is worth noting that for some analyzed solutions this concentration is insufficient, so 1% or 2% solutions can be prepared. This is typical for 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 carrier through a column, can be prepared in alcohol, dimethyl sulfoxide, acetone and other polar solvents - everything will depend on the solvent of certain amino structures.
Application
The ninhydrin reaction makes it possible to detect many amino compounds in solution, which made it one of the first to be used in the 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 forensic science, this method is widely used to determine the presence of sweat marks on any surface.
Even despite the non-specificity of the reaction, the withdrawal of the ninhydrin reaction from chemical practice is impossible, sincereplacing this substance with less toxic analogues (for example, oxolin) proved that they have poorer sensitivity to amino groups and do not give good results in photometric analyses.
