Ninhydrin colored product

Ninhydrin (1,2,3-indantrione, 100 mg dissolved in 50 ml of methanol) transforms all samples containing molecules with NHj groups, such as amino acids, peptides, or amines, into red- or purple-colored products. To perform a reaction, at least 5 to 10 min heating at 120°C is necessary. 

Schematic diagram of an amino acid analyzer. The amino acids are passed through an ion-exchange column and thereby separated. Eluted fractions are mixed and reacted with ninhydrin. The intensity of the resulting colored product is measured in a spectrophotometer, and the results are displayed on a recording chart.

Usually the amino acid analyzer is first standardized by running through it a sample containing known quantities of amino acids to account for any differences in their ninhydrin reaction properties. In this way it is possible to relate directly the amount of amino acid present to the amount of colored product formed, as measured by the area under the peak produced on the strip-chart recorder (see fig. 3.15). Similarly, the amino acid hydrolysate of a protein of unknown composition can be run through the analyzer, and the relative peak areas can be used to estimate the ratios of the different amino acids present. 

Only a few amino acids are detected by the UV or visible spectrophotometers, fluorometers, or electrochemical detectors that are routinely used with HPLC analyzers. Consequently, amino acids typically are postcolumn derivatized for analysis by HPLC. The most widely used reagent for this purpose is ninhydrin. A number of colored products are formed, but the major one is presumed to result from deamination and condensation as follows ... 

Ammonia, some amines, and some proteins and peptides will also yield a colored product but will not generate CO2. Thus, the colorimetric analysis is not specific for amino acids unless CO2 release is measured or the amino acids are purified and freed from interfering materials (the usual procedure). The color reaction with ninhydrin is used extensively in manual and automated procedures. 

Since amino acids are colorless, how can we detect that they have been separated When amino acids are heated with ninhydrin, they form a colored product. After electrophoretic separation of the amino acids, the filter paper is sprayed with ninhydrin and dried in a warm oven. Most amino acids form a purple product. The number of different kinds of amino acids in the mixture is determined by the number of colored spots on the filter paper (Figure 23.1). The individual amino acids are identified by their location on the paper compared with a standard. 

Detection, After separation, the solvent front is marked and the sheet is dried. The separated compounds are then detected in a variety of ways, chemical or physical. If they are colored, detection presents little or no problem usually, however, the substances are colorless. In the latter case, the sheet is sprayed with or dipped in a chemical reagent to produce a colored product. There are a large variety of such visualization reagents [2] for various classes of compounds. For example, amino acids (colorless) are easily detected as a pale blue-violet product by treating the paper with a 0.2% solution of colorless ninhydrin. A number of unsaturated organic compounds fluoresce and can readily be detected under an ultraviolet lamp, and labeled (radioactive) compounds can be detected using a radiation counter. 

Reaction of acetohexamide with 2,4-dinitrophenyl-hydrazine to produce the colored hydrazone was used by Amer and Walash (19) to determine the drug colorimetri-cally. The colored product was dissolved in KOH and determined at 480 nm. The accuracy of the method was claimed to be 100. A ninhydrin colorimetric method for some oral hypoglycemic agents was also reported (20b). 

Derivatization reactions are also commonplace in the determination of amino acids. One such reaction is that with ninhydrin-ascorbate. In the presence of amino acids, ninhydrin (triketohydrindane hydrate) is reduced to hydridantin by ascorbic acid while the amino acids undergo oxidative deamination with the formation of ammonium ion, which is condensed with hydridantin to yield a colored product with maximal absorbance at 405 and 575 nm. The procedure is often used for the determination of amino acids in protein hydrolysates. 

Ion-exchange chromatogram from Beckman-Spinco model 121MB amino acid analyzer introduced in 1969. After separation, amino acids were derivatized with ninhydrin to form colored products detected by visible absorption. [Courtesy Beckman-Coulter, Fullerton CA.1... 

The spectrophotometric detection of free and available cyanide can be achieved by the online reaction of the analyte with ninhydrin in carbonate medium to form a colored product (510 nm Xmax). Cyanides are removed from sample matrix by acidification through a gas-diffusion step incorporated in the SIA manifold shown in Figure 7.13. The assay was validated in terms of linearity (up to 200 pg/1), limit of detection (2.5 pg/1), limit of quantitation (7.5 pg/1), precision (RSD < 2.5% at 100 pg/1), and selectivity. All interferences related to ionic species are removed because just gaseous molecules diffuse. High tolerance against critical species such as sulfides and thiocyanates was achieved. The method was applied to the quantitation of cyanide in tap and mineral waters [30]. 

STEPS IN THE REACTION OF AN AMINO ACID WITH NINHYDRIN TO FORM A COLORED PRODUCT... 

Reaction of this amine with another molecule of ninhydrin forms an imine. Loss of a proton forms a highly conjugated (colored) product (Section 14.21). 

Ninhydrin to Detect Lipids on TLC Plates Ninhy-drin reacts specifically with primary amines to form a purplish-blue product. A thin-layer chromatogram of rat liver phospholipids is sprayed with ninhydrin, and the color is allowed to develop. Which phospholipids can be detected in this way ... 

Boil a few mg each of the product and of ninhydrin (Expt 5.99) in 0.5 ml of water the absence of a blue-purple coloration shows that the product is free from contaminating peptide material. 

Add 10 mL of distilled water to the reaction flask, grasp this with a clamp, swirl the mixture over a free flame to dissolve all the product, and cool under the tap. Remove a drop of the solution on a stirring rod, add it to 0.5 mL of a 0.3% solution of ninhydrin in water, and heat to boiling. If any unacetylated DL-alanine is present a purple color will develop and should be noted. Pour the solution into a 20 x 150-mm test tube and rinse the flask with a little water. Add 1.5 mL of concentrated ammonia solution, stir to mix, check the pH with Hydrion paper, and, if necessary, adjust to pH 8 by addition of more ammonia with a Pasteur pipette. Add 10 mg of commercial acylase powder, or 2 mL of fresh acylase solution, mix with a stirring rod, rinse the rod with distilled water, and make up the volume until the tube is about half full. Then stopper the tube, mark it for identification (Fig. 3), and let the mixture stand at room temperature overnight, or at 37°C for 4 h. 

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