Amino acid ninhydrin reaction

The peptide/polypeptide product is usually hydrolysed by incubation with 6 mol l-1 HC1 at elevated temperatures (110 °C), under vacuum, for extended periods (12-24 h). The constituent amino acids are separated from each other by ion-exchange chromatography and identified by comparison with standard amino acid preparations. Reaction with ninhydrin allows subsequent quantification of each amino acid present. 

Figure 8.2— Visualisation of proteins and amino acids by reaction with ninhydrin. This reagent, after a series of reactions, yields a Ruhemann scolouration. This is one of the reagents that can be used to determine the position of compounds that have migrated on the electrophoretic gel.

The column eluate is mixed with ninhydrin reagent and the mixture passed through the high-temperature reaction coil where the amino acid-ninhydrin complex is formed. (The amount of the coloured ninhydrin complex formed is directly proportional to the quantity of amino acid present in the eluate.)... 

The simplest method for the determination of amino acids is reaction with ninhydrin. Ninhydrin reacts with both primary and secondary amino acids to produce Ruhemann s purple, which can be detected by ultraviolet (UV)-visible spectroscopy. The reaction requires heat, and a reducing agent is generally added to stabilize the color formation. Primary amines are detected with the greatest sensitivity at 570 nm, while the absorption maximum for secondary amines is 440 nm. If both primary and secondary amines are to be determined, a common absorption wavelength of 500 nm is employed however, this leads to decreased sensitivity. Under optimal... 

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. 

Since the amino acid is destroyed during ninhydrin derivatization and forms the same reaction product regardless of the original amino acid (except for the secondary amino acids), ninhydrin derivatization wdl only work as a postcolumn method. 

Much more important than these reactions is the color test of amino acids with ninhydrin (78 — 81). The reactions with isatin or alloxan (82) belong to the same group. The course of the reaction of amino acids with ninhydrin is not yet completely understood. At present it is supposed that in the first step Strecker s degradation of amino acids takes place accompanied by the formation of carbon dioxide, ammonia, and an aldehyde possessing one carbon atom less than the original amino acid. Ninhydrin (I) is supposed to be reduced simultaneously to diketohydrindol (II), which reacts with the liberated ammonia and a second molecule of ninhydrin to form the intensely blue anion of diketohydrindenediketohydrindamine (III). 

The automated amino acid analy2er depends on ion-exchange chromatography (117) and is now a routine tool for the analysis of amino acid mixtures (118). This most advanced machine can detect as Htde as 10 pmol in ninhydrin reaction analysis. One-half to two hours are required for each analysis. An analysis chart is shown in Figure 2. 

It is known that not all reactions proceed in the same manner on all adsorbent layers because the material in the layer may promote or retard the reaction. Thus, Ganshirt [209] was able to show that caffeine and codeine phosphate could be detected on aluminium oxide by chlorination and treatment with benzidine, but that there was no reaction with the same reagent on silica gel. Again the detection of amino acids and peptides by ninhydrin is more sensitive on pure cellulose than it is on layers containing fluorescence indicators [210]. The NBP reagent (. v.) cannot be employed on Nano-Sil-Ci8-100-UV2S4 plates because the whole of the plate background becomes colored. 

Differences in the materials employed for the layers can also become evident when chemical reactions are performed on them. Thus, Macherey-Nagel report that the detection of amino acids and peptides by reaction with ninhydrin is less sensitive on layers containing luminescent or phosphorescent indicators compared to adsorbents which do not contain any indicator [7]. 

In the ion-exchange technique, separated amino acids exiting (eluting) from the end of the chromatography column mix with a solution of ninhydrin and undergo a rapid reaction that produces an intense purple color. The color is detected by a spectrometer, and a plot of elution time versus spectrometer absorbance is obtained. 

Because the amount of time required for a given amino acid to elute from a standard column is reproducible, the identities of the amino acids in a peptide can be determined. The amount of each amino acid in the sample is determined by measuring the intensity of the purple color resulting from its reaction with ninhydrin. Figure 26.3 shows the results of amino acid analysis of a standard equimolar mixture of 17 a-amino acids. Typically, amino acid analysis requires about 100 picomoles (2-3 /xg) of sample for a protein containing about 200 residues. 

The reaction of ninhydrin with an < -amino acid occurs in several steps. 

Draw resonance forms for the purple anion obtained by reaction of ninhydrin with an n-amino acid (Problem 26.53). 

Nicotine, structure of, 30, 916 Ninhydrin, reaction with amino acids, 1030... 

Reagents which form a derivative that strongly absorbs UV/visible radiation are called chromatags an example is the reagent ninhydrin, commonly used to obtain derivatives of amino acids which show absorption at about 570 nm. Derivatisation for fluorescence detectors is based on the reaction of non-fluorescent reagent molecules (fluorotags) with solutes to form fluorescent... 

Post-column reaction is a common feature of many special types of analyses, the most well-known being the amino acid analyzer that uses ninhydrin with a post-column reactor to detect the separated amino acids. In general, derivatization and post-column reactor systems are techniques of last resort. In some applications they are unavoidable, but if possible, every effort should made to find a suitable detector for the actual sample materials before resorting to derivatization procedures. 

Tible 1 Color reactions of amino acids with fluorescein isothiocyanate — ninhydrin (extract from original table). 

Note It is possible to differentiate amino acids by color on the basis of the markedly different shades produced [2, 3]. Proline and hydroxyproline, that only react weakly with ninhydrin, also yield pink-red colored derivatives [2]. Ergot alkaloids and LSD are detected by spraying with 10% hydrochloric add and then heating to 110°C for 20 min after they have been treated with the reagent [9]. Ergot alkaloids and LSD yield red to purple zones when treated in this manner other alkaloids, e.g. reserpine, emetine, quinine, strychnine, pilocarpine, atropine, scopolamine, cocaine and opium alkaloids, do not give a reaction [9]. 

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. 

There are many proteins in the human body. A few hundreds of these compounds can be identified in urine. The qualitative determination of one or a series of proteins is performed by one of the electrophoresis techniques. Capillary electrophoresis can be automated and thus more quantified (Oda et al. 1997). Newer techniques also enable quantitative determination of proteins by gel electrophoresis (Wiedeman and Umbreit 1999). For quantitative determinations, the former method of decomposition into the constituent amino acids was followed by an automated spectropho-tometric measurement of the ninhydrin-amino add complex. Currently, a number of methods are available, induding spectrophotometry (Doumas and Peters 1997) and, most frequently, ELISAs. Small proteins can be detected by techniques such as electrophoresis, isoelectric focusing, and chromatography (Waller et al. 1989). These methods have the advantage of low detection limits. Sometimes, these methods have a lack of specifidty (cross-over reactions) and HPLC techniques are increasingly used to assess different proteins. The state-of-the-art of protein determination was mentioned by Walker (1996). 

The analysis of amino acids involves chromatographic issues similar to those encountered in analysis of simple amines. Underivatized amino acids have, with a few exceptions, weak UV absorbance and a strong tendency to interact with stationary phases in undesirable ways. Underivatized amino acids are normally separated with ion exchange chromatography, then visualized post-column by reaction with ninhydrin, o-phthaladehyde (OPA), or other agents. Underivatized tryptophan and the metabolites kynurenine, 3-hydroxykynurenine, kynurenic acid, and 3-hydroxyanthranilic acid, were separated on a Partisphere 5-p ODS column with fluorescent detection.121... 

Ninhydrin Reaction with Amino Acids, Biochemistry Survey Lecture. D. S. Moore, Department of Chemistry, Howard University,... 

A systematic investigation of the free amino acids of the Leguminosae led to the isolation of a novel ninhydrin-positive compound from the leaves of Derris elliptica Benth. (Papilionidae) (93). This substance was analyzed as C6H,3N04 (microanalysis and high resolution mass spectrometry) and was shown to be an amino alcohol. The absence of a carbonyl in the 1R, the loss of 31 mass units in the mass spectrum, and a positive periodate cleavage reaction were best embodied into a dihydroxydihydroxymethylpyrrolidine structure. The relative simplicity of the NMR spectra (three peaks in the 13C spectrum four spin-system in the H spectrum) pointed out a symmetrical structure. Inasmuch as the material was optically active ([a]D 56.4, c = 7, H20), meso structures were ruled out, and the 2R, 3R, 4R, 5R relative configuration was retained (93). This structure (53) was further confirmed by an X-ray determination (94). 

The improved methods introduced in the years 1940 to 1945 created new possibilities in this field. The Van Slyke method for quantitative determination of amino acids, based on the measurement of the volume of carbon dioxide evolved in the course of the reaction between amino acids and ninhydrin (V2), was much more reliable than the older methods. At the same time the microbiological methods designed for amino acid determinations (D3, S6) made possible the detection of very small concentrations of these compounds. The application of these... 

Ninhydrin, 22 101 Ninhydrin-color reaction amino acids, 2 570 Niobates, 27 152-153 24 315 Niobia-phosphate catalytic aerogels, 2 763t Niobic acid, 27 152 Niobic salts, 27 152-153 Niobium (Nb), 27.132-157 24 313, 315. See also Nb-Ti entries Niobium compounds Niobium metal analytical methods for, 27 142-144 dissolution methods for, 27 142 economic aspects of, 27 140-142 effect on stainless steel corrosion resistance, 7 809... 

The post-column ninhydrin reaction proceeds in a reaction coil at elevated temperatures (ca. I30-I35°C). Ninhydrin reacts with primary amino acids to give a chromophore with a wavelength of maximum absorption of 570nm. 

Ninhydrin Amino acid Ruhemann s purple Simplified reaction between ninhydrin and primary amino acids... 

Ninhydrin (a chromatag is commonly employed to yield corresponding derivatives of amino acids that show absorption specifically at about 570 nm as shown in the following reaction ... 

Ninhydrin (triketohydrindene hydrate) reacts with an amino acid when heated under acidic conditions (pH 3-4) to produce ammonia, carbon dioxide and a blue-purple complex. This reaction forms the basis of many widely used methods (Figure 10.11). One mole of carbon dioxide is liberated from each mole of amino acid, exceptions being the dicarboxylic amino acids, which produce two moles of carbon dioxide, and the a-imino acids, proline and hydroxyproline, which do not produce carbon dioxide. Although this formed the basis of a gasometric technique, colorimetric methods are now the most common. 

Amines other than a-amino acids will also give a colour reaction with ninhydrin but without the production of carbon dioxide. Thus /3-, y-, 8- and e-amino acids and peptides react more slowly than a-amino acids, to give the blue complex, while imino acids result in the formation of a yellow-coloured product which can be measured at 440 nm. Removal of substances such as protein, ammonia and urea from biological samples may be necessary in quantitative work because they also react in a similar manner. 

The ninhydrin colour reaction has proved very useful in qualitative work and is widely used in the visualization of amino acid bands after electrophoretic or chromatographic separation of mixtures. The reagent used in such circumstances is usually prepared in ethanol and, if 2,4,6-collidine is added, the variations in colour produced by different amino acids will aid their identification (Table 10.6). 

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