Proline ninhydrine reaction

All primary amines react with fluorescamine under alkaline conditions (pH 9-11) to form a fluorescent product (Figure 10.12) (excitation maximum, 390 nm emission maximum, 475 nm). The fluorescence is unstable in aqueous solution and the reagent must be prepared in acetone. The secondary amines, proline and hydroxyproline, do not react unless they are first converted to primary amines, which can be done using A-chlorosuccinimide. Although the reagent is of interest because of its fast reaction rate with amino acids at room temperature, it does not offer any greater sensitivity than the ninhydrin reaction. 

Ninhydrin Reaction. A neutral solution of an amino acid will react with ninhydrin (triketohydrindene hydrate) by heating to cause oxidative decarboxylation. The central carbonyl of the tnketone is reduced to an alcohol. This alcohol further reacts with ammonia formed from the amino add and causes a red-purplish color. Since the reaction is quantitative, measurement of the optical density of the color produced is an indication of amino add concentration. Imino acids, such as hydroxyproline and proline, develop a yellow color in the same type of reaction. 

Keywords large scale, ninhydrin, L-proline, cascade reaction, zwitterion, waste-free, solid-solid reaction... 

Two types of detection system are in common use with amino acid analysers, ninhydrin detection and fluorescence detection. The two systems differ in that fluorescence detection is more sensitive than ninhydrin detection, but it is more specific in that it does not detect amino acids such as proline. The detection reagent is mixed with the eluate from the column and the mixture passes into the fluorimeter or spectrophotometer. The system described here is based on the ninhydrin reaction with the separated amino acids. 

Al. Alexeenko, L. P., and Orekhovich, V. N., A method for proline and hydroxyproline determination by means of ninhydrin reaction in acid medium after starch chromatography. Dokl. Akad. Nauk SSSR 133, 690-693 (1960). 

The ninhydrin reaction is not restricted to a-amino acids, however. It is well known that imino acids, notably proline, but also for example pipecolic acid, in solution produce a different color with an absorption maximum at 440 nm. Primary aliphatic amines react likewise with ninhydrin to give Ruhe-man s purple but the color yield is lower than for a-amino acids. Peptides, amino acids with secondary amino groups, mainly N-methylamino acids, and secondary amines also react with ninhydrin, although often more slowly and with smaller yield than the a-amino acids. Tertiary amines and all aromatic amines do not react with ninhydrin. Ammonia itself reacts to give a color with ninhydrin, although with a rather low yield. 

Six to eight standard dilutions in an appropriate concentration range for each amino acid are prepared 2 ml of amino acid solution and 2 ml of buffered ninhydrin are mixed in a test tube, heated in a boiling water bath for 15 min, cooled to room temperature, and 3 ml of 50% ethanol added. The extinction is read at 570 nm (or 440 nm for proline) after 10 min. Standard plots of concentration versus absorbance are drawn for each amino acid. The scraped layer corresponding to each spot is extracted with 70% ethanol in a known minimum volume, and ninhydrin reaction is performed followed by spectrophotometry. The concentration of unknown samples is read from the standard plots. TLC/den-sitometry was used to determine 0.5 mg/L of phenylalanine in blood serum as an indicator of phenylketonuria (181). 

Proline, in which the a-fflnino group is secondary, gives an orange compound on reaction with ninhydrin. 

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 Proline Yellow chromophore Simplified reaction between ninhydrin and secondary amino adds... 

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. 

Quantitation of the amino acids proceeds through the postcolumn reaction with ninhydrin at 120-135°C, yielding a purple complex for the primary amino acids (absorbance measured at 570 nm) and a yellow complex for the amino acids proline and hydroxyproline (absorbance at 440 nm). 

Figure B2.2.2 Reaction of ninhydrin with proline or hydroxyproline, producing the yellow product.

Detection of amino acids is typically by UV absorption after postcolumn reaction with nin-hydrin. Precolumn derivatization with ninhydrin is not possible, because the amino acids do not actually form an adduct with the ninhydrin. Rather, the reaction of all primary amino acids results in the formation of a chromophoric compound named Ruhemann s purple. This chro-mophore has an absorption maximum at 570 nm. The secondary amino acid, proline, is not able to react in the same fashion and results in an intermediate reaction product with an absorption maximum at 440 nm. See Fig. 5. Detection limits afforded by postcolumn reaction with ninhydrin are typically in the range of over 100 picomoles injected. Lower detection limits can be realized with postcolumn reaction with fluorescamine (115) or o-phthalaldehyde (OPA) (116). Detection limits down to 5 picomoles are possible. However, the detection limits afforded by ninhydrin are sufficient for the overwhelming majority of applications in food analysis. 

A stoichiometric mixture of ninhydrin and L-proline 2 (200 g) was milled in a 2-L horizontal ball-mill (Simoloyer ) with steel balls (100Cr6, 2 kg, diameter 5 mm) at 1100 min 1 for 40 min until the liberation of C02 was complete. The temperature varied from 15 °C at the water cooled walls to 21 °C in the center. The power was 800 W. Quantitative reaction to give 3 was secured by weight (146 g, 100%) and by spectroscopic techniques. The product was not separated in a cyclone but the milling-out towards the end was completed with 4 times 250 mL of water, each. This part of the highly disperse (< 1 pm) pure azo-methine ylide 3 was obtained after centrifugation and drying in a vacuum. The combined water phase contained 0.2 g of 3. 

Spray the paper chromatogram lightly with ninhydrin reagent and heat for 5 min in a 100°C oven. The amino acids will appear as blue or purple spots (except proline, which will appear yellow). Figure 6-9 shows the reaction that amino acids undergo with ninhydrin. 

Condensation with ninhydrin. Proline condenses with one equivalent of ninhydrin to give proline yellow (3) and with two equivalents to give proline red (4), which forms a deep blue anion. In the first case, the reaction mixture smells strongly of pyrrolidine. An identical red pigment results from condensation of ninhydrin with pyrrolidine. 

Ninhydrin (triketohydrindene hydrate) reacts with a-amino acids to produce CO2, NH3, and an aldehyde with one less carbon than the parent amino acid. In most cases, a blue or violet compound (proline and hydroxyproline give a yellow color) is formed owing to reaction of the liberated NH3 with ninhydrin, as shown in Figure 2-11. Color and CO2 production provide a basis for the quantitative determination of amino acids. 

The reaction with the amino acid proline results in a yellow derivative. The amount of each amino acid is then determined by measuring the absorption of light by the ninhydrin derivatives. 

Secondary amines and amino acids react in an entirely different way. Indeed, L-proline forms a mixture of the four possible tautomers, but, after a time, ninhydrin-positive substances may be formed by breakdown of the proUne molecule. (Other ketoses do not give this reaction.) Secondary amines, such as piperidine, will not react at 0°, but, at higher temperatures (40-60°), some D-fructosylamine is formed, together with a higher proportion of D-glucose by isomerization. Morpholine or dicyclohexylamine give a 20 to 25% yield of D-n6o-hexulose, and may be used on a preparative... 

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