Ninhydrin with amino acids

Ninhydrin Assay for Adsorbed Proteins. Measurements were made by a colorimetric procedure based on the reaction of ninhydrin with amino acids (25). The films were hydrolyzed in 5 ml of 2.5N NaOH for 2 hrs in capped plastic tubes in a boiling water bath. Then 1.5 ml of glacial acetic acid was added and mixed next I ml of ninhydrin reagent was added and mixed. [The reagent was three times more concentrated in ninhydrin, SnCb, and citrate than prescribed by Moore and Stein (25)]. The tubes were capped and boiled 20 mins more. The solution was clarified by centrifugation, and the absorbance read immediately at 570 nm on a Beckman DB spectrophotometer. If necessary, the sample was diluted with 50-50 2-propanol-water. Calibration curves (absorbance vs. fig of protein) were constructed in the 0-30 and 0-100 fig range with known amounts of each type of protein subjected to this same analysis procedure. 

The reaction itself is analogous to the reaction of ninhydrin with amino acids ... 

Very few ions are directly detectable by uv/vis absorption spectroscopy. However, if a postcolumn reaction is performed to chelate a cation with a chromophore (color producing group), to add a fluorescing agent, or to simply react the compound with another compound, such as using ninhydrin with amino acids, then uv/vis spectroscopy can be used. An example of such a system is the separation of lanthanides shown in the cation section. 

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

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

Example Mixture of amino acids obtained from protein hydrolysates are separated by this method and spots located by using Ninhydrin Reagent that forms a pink to purple product with amino acids. 

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. 

Part of the high molecular weight fractions contained ninhydrin-positive compounds displaying Rf 0 on TLC-plates (BUOH/HAC/H2O), characteristic of cross-links. To remove contaminating common amino acids, the selected fractions were further purified by adsorption chromatography on cellulose. Most of the amino acids eluted with BUOH/HAC/H2O, accounting for about three quarters of the total reactivity towards ninhydrin. The amino acids eluted with BUOH/HAC/H2O displayed spots with widely varying mobilities after TLC separation with the same eluent, whereas those eluted with water showed zero and very low mobility. 

Solvent systems used for thin layer chromatography were 1) n-butanol acetic acidiwater (4 1 5 upper phase), 2) acetic acid water (15 85), 3) ethyl acetate pyridine water (12 5 4), and 4) chloroform acetic acid water (50 45 5). Silica gel plates were used for chromatography of flavonoid aglycones and cellulose plates for all other components. Aluminum chloride was used for detection (under long UV light) of flavonoids, aniline phthalate for sugars, ninhydrin for amino acids and iodine for other components. Cellulose thick layer plates were developed with solvents 1 or 2. 

Specific reagents react with a particular class of compound, For example, rhodamine B is often used for visualization of lipids, ninhydrin for amino acids, and aniline phthalate for carbohydrates. 

The ninhydrin test. Amino acids with a free — NH2 group and proteins containing free amino groups react with ninhydrin to give a purple-blue complex. 

The postcolumn methods usually provide detectivity and are run online. The classic example, which has been mentioned several times, is the ninhydrin reaction with amino acids. For best results, the reaction must be fast and the mixing chamber efficient without introducing excessive dead volume. Most of the examples are in LC. 

DFP inhibits serum proteinase group of enzymes, including choline esterase. Ninhydrin deaminates amino acids and forms a blue complex with the NH3 released. 

In the amino acid analyzer, the components of the hydrolysate are dissolved in an aqueous buffer solution and separated by passing them down an ion-exchange column. The solution emerging from the column is mixed with ninhydrin, which reacts with amino acids to give the purple ninhydrin color. The absorption of light is recorded and printed out as a function of time. 

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. 

Reaction with ninhydrin Ninhydrin is a strong oxidizing agent. When a solution of amino acid is boiled with ninhydrin, the amino acid is oxidatively deaminated to produce ammonia and a ketoacid. The keto acid is decarboxylated to produce an aldehyde with one carbon atom less than the parent amino acid. The net reaction is that ninhydrin oxidatively deaminates and decarboxylates a-amino acids to C02, NH3 and an aldehyde. The reduced ninhydrin then reacts with the liberated ammonia and another molecule of intact ninhydrin to produce a purple coloured compound known as Ruhemann s purple. 

Aminoacylation A portion of the resin (0.9 g, 0.44 mmol) was transferred into a Wheaton glass vial. To a solution of a protected Fmoc-amino acid (1.5 mmol) and HOBt (203 mg, 1.5 mmol, 3.41 equiv.) in DMF (5 mL) was added DIG (237 pL, 1.5 mmol, 3.41 equiv.) and the reaction mixture was shaken for 20 min at rt. The activated amino acid solution was then added to the resin, and the mixture was shaken at rt for 3 h, or until a ninhydrin test was negative. The resin was then washed with DMF (twice), CH2GI2 (once), and DMF (once). The Fmoc group was removed by treatment with 50% piperidine in DMF (twice, 1 -r 15 min), and the resin was washed with DMF (four times). The substitution with amino acid, as determined by UV measurement of the dibenzofulvene-piperidine adduct (Xmax 302 nm) formed during the deprotection, was approximately 0.41 mmol g f... 

Figure 9.4 Hydrolysis of hippuric acid and estimation of the glycine released by ninhydrin. Ninhydrin reacts quantitatively with amino acids to give a purple colour which can be measured spectrophotometrically...

A principal question, the monitoring of the effluent from the column remains to be discussed here. Small amounts of the individual amino acids emerging in the sequence of their elution have to be revealed and quantitated. In earlier procedures these amounts were in the range of 10 to 100 nanomoles but with improvements in the methodology much smaller samples can now be applied. The classical color reagent for the detection of nanomole quantities is ninhydrin, which reacts with amino acids in a transamination-decarboxylation reaction to yield Ruhemann s purple ... 

The simpler nitrogenous constituents of wort consist principally of a-amino acids which can be estimated by the colour reaction either with indane-1,2, 3-trione hydrate (ninhydrin) or 2,4,6-trinitrobenzenesulphonic acid. With ninhydrin, a-amino acids develop a violet colour which can be measured at 570 nm while proline, an important imino acid in wort and beer, gives a yellow colour measured at 440 nm. 2,4,6-Trinitrobenzenesulphonic acid is more specific, for amino acids and does not react with proline or ammonia. It forms yellow derivatives which can be estimated colorimetrically at 340 nm. 

Earlier methods of estimating the a-amino nitrogen, due to Van Slyke, consisted of gasometric analysis of the nitrogen evolved when amino compounds were treated with nitrous acid or, alternatively of the carbon dioxide evolved on treatment with ninhydrin. The amino acids are readily absorbed on ion-exchange resins from which they can be eluted quantitatively either as a group... 

Some of the innumerable reagents used in TEC are based on reactions with more or less well-established mechanism. Eor example, sodium iodobismuthate (Dragendorff reagent) is widely used among others for alkaloids and quaternary ammonium compounds, 4-dimethylaminobenzaldehyde for primary amines and amino acids, 2,4-dinitrophenylhydrazine for aldehydes and ketones, ninhydrin for amino acids and some antibiotics, fluorescamine for primary and secondary amines, phosphomolybdic acid for lipids, various steroids, and other compounds, chlorine vapor followed by Kl/starch for amines and amides. More complex is the mechanism of the reactions with some other reagents, containing high concentrations of sulfuric acid, vanillin/sulfuric acid, phosphoric acid, aluminum chloride, antimony(III)... 

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