Ninhydrin react with amino acids

Amino acids are colorless, so a detection technique is necessary in order to visuafize the location of the various spots. The most common method involves treating the filter paper or gel with a solution containing ninhydrin followed by heating in an oven. Ninhydrin reacts with amino acids to produce a purple product. 

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. 

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 ... 

Amino add raagants reagents for the colorimetric identification and quantitation of amino acids. One of the most important is ninhydrin (2,2-dihy-droxy-lH-indene-13(2H)-dione), which reacts with amino acids to form a blue-violet dye called Ruhe-mann s purple (absorbance maximum 570 nm). With the imino acid, proline, ninhydrin forms a yellow product, absorbance maximum 440 nm. 

Ninhydrin (Section 23.3) The hydrate of indan-l,2,3-trione, a molecule that reacts with amino acids to give the purple dye used in quantitative analysis of amino acids. 

Ninhydrin is a compound that reacts with amino acids and proteins to produce a dark-colored complex. It is used by forensic chemists and detectives to see fingerprints that might otherwise be invisible. Ninhydrin s composition is 60.68% carbon, 3.40% hydrogen, and 35.92% oxygen. What is the empirical formula for ninhydrin ... 

The amino acid analyzer (Section 26-5) detects the elution of any amino acid by the presence of an indicator that turns a deep violet color. This indicator is ninhydrin A, which reacts with amino acids to the purple compound B ( Ruhemaim s purple ), generating as by-products an aldehyde and CO2. 

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. 

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. 

Fluorimetric methods for the determination of amino acids are generally more sensitive than colorimetric methods. Fluorescamine (4-phenyl-spiro[furan-2(3H),l -phthalan]-3,3 -dione) and o-phthaldialdehyde (OPA) substances are used for protein analysis. Fluorescamine reacts with amino groups to form fluorophores that excite at 390 nm and emit at 475 nm (Weigele et al., 1972). Applications of fluorescamine include monitoring the hydrolysis of K-casein (Beeby, 1980 Pearce, 1979) and quantification of proteins, peptides, amino acids in extracts (Creamer et al., 1985). OPA produces fluorescence on reaction with 2-mercaptoethanol and primary amines, with strong absorption at 340 nm. Lemieux et al. (1990) claimed that this method was more accurate, convenient, and simple for estimating free amino acids than the TNBS, ninhydrin, or fluorescamine methods. 

Ninhydrin is a common reagent for visualizing spots or bands of amino acids that have been separated by chromatography or electrophoresis. When ninhydrin reacts with an amino acid, one of the products is a deep violet, resonance-stabilized anion called Ruhemann s purple. Ninhydrin produces this same purple dye regardless of the structure of the original amino acid. The side chain of the amino acid is lost as an aldehyde. 

The second method involves staining larval entry holes in pulse seeds. In the third method, ninhydrin (triketohydrindene hydrate) has been used to react with the body fluids of insects developing inside grains. A purple spot develops when ninhydrin reacts with the free amino acids (and keto acids) present in the body fluids of insects (Dennis and Decker, 1962). Ninhydrin at a 0.3% level in acetone is impregnated into filter paper. When infested grains are crushed between the folds of ninhydrin-treated paper, the body fluids from the crushed insect bodies react with ninhydrin in the paper. Purple spots develop in the paper in 20-30 minutes at room temperature (20-25 °C), the number of spots indicating the number of insects present. 

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. 

Ninhydrin reacts with an amino acid to form a purple-colored compound. Propose a mechanism to account for the formation of the colored compound. 

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...

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... 

A dye commonly used to detect amino acids is ninhydrin (1,2,3-indanetrione monohydrate). Ninhydrin reacts with a-amino acids to produce an aldehyde, carbon dioxide, and a purple-colored anion ... 

Even today, the postcolumn derivatization with ninhydrin introduced by Spademan, Stein, and Moore [29] represents the most common detection method for quantitative amino acid analysis. As a strong oxidant, ninhydrin reacts with the a-amino groups of eluting amino acids at temperatures around 130 °C, according to Eq. (5.3), releasing carbon dioxide. 

On soft materials, chemical treatments are generally used to visualize latent prints. Ninhydrin, 26.35, reacts irreversibly with amino acids to give a dark-purple product and works well on paper and porous surfaces. It is less useful on wetted items or painted materials. Ninhydrin prints may be treated with zinc(II) chloride, which makes them fluoresce under an argon laser. A related dye is l,8-diazafluoren-9-one, 26.36, which is about 2.5 times as sensitive as ninhydrin. 

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