Amino acid ninhydrin

With secondary amino acids, ninhydrin reacts to form a yellow complex with different absorbance characteristics. For this reason, detection occurs at both 570 and 440nm. 

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 mixture thereafter is fed into the photometer unit where the amount of light absorbed at specific wavelengths (see below) indicates the amount of each amino acid-ninhydrin complex. 

Amino acid Ninhydrin- collidine Isatin-Cd, immediate Isatin-Cd, > 12 hr at room temp... 

Electrophoresis, amino acids, ninhydrin, isatin, Sakaguchi reagents. Introduction... 

The analytical tools available in the early history of peptide research were rather poor. The starting point for analytical, at first qualitative, studies was probably the introduction of ninhydrin by S. Ruhemann (cf. p. 51) in 1910. For the determination of small amounts of a-amino acids ninhydrin is far superior to the combustion methods in use until then. 

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. 

Amines (primary), amino acids Ninhydrin Formation of reddish-bluish compounds... 

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 colour test is not specific for a-amino acids other primary amino compounds and also ammonia give a blue colouration with ninhydrin. 

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. 

Fig. 2. Amino acid analysis by automated ion-exchange chromatography. Standard column, 4.6 mm ID x 60 mm Ninhydrin developer. Computer print out indicates retention time (RT), height and area of peaks, and the ratio of the height of an amino acid in the sample to the height of a standard amino acid.

Thin-Layer Chromatography (tic). Tic (126) is used widely for quahtative analysis and micro-quantity separation of amino acid mixtures. The amino acids detected are developed by ninhydrin coloring, except for proline and hydroxyproline. Isatia has been recommended for specific coloring of pToline (127). 

The most widely appHed colorimetric assay for amino acids rehes upon ninhydrin-mediated color formation (129). Fluorescamine [38183-12-9] and (9-phthalaldehyde [643-79-8] are popular as fluorescence reagents. The latter reagent, ia conjunction with 2-mercaptoethanol, is most often used ia post-column detection of amino acids separated by conventional automated amino acid analysis. More recently, determiaation by capillary 2one electrophoresis has been developed and it is possible to determine attomole quantities of amino acids (130). 

Ninhydrin amino acids, and amines 0.3% ninhydrin in n-BuOH with 3% AcOH, followed by heating to 125°C/10 min blue spots... 

Amino acids have high melting or decomposition points and are best examined for purity by paper or thin layer chromatography. The spots are developed with ninhydrin. Customary methods for the purification of small quantities of amino acids obtained from natural sources (i.e. l-5g) are ion-exchange chromatography (see Chapter 1). For general treatment of amino acids see Greenstein and Winitz [The Amino Acids, Vols 1-3, J.Wiley Sons, New York 1961] and individual amino acids in Chapters 4 and 6. 

RS- P-Aminoisobutyric acid (a-methyl-P-alanine) [10569-72-9] M 103.1, m 176-178 , 178-180 , 181-182 , R -(-)- isomer [144-90-1] m 183 , [a] -21 (c 0.43, HjO), pKes,(,) 3.7, pKEst(2) 10.2. Colorless prisms from hot H O, were powdered and dried in vacuo. The purity is checked by paper chromatography (Whatman 1) using ninhydrin spray to visualise the amino acid Rp values in 95% MeOH and n-PrOH/5N HCOOH (8 2) are 0.36 and 0.50 respectively. [Kupiecki and Coon Biochem Prep 7 20 7960 Pollack J Am Chem Soc 65 1335 7943.] The R-enantiomer, isolated from iris bulbs or human urine was crystd from H2O and sublimed in vacuo [Asen et al. J Biol Chem 234 343 7959]. The RS-hydrochloride was recrystd from EtOH/Et20 m 128-129 , 130° [Bbhme et al. Chem Ber92 1258, 1260, 1261 7959]. 

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]. 

The blue-violet stain which forms on thin-layer chromatograms when amino acids are stained with ninhydrin is only stable for a short time. It rapidly begins to fade even on cellulose layers. The stability can be appreciably enhanced by complex formation with metal ions [3]. 

The chromatograms stained with ninhydrin are immersed in the reagent solution for 1 s or sprayed evenly with it and then placed in the free half of a twin-trough chamber containing 25% ammonia solution. Apart from proline and hydroxyproline, which yield yellow copper complexes, all the amino acids yield reddish-colored chromatogram zones [3],... 

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... 

The percentages of amino acids in silk fibroin which Poison et al. (224) found by direct visual and indirect photometric analysis of ninhydrin paper-partition chromatograms are shown in Table VII. The percentages obtained for alanine, glycine, and serine appear to be reasonably accurate, inasmuch as they agree closely with those found by other methods. It would be of interest to determine alanine by the microbiological method reported recently by Sauberlich and Baumann (238), in view of the widely different values found for this amino acid by the described ninhydrin-chromatographic procedure and the selec-... 

Amino Acids. Early observations on the liberation of amino acids by plant roots were reviewed by Loehwing (94), Rademacher (121), and Borner (12). Free amino acids have been isolated from soil fractions (119), and the excretion of a variety of ninhydrin-positive compounds by plant roots has been demonstrated under controlled conditions by Katznelson et al. (18), Rovira (121), and Pearson and Parkinson (115). 

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. 

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