Ninhydrine

D) Spraying solution. A solution of 0-2 g. of ninhydrin in a mixture of 99 ml. of redistilled n-butanol and i ml. of glacial acetic acid. 

Then remove the strip and dry it in a stream of cold air, either from a blower, or by pinning it to the lower edge of a fume-cupboard window having a vigorous draught already in operation. Then spray the strip lightly but uniformly with the ninhydrin solution (D) in a fume-cupboard, and dry as before. 

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

Ninhydrin (also named 1 2 3-triketoindane or 1 2 3-triketohydrindene hydrate) is prepared most simply from the inexpensive phthahc anhydride (I). The latter is condensed with acetic anhydride In the presence of potassium acetate to give phthalylacetlc acid (II) reaction of the latter with sodium methoxide in methanol yields 1 3-indanedionecarboxyhc acid, which is decomposed upon warming with dilute hydrochloric or sulphuric acid to indane-1 3-dione (or 1 3-diketohydrindene) (HI). Selenium dioxide oxidation of (III) afibrds indane-1 2 3-trione hydrate (ninhydrin) (IV). 

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]. 

Diisopropyiethyiamine [7087-68-5] M 129.3, b 127 , pKe i -10.9. Distd from ninhydrin, then from KOH [Dryland and Sheppard, J Chem Soc, Faraday Trans I 125 1986]. It is a strong base and should be stored in the absence of carbon dioxide. 

Ninhydrin (1,2,3-triketohydrindene hydrate) [485-47-2] M 178.1, m 241-243 (dec), pK 8.82. Crystd from hot water (charcoal). Dried under vacuum and stored in a sealed brown container. 

Reactions can also occur during chromatographic development. These can either be undesired reactions or planned derivatizations. Thus, Weicker and Brossmer [11] have reported, for example, that hexoses, pentoses and disaccharides can be ammated when ammonia-containing mobile phases are employed on silica gel G layers. On the other hand, fluorescamine or ninhydrin have been added to the... 

Smith [203] has described a special procedure for distilling reagents homogeneously onto a TLC plate. Ripphahn [179] later started with a TLC plate that had been dipped into ninhydrin solution, laid a 0.1 mm thick terephthalate... 

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],... 

When staining with ninhydrin the appearance of colors of various hues on TLC layers with and without fluorescence indicators is probably a result of complex formation between the ninhydrin zones and the cations of the inorganic fluorescence indicators. 

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