Bratton

Bratton, D. 1.., Tanaka, D. T, and Gnmstein, M. M. (1987). Effects of temperature on cholinergic contractility of rabbit airway smooth muscle. /. Appl. Physiol. 63, 1933-1941. 

Folic acid is detected by irradiating the chromatogram with broad-spectrum UV light for 30 min before reaction with Bratton-Marshall reagent (detection limit 200ng)[12]. 

For quantitative work, it is necessary to estimate the concentration of 5-amino-l-(P-D-ribofuranosyl)imidazole in aqueous solution. It seems that the only available method is the Bratton-Marshall assay, which was originally developed for the estimation of arylamines in biological fluids. The principle of the method is the spectrometric estimation of a salmon-pink colored dyestuff obtained by diazotation in situ, followed by coupling with /V-( 1 -naphthyl)ethyl-enediamine.65 The only remaining problem then is to know the molar extinction of this dye because pure samples of AIRs are not available. A value of 16800 at 520 nM was obtained for the dyes prepared from a model compound, 5-amino-l-cyclohexylimidazole-4-carboxylic acid (54), which is crystalline. A comparable molar extinction can be expected for the dye prepared from imidazole 55, if the carboxyl group does not exert too much influence on the chromophore. Actually, its influence is perceptible even with the naked eye, the dyestuff prepared from 53 having a somewhat different, wine-red color, with max>520 nM. The molar extinction for 55 is 17400 at 500 nM. When the decarboxylation of 54 was conducted under mild acidic conditions (pH 4.8, 50°C, 1 hour), estimation of 5-aminoimidazole 55 by the Bratton-Marshall method led to the conclusion that the reaction was almost quantitative.66 Similar conditions for the final decarboxylation were adopted in the preparation of samples of AIRs labeled with stable isotopes.58... 

However, more-rigorous treatment (5% acetic acid, 100°C, 17 hours) opened the imidazole ring and produced /V -cyclohexyl-a-formylaminoacetamidine (57), characterized as the crystalline picrate. Amidine 57 produced no dye in the Bratton-Marshall assay. The same behavior can be expected from AIR (46), although the product of hydrolytic ring-opening was not actually isolated. On the other hand, it was observed that a solution of AIRs (0.2 mM in 0.01-M ammonium hydroxide) prepared by biosynthesis, when stored at 4°C, did not change appreciably within a day. A decrease in the concentration of AIRs of about 30% occurred within a month. 

Sodium nitrite -l-a-naphthol or Bratton-Marshail reagent... 

The specific detection of aromatic nitro compounds is a second example. These can be converted by reduction to primary amines, which are then diazotized and coupled to yield azo dyes (cf. reagent sequence Titanium(III) chloride — Bratton-Marshall reagent ). Sodium nitrite —naphthol reagent, diazotized sulfanilic acid and other reagents specific for amino groups (e.g. ninhydrin, fluorescamine, DOOB, NBD chloride [9]) can also be used in the second stage of the reaction (Fig. 21). 

This combination of the modified Bratton-Marshall reaction with prechromato-graphic derivatization with N-methyl-N-(4-aminophenyl)-amine allows specific detection of isocyanates, that is especially applicable to aliphatic isocyanates. 

Primary aromatic amines are first diazotized by the action of sodium nitrite in acid solution and then coupled, for instance, with 1-naphthol to form azo dyes (cC Bratton-Marshall reagent, Vol. 1 a). ... 

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