Diphenylamine, color reactions

The well-known diphenylamine color reaction of nitric acid depends on the oxidation of diphenylamine (I) through colorless N,N -diphenylbenzidine (II) to the blue quinoid imonium ion (III) ... 

In the derivatization of sugars with aniline-diphenylamine reagent for example, this leads to unsatisfactory irregular coloration. The standard deviation for the method deteriorates from 2 to 3% to 5 to 8%. For this reason color reactions should be avoided for direct quantitation if it is possible to scan in the UV range without derivatization. 

The total content of RNA + DNA in tissues may be estimated from the phosphorus content or by color reactions of the sugars.37 545 These reactions depend upon dehydration to furfural or deoxyfurfural by concentrated sulfuric acid or HC1 (Eq. 4-4). Furfural formed from RNA reacts with orcinol (3,5-dihydroxy-toluene) and ferric chloride to produce a green color useful in colorimetric estimation of RNA. A similar reaction of DNA with diphenylamine yields a blue color. 

TNT forms charge-transfer, or 7r, complexes with polycyclic aromatic hydrocarbons, aromatic amines, and aromatic nitro compds a number of these are listed below in Table 2. The complexes with three amines (diphenylamine, diethyl-aniline, p-anisidine) have characteristic colors this forms the basis for a rapid and convenient thin-layer chromatographic analytical procedure (Ref 34) for the identification of very small amounts of TNT. (For a discussion of the many color reactions of TNT, and of composite expls containing it, see Vol 3, C405-L ff)... 

Bailey, R.W. and Bourne, E.J., Color reactions given by sugars and diphenylamine-aniline spray reagents on paper chromatograms, J. Chromatogr., 4, 206, 1960 Chem. Abs., 55, 4251c, 1961. 

The detection of nitrates and nitrites present in FDR using a color reaction with diphenylamine in sulfuric acid was first applied to firearms related examinations about 1911, and in 1914 Doctor Iturrioz used paraffin wax as a lifting medium for propellant residue on clothing prior to treatment with... 

Deveaux et al. [21] reported that mefenamic acid, being an N-aryl derivative of anthranilic acid, can be characterized by two color reactions. The color reactions, negative with N-aryl derivatives of aminonicotinic acid, are associated with the diphenylamine structure. For the first color reaction, add to a test tube approximately 0.5 g of oxalic acid dihydrate and at least 1 mg of the test material. Place the tube into an oil bath at 180-200°C for 4-5 min. After cooling, dissolve the residue in 10 mL of 95% ethanol to obtain a stable, intense blue solution (absorption maximum at 586-590 nm). To use the reaction for capsule formulations, extract the active ingredient with acetone and filter prior to the assay. For the second color reaction, add mefenamic acid (100-800 pg) in 1 mL of H0Ac-H2S04 (98 2, v/v), 5 mL of HOAc-HCl (50 50, v/v) and 1 mL of 0.10% (w/w) aqueous levulose. Heat the mixture for 25 min at 100°C, and after cooling, measure the absorbance at 597 nm. 

Qualitatively, the presence of the nonulosaminic acids is best indicated by the brilliant-red coloration formed on addition of an acidic solution of p-dimethylaminobenzaldehyde (the so-called direct Ehrlich reaction ), and by the bright-purple coloration which develops upon boiling with Bial s reagent for several minutes at 100°. In addition, the nonulosaminic acids give a blue-violet coloration with Dische s diphenylamine reagent for deoxypentoses and a positive reaction in the tryptophan-perchloric acid test. Since no single one of these color reactions is absolutely specific for a nonulosaminic acid, it is advisable to carry out at least two of these for a qualitative analysis. For quantitative determinations, all four reactions have been employed, using either A/ -acetylneuraminic acid (m. p., 183-185° [a] —32.0°) or methoxyneuraminic acid [m. p., 200° (dec.) [a]o —55.0°] as colorimetric standards. 

A more sensitive test for nitrocellulose is provided by an intense blue color reaction when a drop of a solution of diphenylamine in concentrated H2SO4 (5% w/v) is added to the sample in the absence of other oxidizing agents. 

The color reaction of triphosgene with Harrison s reagent (a mixture of 4-(N,N-dimethylamino)benzaldehyde and diphenylamine in a non-aqueous solvent) yields a yellow to deep-orange hue, according to the concentration [19]. 

Indole has been separated from carbazole and diphenylamine on silica gel, copper sulphate-impregnated silica gel, on alumina and on cellulose with aqueous and non-aqueous systems (212). The color reactions with six different spray reagents following two-dimensional separations on silica gel H have been studied (213) for a wide range of indoles and skatole on cellulose detected by its fluorescence with 2,4-dinitrophenylpyridinium chloride (213a). Indole and oxindole alkaloids have been separated in 10 neutral and 10 alkaline solvents (214). 

Diphenylamine or N,N-diphenylbenzidine dissolved in concentrated sulfuric add, is oxidized by antimony pentoxide to blue quinimine dyestuffs. The chemistry of the color reaction is the same as that of these amines by nitric acid (see page 359). 

Chlorine dioxide is produced if chlorates are warmed with concentrated sulfuric acid. Like free chlorine, this product converts diphenylamine (dissolved along with trichloroacetic acid in ethyl acetate) into a blue-green oxidation product (compare page 359). This color reaction is not shown by bromates and iodates because they are weaker oxidants than chlorates. 

The sensitive color reactions for nitrates with brucine or diphenylamine are not applicable in the presence of nitrites which give similar reactions. Hydrazoic acid can be used to destroy the nitrite beforehand ... 

Perhaps the main disadvantage of the diphenylamine reaction for de-soxypentose is the lack of sensitivity. In addition, interference in the test by some substances has been shown by certain investigators (17,21,23,44). Three recently proposed methods deserve serious consideration. Cohen (23) has developed a color reaction with tryptophane, while Stumpf (98) has standardized the cysteine reaction originally proposed by Dische (40). Both methods are accurate in the range of 50-500 / g. of DNA. Ceriotti (21) has developed a micromethod (2.5-15 /xg. of DNA) using the reaction of indole with DNA, followed by extraction of the yellow pigment with chloroform. 

Caldwell and Hinshelwood (17) have applied the orcinol and diphenylamine reactions to whole bacterial suspensions or their hot tridiloroacetic acid extracts and have shown that, by extrapolation of the time curves of the color reactions, the additional amount of coloration due to interfering material can be eliminated. 

Color reactions. Two very old methods, which are still widely used, depend on the hydrolysis of the glycosidic linkage of purine nucleosides by hot concentrated acids, with the subsequent transformation of the sugar into furfural derivatives. These derivatives, in turn, give specific color reactions with such aldehyde specific reagents as orci-nol, which reacts with furfural (from ribose) and ferric chloride to give a green color the intensity of this color is proportional to the ribose concentration. A blue color is obtained with deoxyribose in concentrated sulfuric acid with diphenylamine (7). 

Dialkylanilines and diphenylamines can also be detected by reaction with 5-nitroisatin (26). Many color reactions are based on oxidation with inorganic reagents FeCla (27, 28), potassium dichromate, and sxilfuric acid (28 — 30), and hydrogen peroxide in the presence of V2O5 (31), or a number of other reactions (35 — 53). 

Organic esters of nitric acid give a color reaction with ferrous sulfate and cone, sulfuric acid [this reaction is also given by nitramines (30, 31)] or with phenoldisulfonic acid (32), and also with diphenylamine in sulfuric acid [see also nitramines (33, 34)]. 

Ayala, W., Moore, L. V., and Hess, E. L., 1951, The purple color reaction given by diphenylamine reagent. I. With normal and rheumatic fever sera, J. Clin. Invest. 30 781. 

Note This reagent sequence is a modiflcation of the reagent chlorine — potassium iodide — starch . Mobile phases containing ammonia must be removed completely before treatment with the reagent sequence, since otherwise the background will be colored too. Some secondary amines (e.g. diphenylamine) and some amides (e.g. 2,4-dinitrobenzamide) and methionine sulfoxide do not give reactions even in quantities of up to 1 to 2 (ig. 

The mechanism of the reaction has not been elucidated. Presumably several reactions occur simultaneously. Thiocyanates react with iron(III) salts with the formation of red-colored complexes. In sulfuric acid medium nitrate or nitrite coddize diphenylamine to... 

Stability. In order to have maximum effectiveness over long periods of time, an antioxidant should be stable upon exposure to heat, light, oxygen, water, etc. Many antioxidants, especially in the presence of an impurity when exposed to light and oxygen, are subject to oxidation reactions with the development of colored species. Alkylated diphenylamines are least susceptible and the />-phenylenediamine derivatives the most susceptible to direct oxidation. 

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