Benzidine blue

Identification of free radicals. The existence of free radicals in carbon has been shown by electron spin resonance (e.s.r.) techniques by a large number of scientists, notably by Bennett, Ingram, and Tapley (95), Etienne and Uebersfeld (96a), Uebersfeld etal. (966),andMrozowski and Andrew (97). It is not possible to summarize here all publications dealing with e.s.r. measurements on carbon. Attempts at chemical identification by combination with free radicals were made. The reaction with benzidine blue was used by Garten and Sutherland (98). The reagent is obtained by controlled oxidation of benzidine. It is very unstable, however, and other methods proved to be better suited. 

Nickel(II) hydroxide test The auto-oxidation of sulphur dioxide (or sulphurous acid) induces the oxidation of green nickel(II) hydroxide to the black nickel(III) hydroxide. The colour change is quite distinct, but for very small amounts of sulphur dioxide use may be made of the conversion of benzidine acetate to benzidine blue by the nickel(III) hydroxide. (DANGER THE REAGENT IS CARCINOGENIC.) Thiosulphates give a similar reaction and must therefore be absent sulphides also interfere. 

CARCINOGENIC.) In this test use is made of the fact that benzidine, which is unaffected by normal molybdates and by free molybdic acid, is oxidized in acetic acid solution by phosphomolybdic acid or by its insoluble ammonium salt (see reaction 4 above). This reaction is extremely sensitive two coloured products are formed, viz. the blue reduction product of molybdenum compounds ( molybdenum blue ) and the blue oxidation product of benzidine ( benzidine blue ). Moreover, solutions of phosphates which are too dilute to show a visible precipitate with the ammonium molybdate reagent will react with the molybdate reagent and benzidine to give a blue colouration. 

Blood is identified in most cases on the basis of the peroxidase-like activity of the heme group of hemoglobin. In the classical blood test, benzidine is oxidized to the quinoidal benzidine blue by hydrogen peroxide in the presence of hemoglobin. Since benzidine and its salts are now considered to be carcinogenic, 3,5,3, 5 -tetranethylbenzidine is used instead. 

Benzidine is converted into benzidine blue by a number of oxidizing agents and autoxidizable substances (see page 282). Both hydroxide and the autoxidizable Ce hydroxide react in this way. The reaction, applied as a spot test, is characteristic for cerium among the rare earths. 

Procedure. A drop of a freshly prepared moderately concentrated sodium peroxide solution and a drop of the test solution are successively placed on filter paper. The resulting chromate diffuses through the fibers of the paper into the outer zone of the moist fleck. It can be detected there by spotting with a drop of an acetic acid-benzidine solution. A blue ring (benzidine blue) is formed. 

According to L. KuPberg, MikrochemUy 20 (1936) 244, the sensitivity of this test for chromates with benzidine may be doubled by using a freshly prepared mixture of equal parts of a 1 % alcoholic solution of benzidine and 20 % Hj02. Production of perchromate is responsible for this enhancement, since perchromic acid would convert the reagent into benzidine blue more completely than chromic acid. 

Benzidine blue is produced by oxidation when even very dilute solutions of copperii united with benzidine and alkali cyanide (see page 348)... 

Gold salts oxidize acetic acid solutions of benzidine to benzidine blue which is a quinoneimine type of dyestuff (compare page 283). Colored gold hydrosols are produced at the same time. Considerable quantities of heavy metals or alkali salts flocculate the reaction products and so prevent the formation of the sols and consequently impair the test. Platinum salts and oxidants which act on benzidine must be absent. 

Benzidine acetate is oxidized by soluble ferricyanides, with formation of insoluble blue meri-quinoid compounds (see page 283). This redox reaction permits the detection of ferricyanides in the absence of other oxidizing compounds (chromates, peroxo-compounds, etc.). The test can also be used in the presence of ferrocyanides. However, it should be noted that the benzidine salt of ferrocyanic acid separates as a white precipitate, similar to benzidine sulfate. More reagent is consumed, and the detection of very small amounts of ferricyanide is rendered more difficult. To detect very small amounts of ferricyanide in the presence of large amounts of ferrocyanide, it is necessary to add sufficient lead salt to precipitate lead ferrocyanide ferricyanides remain in solution. Addition of benzidine, then causes the white Pb2[Fe(CN)e] to turn blue, because of the formation and adsorption of benzidine blue. 

Lead peroxide, in common with a number of other oxidizing agents, can oxidize benzidine (I) to benzidine blue (II). The latter is a meriquinoid oxidation product consisting of one molecule of the amine, one molecule of the imine, and two equivalents of a monobasic acid ... 

This test for lead is interfered with by cerium, manganese, bismuth, cobalt, nickel, silver and thallium salts. Under the same conditions, these metal ions also give higher oxides capable of oxidizing benzidine to benzidine blue. If, however, the lead test is carried out in an alkaline extract (plumbite solution), only thallium offers interference. All the other metal ions are precipitated as hydrous metal oxides and so do not enter the alkaline extract. When only bismuth salts are present, it is sufiftcient, before the addition of bromine water, simply to heat with the alkali, BiO(OH) is formed which is not converted into the higher bismuth oxide by hypobromite. 

This product reacts with benzidine to jaeld benzidine blue (see page 261). The reaction sequence precipitation— autoxidation—formation of benzidine blue, can be used in the form of a spot reaction on filter paper to detect quantities of MnOg that are too slight to be seen. It is interesting to note that the test is not sensitive if carried out on a spot plate or in a test tube. The paper functions somewhat as a participant in the reaction, probably through its capillary action and the resultant high dispersion of the MnOg. 

Copper salts form copper hydroxide and basic copper acetate, which can hide the benzidine blue. When small amounts of manganese are to be detected in the presence of large amounts of copper, the test should be carried out as in the presence of cobalt. Cuprous cyanide is formed it should be filtered off before proceeding with the test for manganese. 

This reaction can be used as a sensitive test for ammonia, since a neutral solution of manganese and silver salts (sulfate or nitrate) on being treated with a drop of ammonia, or exposure to ammonia gas, gives a black precipitate or dark color, according to the amount of ammonia involved. The sensitivity can be increased if the precipitate is spotted with a drop of an acetic acid solution of benzidine. The resulting benzidine blue will plainly reveal traces of a precipitate that was practically invisible. 

The oxidation of Ni(OH)2 by contact with SO2 and atmospheric oxygen may be applied for the detection of sulfites the change of color is very distinct when the experimental conditions are controlled. The formation of benzidine blue from benzidine acetate by means of higher oxides of metals (see page 283) may be used for smaller quantities of sulfite, because the reaction succeeds even when no more than traces of Ni(OH)4 are formed. 

Chromates, permanganates, ferricyanides and hypohalogenites should not be present, because they also oxidize benzidine to benzidine blue. Chlorates, perchlorates, bromates, iodates and nitrates do not react under the conditions of the test, but they do reduce the sensitivity of the persulfate-benzidine reaction. Periodates give a brown coloration. This is also true with o-tolidine. 

When thallium is present in the Tl state, it can be detected, after conversion into TIO(OH), by the oxidation of benzidine to benzidine blue (see page 283). Thallous salts must be oxidized beforehand. This is accomplished by means of potassium ferricyanide in alkaline solution... 

When solutions of lead or bismuth salts are treated with alkali hypo-bromite, lead peroxide and higher oxides of bismuth are formed. These products react with benzidine acetate to give benzidine blue (seepage 282). However, if bismuth salts are heated alone with alkali hydroxides, a yellow precipitate forms, [probably BiO(OH)] and, when once formed, is not converted into higher bismuth oxides on treatment with h3q)obromite. This resistance is applied in the detection of lead in bismuth salts. The following procedure is recommended in the examination of pharmaceutical bismuth preparations (e.g., bismuth sub-gallate, -tannate, -salicylate). 

The oxidation of benzidine to benzidine blue has been used for peroxidase tests by Madelung (234), Begemann (58), Zinn el al. (382), and Farrell (145). But this reaction can hardly be regarded specific and reliable since numerous compounds give the blue color, such as Cu salts (245), alkali metal halides (302), benzoyl peroxide (119), and others. A compound closely related to benzidine, 2,7-diaminofluorene, has been suggested as a donor by Schmidt and Hinderer (306) and is said to be even more sensitive than benzidine itself. The structures of the two compounds are ... 

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