Nitrous acid test

Dissolve 36 g. of p-toluidine in 85 ml. of concentrated hydrochloric acid and 85 ml. of water contained in a 750 ml. conical flask or beaker. Cool the mixture to 0° in an ice-salt bath with vigorous stirring or shaking and the addition of a httle crushed ice. The salt, p-toluidine hydrochloride, will separate as a finely-divided crystalline precipitate. Add during 10-15 minutes a solution of 24 g. of sodium nitrite in 50 ml. of water (1) shake or stir the solution well during the diazotisation, and keep the mixture at a temperature of 0-5° by the addition of a httle crushed ice from time to time. The hydrochloride wUl dissolve as the very soluble diazonium salt is formed when ah the nitrite solution has been introduced, the solution should contain a trace of free nitrous acid. Test with potassium iodide - starch paper (see Section IV,60). 

While the cuprous cyanide solution is warmed gently (to 60°-70°) on the water bath, a solution of p-tolyldiazonium chloride is prepared as follows Heat 20 g. of p-toluidine with a mixture of 50 g. of concentrated hydrochloric acid and 150 c.c. of water until dissolution is complete. Immerse the solution in ice-water and stir vigorously with a glass rod so that the toluidine hydrochloride separates as far as possible in a microcrystalline form. Then cool the mixture in ice and diazotise with a solution of 16 g. of sodium nitrite in 80 c.c. of water, added until the nitrous acid test with potassium iodide-starch paper persists. The diazonium chloride solution so obtained is poured during the course of about ten minutes into the warm cuprous cyanide solution, which is meanwhile shaken frequently. After the diazo-solution has been added the reaction mixture is heated under an air condenser on the water bath fox a further quarter of an hour, and then the toluic nitrile is separated by distillation with steam (fume chamber, HCN ). The nitrile (which passes over as a yellowish oil) is extracted from the distillate with ether, the p-cresol produced as a by-product is removed by shaking the ethereal extract twice with 2 A-sodium hydroxide solution, the ether is evaporated,... 

Extract the oil obtained in the nitrous acid test with about 5 ml of ether and wash the extract successively with water, dilute sodium hydroxide and water, and evaporate off the ether. Apply Liebermann s nitroso reaction to the residual oil or solid. Place 1 drop of 0.01-0.02 g of the nitroso compound in a dry test tube, add 0.05 g of phenol and warm together for 20 seconds cool, and add 1 ml of concentrated sulphuric acid. An intense green (or greenish-blue) coloration will be developed, which changes to pale red upon pouring into 30-50 ml of cold water the colour becomes deep blue or green upon adding excess of sodium hydroxide solution. 

Nitrous acid test. The conditions of the test are similar to those described for the classification of primary amines (Section 9.5.2, p. 1215), but using acetic acid in place of hydrochloric acid. An a-amino acid yields nitrogen and an a-hydroxy acid. 

Residues from the nitrous acid test should be poured into a waste container containing 6 M hydrochloric acid. Dispose of all aqueous solutions in the container designated for aqueous waste. Any remaining organic compounds must be disposed of in the appropriate organic waste container. 

Two common qualitative tests for amines are the Hinsberg test and the nitrous acid test. The nitrous acid test is not included here because the N-nitroso derivatives of some secondary amines are carcinogenic. The risk of producing an as-yet-unrecognized carcinogenic material in this procedure outweighs any possible benefit of a test that can also be misleading and difficult to interpret. The modified sodium nitroprusside test has been included as an alternative. 

Some acylated arylamines (e.g. anilides) give misleading results in the nitrous acid test for amines (page 69). 

The Reaction has the following limitations (i) a compound that can liberate nitrous acid in acid solution is required (e.g., a metallic nitrite or a nitroso-amine, p. 204). (2) Nitrophenols and />-substituted phenols do not give the test. (3) Among the dihydroxyphenols. only resorcinol gives a satisfactory positive test. 

The evolution of nitrogen is not always entirely satisfactory as a test owing to the possible evolution of gaseous decomposition products of nitrous acid itself. The test may be performed as follows. To i ml. of chilled concentrated sodium nitrite solution add i ml. of dilute acetic acid. Allow any preliminary evolution of gas to subside, and then add the mixed solution to a cold aqueous solution (or suspension) of the amide note the brisk effervescence. 

Action of nitrous acid. To a few ml. of 20% NaNO, solution add a few drops of cold dil. acetic acid. Pour the mixture into a cold aqueous solution of glycine, and note the brisk evolution of nitrogen. NH CH COOH -h HNO2 = HO CH2COOH + N + H O. Owing to the insolubility of cystine in acetic acid use a suspension in dU. acetic acid for this test. In each case care must be taken not to confuse the evolution of nitrogen with any possible thermal decomposition of the nitrous acid cf. footnote, p, 360). 

Primary aromatic amines differ from primary aliphatic amines in their reaction with nitrous acid. Whereas the latter yield the corresponding alcohols (RNHj — ROH) without formation of intermediate products see Section 111,123, test (i), primary aromatic amines 3neld diazonium salts. Thus aniline gives phcnyldiazonium chloride (sometimes termed benzene-diazonium chloride) CjHbNj- +C1 the exact mode of formation is not known, but a possible route is through the phenjdnitrosoammonium ion tlius ... 

The experimental conditions necessary for the preparation of a solution of a diazonium salt, diazotisation of a primary amine, are as follows. The amine is dissolved in a suitable volume of water containing 2 5-3 equivalents of hydrochloric acid (or of sulphuric acid) by the application of heat if necessary, and the solution is cooled in ice when the amine hydrochloride (or sulphate) usually crystallises. The temperature is maintained at 0-5°, an aqueous solution of sodium nitrite is added portion-wise until, after allowing 3-4 minutes for reaction, the solution gives an immediate positive test for excess of nitrous acid with an external indicator—moist potassium iodide - starch paper f ... 

Dissolve 20 g, (19 -6 ml.) of anihne in a mixture of 55 ml. of concentrated hydrochloric acid (1) and 55 ml. of water contained in a 350 ml, conical flask. Place a thermometer in the solution and immerse the flask in a bath of crushed ice (2) cool until the temperature of the stirred solution falls below 5°, Dissolve 16 g. of sodium nitrite in 75 ml. of water and chUl the solution by immersion in the ice bath add the sodium nitrite solution (3) in small volumes (2-3 ml. at a time) to the cold anihne hydrochloride solution, and keep the latter weh stirred with the thermometer. Heat is evolved by the reaction. The temperature should not be allowed to rise above 10° (add a few grams of ice to the reaction mixture if necessary) otherwise appreciable decomposition of the diazonium compound and of nitrous acid wih occur. Add the last 5 per cent, of the sodium nitrite solution more slowly (say, about 1 ml. at a time) and, after stirring for 3-4 minutes, test a drop of the solution diluted with 3-4 drops of water with potassium iodide - starch paper (4) if no immediate blue colour... 

I) An alternative procedure is to cool the solution containing the sodium sul. phanilate and sodium nitrite in a bath of crushed ice to about 5° and then add 10-5 ml. of concentrated hydrochloric acid diluted with an equal volume of water slowly and with stirring the temperature must not be allowed to rise above 10 and an excess of nitrous acid should be present (the solution is tested after standing for 5 minutes). The subsequent stages in the preparation—addition of dimethyl-aniline solution, etc.—are as above. 

Place 130 ml. of concentrated hj drochloric acid in a 1 - 5 litre round-bottomed flask, equipped ith a mechanical stirrer and immersed in a freezing mixture of ice and salt. Start the stirrer and, when the temperature has fallen to about 0°, add 60 g. of finely-crushed ice (1), run in 47 5 g. (46 5 ml.) of pure aniline during about 5 minutes, and then add another 60 g. of crushed ice. Dissolve 35 g. of sodium nitrite in 75 ml. of water, cool to 0-3°, and run in the cold solution from a separatory funnel, the stem of which reaches nearly to the bottom of the flask. During the addition of the nitrite solution (ca. 20 minutes), stir vigorously and keep the temperature as near 0° as possible by the frequent addition of crushed ice. There should be a slight excess of nitrous acid (potassium iodide-starch paper test) at the end of 10 minutes after the last portion of nitrite is added. 

Qualitative Analysis. Nitric acid may be detected by the classical brown-ring test, the copper-turnings test, the reduction of nitrate to ammonia by active metal or alloy, or the nitrogen precipitation test. Nitrous acid or nitrites interfere with most of these tests, but such interference may be eliminated by acidifying with sulfuric acid, adding ammonium sulfate crystals, and evaporating to alow volume. 

Nitric acid may be precipitated by nitron [2218-94-2] (4,5-dihydro-l,4-diphenyl-3,5-phenylimino-l,2,4-triazole). The yellow precipitate maybe seen at dilutions as low as 1 60,000 at 25°C or 1 80,000 at 0°C. To prevent nitrous acid from interfering with the test results, it may be removed by treating the solution with hydrazine sulfate, sodium azide, or sulfamic acid. 

The Deamination of Adenosine 20 g of adenosine are dissolved in one liter of water by warming, and after cooling to room temperature 120 g of barium nitrite (monohydrate) are added to the solution. Under stirring there is added in time intervals of one hour 160 cc of 2N sulfuric acid after each time interval. After the third addition, the reaction mass is allowed to stand for 3 hours at room temperature. The solution is then tested for barium, and if some barium is still present a slight excess of sulfuric acid is added. 300 cc of methanol is then added. In order to drive off the excess of nitrous acid, CO is conducted... 

To detect nitrous acid, a drop of the mixture is diluted with water and tested with starch iodide paper. 

A mixture of 280 g. (1.52 moles) of commercial benzidine and 880 cc. (10.23 moles) of concentrated hydrochloric acid (sp. gr. 1.182) is placed in a 5-I. round-bottomed flask and wanned on a steam bath for one to two hours, with occasional shaking, to form the dihydrochloride. The flask is then equipped with a mechanical stirrer and a dropping funnel, and cooled, with stirring, to — ro° in an ice-salt bath. When this temperature has been reached, the benzidine dihydrochloride is tetrazotized over a period of two hours with a solution of 232 g. (3.19 moles) of 95 per cent sodium nitrite in 800 cc. of water, until a faint test for nitrous acid with starch-iodide paper is obtained after twenty minutes. During this reaction, the temperature is kept below —5 °. 

Testing for excess of nitrous acid at the end of the reaction. For this purpose starch-potassium iodide papers are best used, and these indicate nitrite in acid solution by turning blue instantaneously. With some practice, the nitrite reaction can be clearly distinguished from the coloration caused by certain diazo compounds, such as those bearing nitro substituents. The latter react only after 0.5 to 2 seconds. Often the difference becomes more marked after dilution of the diazo solution with concentrated hydrochloric acid. A properly conducted diazotization should exhibit on completion a very weak nitrite reaction, corresponding to an excess of about 10 4 m. 

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