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I N D U S T R I A L A X D ENGINEERING C H E M I S T R Y
VOl. 15, K O . 7
Preparation of Methyl Redl By A. W. Schorger C . F. BURGESS LABORATORLES, MADISON. Wrs.
ETHYL RED, p-dimethylaminoazobenzene-o-carboxylic acid, has become so well known in recent years t h a t a discussion of its value as an indicator would be superfluous. It was first prepared by Rupp and Loose2 in 1908, by diazotizing anthranilic acid in alcoholic solution, and coupliiig with dimethylaniline likewise dissolved in alcohol. Aside from the use of a n expensive solvent, the method gives poor yields. The method given by Tizard and Winmill,3 using aqueous solutions, is a decided improvement. The indicator is described in various catalogs as consisting of violet crystals; yet, most of the samples examined by the author which had been imported prior to the war were steelblue in color. It was found t h a t the violet crystals consisted of free methyl red, and the blue crystals of the hydrochloride. The methods described below were found to give products superior both in appearance and purity t o any hitherto obtainable. Numerous small-scale experiments were made using 10 g. of anthranilic acid, which theoretically should give 19.6 g. of free methyl red. Tizard and Winmill state t h a t the yield is almost quantitative. Following their directions, a t room temperature t h e best yield obtainable with reagents of high purity was only about 80 per cent. By diazotizing and coupling in the cold, and allowing the reaction mixture to stand for several hours before warming, yields of about 95 per cent could be obtained. Rupp and Loose, as well as Tizard and Winmill, use over twice the amount of hydrochloric acid theoretically necessary for the reaction. Experiment has shown t h a t the use of the theoretical amount of hydrochloric acid gives fully as high yields of methyl red, and with greater economy of materials. It might also be mentioned t h a t the methyl red can be precipitated with sodium carbonate in place of the more expensive sodium acetate.
M
PRELIMINARY PROCEDURE I n an earthenware jar of 35-gal. capacity, provided with a mechanical stirring apparatus, are placed 100 lbs. of water, 40 lbs. of ice, 5 lbs. of anthranilic acid, and hydrochloric acid equivalent to 2.5 lbs. of the anhydrous acid, and the whole stirred until the anthranilic acid has dissolved, when 2.5 lbs. of sodium nitrite are added. After being stirred the mixture is allowed to stand half an hour. I n the meantime 4.6 lbs. of dimethylaniline are dissolved in a mixture of 30 lbs. of water, 15lbs. of ice, and an amount of hydrochloric acid equivalent to 1.4 lbs. of the anhydrous acid. This solution is added to the diazotized anthranilic acid with stirring, allowed to stand 15 to 20 min.; then 3.5 lbs. of sodium carbonate followed by 1 lb. of sodium acetate are added gradually with stirring. It is preferable to replace a portion of the sodium carbonate with sodium acetate t o avoid the risk of using a n excess of the former, whereby the soluble sodium salt of methyl red is formed. It has been found advantageous t o allow this mixture t o stand several Hours, usually until the following morning. 1
*
3
Received October 20, 1922. B e y , 4 1 (1908), 3905. J . Chem SOC ( L o n d o n ) , 97 (1910), 2485.
Steam is then introduced to raise the temperature to 4050" C . , when the mass is ready for filtering. It is essential t h a t a good quality of anthranilic acid b e employed, and t h a t no free nitrous acid remain in t h e diazotized solution. If nitrous acid is present after the solution has stood half an hour, small amounts of anthranilic acid are gradually added until the nitrous acid is consumed; this will prevent the possible formation of nitrosodimethylaniline. The red, amorphous product is filtered on a large stoneware suction filter and washed with water. This is usually a slow procedure and it is preferable to use a filter press. CRYSTALLIZATION STEEL-BLUECRYSTALS-The amorphous methyl red is dissolved by using 2 lbs. of caustic soda in sufficient water t D give a final solution of the methyl red weighing about 30 lbs. Gentle heat may be used to effect solution. The solution is then filtered. One volume of strong hydrochloric acid and three volumes of water are heated t o boiling and one volume of the 61tered solution of methyl red is gradually added with stirring, care being taken t h a t all the methyl red is dissolved. Separation of brilliant steel-blue crystals of the hydrochloride of methyl red begins a t once and the size of the crystals may be regulated by the rate of cooling of the solution. After crystallization is complete, the crystals are filtered off a n d washed with a Iittle 1 : 3 hydrochloric acid. This method gives a n excellent product, since any impurities present, such as anthranilic acid o r dimethylaniline, are dissolved by the hydrochloric acid. The crystallization of the methyl red from the acid solution is almost quantitative. The yield is about 10 lbs. The sulfate of methyl red is much more soluble than the hydrochloride, so t h a t the use of sulfuric acid for crystallization is not feasible. VIOLET CRYSTALS-RUPP and Loose dissolved the crude methyl red in acetic acid, added water until turbidity was produced, and left to crystallize. This method is difficult to control as the methyl red frequently separates as a tar-like mass. The use of glacial acetic acid gives much better results. The dry, amorphous methyl red is dissolved in hot. glacial acetic acid until a strong solution is obtained. On cooling the methyl red crystallizes readily. T o make a pure product it is essential to filter the hot acetic acid before allowing i t to crystallize. The fumes are disagreeable and trouble is encountered by crystallization in the filter. The crystals separating from glacial acetic acid apparently contain two molecules of acetic acid. Crystals after exposure to the air over night were heated a t 105' C . for 15 min., a strong odor of acetic acid being evolved. Per cent
. . . . . . . . . . . . . . . . . . . 9 . 8 4 , 9 87 + 2CH8.COOH. . . . 10.0
CHa. C O O H found. , c d k d for ClaH~sNsOz
The heated crystals had a golden brown color in diffused light, compared with the violet color of crystals obtained by diluting the acetic acid solution with water. Methyl red is easily soluble in chloroform and in warm benzene and acetone, but is sparingly soluble in petroleum ether or carbon tetrachloride. If methyl red is dissolved in chloroform and petroleum ether gradually added with
July, 1923
INDUSTRIAL A N D EAVGILVEERIVGCHEMISTRY
shaking, the methyl red separates immediately as fine violet crystals, A hot, concentrated solution of methyl red in benzene, when allowed to cool slowly, deposits crystals of the indicator having a very deep violet color. Either of these methods of crystallization are preferable to the use of acetic acid. WATER-SOLUBLE METHYL RED-If the alkaline solution obtained by dissolving the methyl red in caustic soda is allowed
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to cool, large, reddish brown, flat plates of the sodium salt of methyl red separate. These crystals, like those of methyl orange, are readily soluble in water and entirely suitable as a titrimetric indicator after a further recrystallization. This form of methyl red has only recently become available to chemists; yet, it has the advantage of being soluble in water, while the violet and steel-blue crystals must be dissolved in alcohol.
T h e Determination of Urea Alone and in the Presence of Cyanamide by Means of Urease' By Edward J. Fox and Walter J.'Geldard FIXEDA-ITROGEN RESEARCHLABORATORY,
WASHINGTON,
D.
c.
since been found in quite N ACCURATE A n accurate and direct method has been developed for the detera variety of beans. It ocmethod for the demination of urea, which is based on the conversion of urea to ammontermination of urea curs principally, however, ium carbonate by the action of the enzyme urease and subsequent in the jack bean (Canavalia was required in connection titration of the ammonia fhusformed. ensiformis), sword bean with a number of investigaThe adaptation of the method to various mixtures containing tions at this laboratory on (Canavalia gladiatum), and cyanamide is described, but the method appears to be of quite soy bean (Soja max or calcium cyanamide and its general application. derivatives. I n the method Glycine m a x ) . According A brief study of the rate of conversion of urea by urease in solutions t o Mateer and Marshal1,G which has been frequently . . .. of various concentrafions has been made. eniployed for its deterthe jack bean contains fifteen times and the sword mination in commercial calcium cyanamide the difference between total nitrogen and bean five times as much urease as the soy bean. The enzyme is extremely sensitive to acids and alkalies. the sum of cyanamide and dicyanodiamide nitrogen is considered as urea. This method does not give reliable results, Hydrochloric acid in concentrations greater than 0.005 iV completely inhibits its action, while sodium hydroxide in for several reasons, as has been previously pointed out.2 Direct methods, such as precipitation by mercuric nitrate concentrations greater than 0.02 N also inhibits its action. or measurement of the nitrogen liberated on treatment with Temperature greatly affects its rate of reaction, each 10sodium h y p ~ b r o m i t e .are ~ not applicable in the presence of degree rise in temperature betyeen 10" and 50" C. causing other nitrogen compounds, which frequently occur in calcium the velocity to nearly double. A temperature of 80" C. or cyanamide. The method proposed by J ~ h n s o n which ,~ is more will destroy its activity. based on the precipitation of urea with oxalic acid in anhyEXPERIMENTAL drous organic solvents, is of limited application, since it can Several methods of preparing and preserving the enzyme be used only on anhydrous samples. The determination by means of xanthydro15 was found unsatisfactory, since in the have been presented by the investigators previously menpresence of strong acetic acid cyanamide is gradually con- tioned. The enzyme is more active, however, when freshly extracted, and for that reason it is the practice a t this laboraverted into urea. From a number of investigations6 on the action of the tory to prepare it each day as needed. A few grams of jackenzyme urease on urea and similar compounds, and on 'the bean flour are ektracted with twenty times its weight of water use of this enzyme in the determination of urea in urine and for 10 to 15 min., exactly neutralized with hydrochloric acid blood, it appeared that a method based on the action of the (about 1 cc. of 0.1 N HCl per gram of jack-bean flour), and enzyme might be developed which would be applicable to filtered. Ten cubic centimeters of this extract are sufficient the determination of urea alone and in various mixtures to convert 0.1 g. urea to ammonia in less than 1hr. The procedure first developed consisted of adding 2 to 3 g. of containing cyanamide and its derivatives. A study of this possibility was therefore made, and a very sat'isfactorymethod jack-bean flour directly to the sample of urea in water solution, adding a few cubic centimeters of potassium acid phosphate as a was developed which has now been in use a t t,his laboratory buffer, digesting in a water hath a t 40" C. for 1 hr., and collecting for nearly three years. the ammonia thus formed in standard acid by aeration. The reI n aqueous solutions the enzyme urease converts urea into sults of urea alone were fairly satisfactory, but when tried in the ammonium carbonate and so far as is known it is specific presence of cyanamide they were usually high. Attempts t o remove the cyanamide previous to treatment with urease, withfor urea. The ammonia thus formed can then be readily out affecting the urea content, proved futile. The volume of determined by titration with an acid. The enzyme was solution after precipitating the cyanamide and subsequent rediscovered in the soy bean by Takeuchi' about 1909, and has moval of the excess silver, was so great, and the concentration of
A
Received November 18, 1922. 2 Prartke, "Cyanamid," 1913, p. 22. 3 Monnier, Chem. Z t g . , 35 (1911), 601. 4 Johnson, THISJOURNAL, 13 (1921). 536. 6 Fosse, Ann. chim., 6 (1916), 13. 8 Mateer and Marshall, J. Biol. Chem., 14 (1913), 283; I b i d . , 26 (1916), 297; Van Slyke and Cullen, Ibid., 19 (1914), 221; Armstrong and Horton, Proc. Roy. Soc.. 85B (1912) 109. 7 J. Coli. Agr., Tokyo, 1 (l909), 1414. 1
inorganic salts so high, t h a t it was almost impossible to get a satisfactory determination of the urea.
As referred to later in connection with the determination of urea in the presence of cyanamide, the stability of cyanamide in aqueous solution decreases rapidly with increasing temperature, particularly in other than neutral solutions. The foregoing procedure was, therefore, modified to overcome this difficulty.
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