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
743
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, i t 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 i t 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'isfactory method jack-bean flour directly to the sample of urea in water solution, a few cubic centimeters of potassium acid phosphate as a was developed which has now been in use a t t,his laboratory adding 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, that 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.
!
I N D LTSTRIALA N D ENGINEERING CHEMISTRY
744
The sample in solution was brought t o exact neutrality, using methyl red as the indicator, 10 cc. of the urease solution were added, and the solution was allowed t o stand for an hour a t room temperature instead of a t 40° C. An excess of standard 0.1 N hydrochloric acid was then added and the solution aerated by passing a current of air, washed free from carbon dioxide and ammonia, through the solution until all of the carbon dioxide was expelled. Five to 10 min. were found to be sufficient for this. The excess acid was titrated with standard 0.1 N sodium hydroxide. The acid used up is a direct measure of the urea originally in solution. Each cubic centimeter of 0.1 N acid is equivalent to 3 mg. of urea. This procedure necessitates the use of a neutral solution of urease, and requires Chat the urea be in a neutral solution free from carbonates or other substances that would affect the final titration. Results by this method are accurate to within a few hundredths of a per cent, the accuracy being limited only by the accuracy of the measurement of the reagents. For samples containing soluble carbonates it is necessary to remove the carbon dioxide previous to the addition of the urease solution. This is readily accomplished by making the solution acid, aerating as described above, and again neutralizing,
I n order to determine safe limits, both as to time of digestion and quantities of urea and urease, with which to work, the following experiments were carried out : A 250-cc. solution containing 2.5000 g. of urea was prepared. The urease solution was made from freshly ground jack-bean flour (200 mesh). Fifteen grams of flour were extracted with 300 cc. of water for 15 min., with frequent stirring, the solution neutralized with 15 cc. of 0.1 N hydrochloric acid, and the insoluble residue allowed t o settle out. The extract was then filtered through a fluted filter paper. In this way quite rapid filtration was obtained. Aliquots containing 50, 100, and 200 mg. of urea were pipetted into tall-form, wide-mouth bottles (capacity 100 cc.) and diluted so that when the urease solution was added the total volume was 50 cc. ; 5, 10, or 20 cc. of the urease solution were then added t o the various solutions, the time noted, and the reaction stopped at various intervals by running in a measured excess of 0.1 A7 hydrochloric acid solution. The sample was then aerated by passing a rapid current of air for 5 min., and the excess acid titrated with 0.1 N sodium hydroxide solution.
The following data were obtained, which clearly show the course of the reaction: RATE O F C O h T E R S I O N
OF
UREA
---
TO .kMMOKIA B Y C R E A S E I N V A R I O U S OF UREA
CON-
CENTRATIONS
Urea Urease Added Added Expt. Mg. Cc. A 100 20 B 100 10 c 100 5 D 50 10 E 200 10
1 66.9 30.0
. ., .
47.4 17.2
Per cent Urea Converted, Min.------. 2 3 5 8 lo 15 95.4 99.9 .. .. .. . . . . . . , . 45.0 . . . 87.0 99.3 99.9 . . . . 23.7 . . 49.2 . . . 7 7 . 4 95.1 84.0 97.2 .. . . .. . .._. 28.5 . . . . 66.2 . . . . 79.9 96.0
. ..
..
. .. . .
.
Experiments A, B, and C show that the rate of reaction is dependent on the concentration of urease and that the rate for the first few minutes varies approximately as the ratio of the concentrations. B, D, and E show the effect of urea concentration. Most salts have a depressing effect on the action of the urease. Sodium chloride in concentration of 0.25 per cent depresses the rate of reaction to about two-thirds normal, while concentration of 1 per cent depresses the rate t o onehalf normal. Barium and calcium chlorides completely inhibit the action of urease after the copversion is about 75 per cent complete. To determine urea in the presence of these salts, therefore, it is necessary to remove them from solution previous to the addition of the urease. This is readily accomplished by precipitating them as carbonates. The excess carbon dioxide is then removed by aeration as described above. DETERMINATION OF UREAALONE
As a result of the preceding experiments the following procedure has been adopted : Weigh out a 0.5-g.sample and dissolve it in 250 cc. of water. Pipet 25 cc. into a tall-form, wide-mouth bottle of about 100-cc.
Vol. 15, No. 7
capacity, add a few drops of methyl red indicator and bring t o exact neutrality, using 0.1 N hydrochloric acid or sodium hydroxide. Add 10 cc. of neutral urease solution and let stand for 1 hr., keeping the mouth of the bottle stoppered. Add from a buret a measured excess of standard 0.1 N hydrochloric acid solution, insert into the boctle a glass tube with a small bulb and fine holes a t its lower end, connect with the air line through a series of acid and alkali gas-washing bottles, and aerate as vigorously as possible without danger of losing some of the solution through spattering, until all the carbon dioxide is removedfrom 5 to 10 min. Two or three drops of caprylic alcohol or liquid petrolatum added to the solution will prevent frothing. After aerating for 5 min., reduce the current of air and titrate the solution to exact neutrality with 0.1 N sodium hydroxide solution. RESULTS OF
D E T E R M I N A T I O N O F UREA Acid Useda Urea Equivalent Per cent Recovered Mg. cc. Mg. 50.0 16.67 50.01 100.02 100.02 60.0 16.67 50.01 100.02 50.0 16.67 50.01 50.0 16.66 49.98 99.96 20.0 6.66 19.98 99.90 100.05 20.0 6.67 20.01 a Since a small difference in the quantity of 0.1 N acid used produces such relatively large variations in t h e calculated result, the use of a more dilute acid mould have been preferable.
Urea Used
DETERMINATION OF UREAI N
THE
PRESENCE OF CYANAMIDE
The applicability of the urease method to the determination of urea in the presence of cyanamide was determined in solutions of known urea and cyanamide content by the method just described. Since the cyanamide contained a small percentage of urea as an impurity, it was necessary t o make a correction for it. The correction was determined by difference between total nitrogen and cyanamide nitrogen, and agreed with the urea nitrogen as found by the urease method. The solutions used and results obtained are shown in the following table: Urea Added ME. 50.0 50.0 20.0 20.0
Added HzCNz
in Urea HzCNz
Used Acid
Equivalent Urea
Per cent
Mg. 50.0 50.0 50.0 50.0
Mg. 0.18 0.18 0.18 0.18
cc. 16.73 16.73 6.73 6.73
Mg. 50.19 50.19 20.19 20.19
Recovered 100.02 100.02 100,05 100,05
From these analyses it is evident that the determination of urea in the presence of cyanamide is entirely reliable. The high results previously obtained by the digestion and aeration method are explained by the fact that cyanamide is partially converted t o urea in the slightly alkaline medium which quickly results from the action of urease on the urea in solution. At 40" C. the conversion of cyanamide to urea is much more rapid than a t room temperature. Furthermore, the solution is made much more alkaline upon the addition of jack-bean flour to the solution than it is by the addition of neutral urease solution. DETERMINATION OF UREAI N CYANAMIDES Extract 2 g . of cyanamide with 400 cc. of water for 2 hrs., add 2 g. of anhydrous sodium carbonate t o precipitate tha calcium, and continue to shake for another half hour. Filter the extract through a dry filter and take 25-cc. aliquots for urea analysis. Pipet the aliquots into the bottles previously mentioned, make the solution distinctly acid with dilute hydrochloric acid and aerate until all the carbon dioxide is expelled, as shown by the sharpness of the end-point in the subsequent neutralization. Then make the solution exactly neutral, using methyl red as indicator, add urease solution, and determine urea as described above.
The reliability of this procedure is shown by the following experiment : A sample of cyanamide was first analyzed for urea and then t o 2-g. samples of the same lot were added 0.1 g. and 0.5 g. of pure urea. The results are shown in the following table: 8
Fertilizer material, hydrated and oiled commercial calcium cyanamide.
INDUXTRIAL A N D ENGINEERING CHEMISTRY
July, 1923 Sample NO.
1 2 3
Cyanamide G. 2.0000 2,0000 2.0000
Urea Added G. None 0.100 0.500
DETERXINATIOK OF UREA
Urea Found
G.
0.01344 0.11328 0.51284
I N PHOsPH.4TE
Per cent Recovered 99:84
99.88
MIXTURES
The method was also applied to the determination of urea in mixtuxs of cyanamide with acid Phosphate and with calcined phosphate. For such mixtures which contain from 1 to 4 per cent nitrogen as cyanamide, an 8-g. sample was extracted with 400 cc. of water the same as for cvanamide. The DhosDhate is then mecipitated completely with barium hydroxide, the CXCCSS barium precipitatctd along with any soluble calcium salts by the addition of a few grams of sodium carbonate, the extract filtered through
745
a dry filter, and aliquots taken for urea determination as for cyanamide. Sample
No. 1 2 3 4
Phosphate G. 8 (acid) 8 (acid) 8 (calcined 8 (calcined)
Urea Added
G. 0.1000 0,5000 0.1000 0.5000
Urea Found G.
0.10030 0.50016 0,10080 0.50016
Per cent Recovered 100.30 100.03
100.80 100.03
I n addition to the various applications of the urease method as shown in this paper, the method has been very satisfactorily employed in a number of other cases and, hence, it appears to be Of quite general application*
ACKSOWLEDGMENT Acknowledgment is made to J. M. Braham for his helpful criticism and cooperation in the preparation of this paper.
Expression of Quantity of Water-Soluble Arsenic Present in Commercial Lead Arsenates’ By C . A. Klein and W. Hulme BRIXSDOWN LEAD Co., LTD., ENFIELDHIGHWAY, MIDDLESEX, ENGLAND
The method now used for the expression of fhe wafer-soluble arsenic content of lead arsenates is considered to be irrational, since it ignores the fact that the quantity of water-soluble arsenic present in a spraying mixture is determined, not solely by the quantity in which it is present in the arsenafe as sold, but by the total quantify of the arsenic present, the latter being the facfor which determines the proportions of commercial lead arsenate and water used in making up spray mixtures. i t is suggested that, in order to estab-
lish a better standard of comparison between the water-soluble contents of lead arsenates of varying composition, the former shall be calculated and expressed as a percentage of the total arsenic present. I t is also suggested that all analytical values shall be determined on the samples as received, but shall be expressed on the basis of dried maferial, and not, as is frequently the case, on the samples as receioed, which may be in the dry or paste form, the water content of the latter being known to vary within wide limits.
I
N THE course of an investigation concerning the preparaA comparison of the two specifications made on the same tion and use of commercial lead arsenates for horti- basis is as below: cultural and agricultural purposes, it appeared that the (CALCULATED O N DRY PRODUCT, ASSUMING 50 PER CENT WATER IN systems now in use for the expression of the water-soluble PASTE) Total AszOs Soluble AszOp arsenic contents are unsatisfactory and require modification. (Minimum) (Maximum) b X 100 It is generally accepted that in order to be effective, the (a) (.b.) a mixture of water and lead arsenates used for spraying must Federal Insecticide Act 1910 U.S.A.. 25.0 1.5 0.0 Ministry of Agriculture‘ and Pisheries, contain not less than a certain minimum quantity of arsenic, Great Britain.. . . . . . . . . . . . . . . . . 28 .O 1.0 3.6 and further, owing to the injury caused to foliage by waterThe following analyses shorn7 t h a t in America and England soluble arsenic, it is necessary to limit the quantity which much of the arsenate sold contains total Asz05 considerably may be permitted. in excess of that laid down as the minimum permissible COMPARISON OF BRITISH AND UNITED STATES SPECIFICATIONS auantitv: The C~S. A. Federal Insecticide Act of 1910 decrees that ENGLISH LEAD ARSENATE “lead arsenate paste must not contain more than 50 per cent (Determined on paste as sold) Total AszOa water or more than 0.75 per cent of water-soluble arsenic Expressed on Dried (expressed as AS~OF,),and not less than 12.5 per cent of total Material SAMPLE % AsZ06.” A 33.04 The limits recommended by the Ministry of Agriculture B 32.93 C 31.96 and Fisheries of Great Britain (Leaflet 363) are that lead D 30.53 E 32.34 arsenate pastes shall contain not less than 14 per cent of Asz06and not more than 0.5 per cent water-soluble arsenic The following analyses published by Robinson2 refer t o (expressed as Asz06),and that the material when dry shall dry material: contain not less than 28 per cent of Asz05. Further, the AMERICANARSENATES substances other than arsenate of lead and water which may Total AszOs be present in the paste as sold shall not exceed 3 per cent. SAMPLE % A 31.80 It is also recommended that the container shall be labeled C 31.65 to indicate in terms of Asz06the actual percentage of arsenic E 31.27 H 30.75 present, together with the dilution required for the production I 32.07 32.94 of a standard spraying mixture containing 0.1 per cent of 31.04 arsenic pentoxide.
JL
1
Received June 22. 1922.
THIS JOURNAX.,14 (1922), 314.
I
