Determination of Ammoniacal and Urea Nitrogen J. Y. YEE
AND
R. 0. E. DAVIS, Bureau of Chemistry and Soils, Washington, D. C.
S
INCE Marshall (IO) first published the clinical methods for the determination of urea in urine and blood by means of urease, there have been a number of improvements and modifications made on the technic and apparatus by Van Slyke and his co-workers ( l a , I S ) , Fisher (S), and Kay aad Sheehan (9). The latter have worked out a very careful technic for the determination of urea in blood. Fox and Geldard (6) modified Marshall's direct titration method for roiitine chemical analysis. This modified method, however, is not applicable to urea samples that are highly colored or that contain substances which interfere with the end point. For example, ammoniated peat extracts are always highly colored and contain urea, ammonium salts, and under certain conditions a little free ammonia. This investigation was undertaken to modify the urease method so that it can be used in analyzing such samples. Most of the improvements made on Marshall's method deal with t#he technic in determining the ammonia liberated by the enzyme action. Since the direct titration method cannot be used, attention is directed to the improvement of the aeration m.ethod, which is used in all the modified clinical analyses for urea. The chief objection to this method is the length of time required to make a determination. The ordinary distillation method with sodium hydroxide cannot be employed because the urease preparation used generally contains a certain amount of nitrogen. The present investigation led to the development of a method whereby ammonia is determined by distillation a t 40" C. with an alcoholic solution of a n alkali under reduced pressure together with aeration. The urease reaction and distillation are carried out at 40" c.,because this temperature is near the optimum point, as demonstrated by Van Slyke and Culler1 (IW), for the enzyme action. Free ammonia or ammonium salts present in the sample can be liberated a t this temperature by alkali without any danger of decomposing the urea. The urea is determined in such samples by difference (total ammoniacal and urea nitrogen minus that for free ammonia or ammonium salts). Complete recovery of the animonia can be accomplished by this method in 10 to 15 minutes.
Apparatus and Procedure The simple apparatus used is shown in Figure 1. About 5 cc. of the urea solution or about 50 mg. of the solid sample dissolved in that amount of water are introduced into the reaction flask, A , of 100-cc. capacity, which has a constriction and indentations along the upper part to prevent any alkaline spray from being carried over to the absorption vessel, B, a 400-cc. cylinder, that contains the necessary amount of 0.1 N standard sulfuric acid. A fritted glass plate, H , is used as the bubbler. After the reaction flask and the absorption vessel have been connected together and stopcock C has been closed, 0.1 gram of urease powder (Arlco-urease) is added to the sample and the reaction flask is placed in the thermostat, G, at 40" C. The urease is allowed to react for 15 minutes. In the meantime the inlet of the reaction flask is connected to an acid bubbler containing dilute sulfuric acid to remove ammonia from the incoming air used for aeration, and the outlet tube, D, of the absorption vessel to a water suction pump. It is advisable to place a 2-liter flask between the apparatus and the pump, so that with pinchcock E closed, this reservoir can be evacueked while the urease reaction is taking place. At the e'nd of the 15-minute period pinchcock E is opened slightly to evacuate
the system partially before introducing into the flask, from funnel F , 20 cc. of alkaline methyl alcohol solution, each liter of which contains 4 grams of sodium hydroxide and 5 cc. of capryl alcohol. The latter prevents any foaming during distillation. If a larger sample than 5 cc. has been taken, a correspondingly larger amount of alcoholic solution should be used. Air is then admitted into the system at the rate of one bubble per second through the acid bubbler. Slowly the system is further evacuated until the solution boils vigorously, but not violently enough to carry spray over to the absorption vessel, nor to bring the foam in the latter too close to the outlet tube, D. Stopcock C usually needs to be regulated again at this point in order to maintain the same rate of air flow through the system.
FIGURE1. DIAGRAM OF APPARATUS
After the first 3 minutes, during which most of the ammonia and alcohol are distilled over, no attention is generally required until the 1.5-minute boiling period is completed. Then more air is admitted and pinchcock E closed. After the vacuum has been completely released, the excess acid in the absorption vessel is titrated with standard 0.1 N sodium hydroxide, using methyl red as indicator. For very accurate analysis, weaker alkali 0.025 N should be used. The carbon dioxide liberated by the enzyme action on the urea has already been removed by the aeration process and can cause no interference with the end point. For ammonia determinations, the same procedure is followed except that no digestion period is necessary. With a 2-cc. sample, complete recovery of the ammonia can be accomplished by boiling the solution for only 10 minutes. Blank determinations should be made on each batch of urease powder. The latter is more convenient to use than the urease tablets, which dissolve slowly.
Experimental Results In order to check the accuracy of this modified method, many attempts were made to prepare urea of very high purity by recrystallizations from water and alcoholic solutions, but a product of about 99.5 per cent purity was usually the result. Other members of this laboratory and Dalman (1) have en259
INDUSTRIAL AND ENGINEERING CHEMISTRY
260
TABLEI. COMPARISON OF XANTHYDROL AND MODIFIED UREASEMETHODS Urea Found by Modified ureaae Xanthydrol method method' Me. Mg, 32.33 32.29 Urea solution A, 2 cc. 32.33 32.32 32.33 32.35 Av. 32.33 Av. 32.32 Urea solution B, 2 cc. 34.96 34.93 34.96 34.90 34.97 34.96 34,96 34.90 Av. 34.96 Av. 34.92 Ammoniated peat extract, 5 eo. 4.02 3.97 3.99 4.01 Av. 4.00 Av. 3.99 5 For best results the weight of xanthydrol used should be fifteen t o twenty times that of the urea. present. Samdes
AND UREANITROGEN DETERTABLE11. AMMONIACAL
MINATIONS
Ordinary Method" New Method and Urea NHs NHs Urea Urea Ammonium salt solutions solution nitrogen nitrogen nitrogen Nitrogen Cc. Cc. Mi. Mi. Mi. Mo. 4.82 4.82 ... (NHa)zSO4 soln. A 4.82 4.82 ... ... aoln. A ... 14.83 14.82 soln. B 14.83 14.83 soln. B 23.69 23.69 *.. soln. C 23.68 23.69 soln. C 16.17 16.17 NHlNOs aoln. ... 16.17 16.17 NHaNOa soln. ... ... 30:OS 5 2 ... 9.51 ("4)Zso; soln. D 2 9.51 ... ... soln. D 2 9.51 5 ... soln. D soln. D 2 5 ... 9.51 soln. D 2 5 ... ... 30:09b 39:60 aoln. D 2 5 ... ... 30.08b 39.59 30.08b 39.59 soln. D 2 5 ... 30.06b 39.57 soln. D 2 5 a Distillation with alkali a t atmospheric presaure. b Urea nitrogen by difference. Samples Taken
... ... ... ... ...
... ... ... ... ...
... ...
... ...
...
... ...
...
... ... ... ..... . ... ... ....* . ...
...
countered similar experiences. Having failed in this, the degree of purity of the urea as determined by the proposed method was compared with that obtained by another analytical procedure. The ordinary Kjeldahl method was found by several members of this laboratory to give inconsistent total nitrogen results for urea, so Fosse's xanthydrol method (4) was tried and the results obtained were found to agree with those secured by the urease method, as shown in Table I. The results for ammoniacal nitrogen determinations were found to agree very closely with those obtained by ordinary distillation with alkali. These, together with the results for urea and ammoniacal nitrogen in the same sample, are tabulated in Table 11.
Inactivation of Urease by Heavy Metals Small amounts of heavy metal salts can inactivate a relatively large amount of enzyme (g, If). Under the conditions employed in the modified method, mercuric and silver ions have about the same toxicity on urease, while cupric ions are not quite so toxic. When such heavy metal ions are present, more urease should be used in order to counteract their effect. Inactivation caused by either mercuric or silver ions can be overcome by a much smaller excess of enzyme than that resulting from cupric ions. Another method for obtaining accurate results under such conditions is t o allow the urease to react for a much longer time. The length of time required to bring about complete decomposition of the urea depends upon the amount and kind of poisons present in the sample. By lengthening the reaction time, accurate determinations can be accomplished in the presence of mercuric and siLver ions; with cupric ions present, however, it is much more difficult to obtain good results, and complete decomposition of urea by the urease, even with much
VOL. 7, NO. 4
longer reaction time, may not be accomplished, although the initial toxic effect is less than that caused by either mercuric or silver ions. Tables 111,N, and V show the results of experiments demonstrating these points. I n these experiments, unless stated otherwise, various amounts of heavy metal salt solutions were added to 2 cc. of urea solution containing 32.24 mg. of urea. Enough water was added, if necessary, to make a total volume of 4 cc. The rest of the experiment was carried out as described in the procedure. The inactivation of urease by mercuric chloride wa? observed to vary according to the length of time during which the urea and the mercuric salt are in contact previous t o the addition of the enzyme. If this time interval is long enough, the toxic effect disappears almost completely. The length of time necessary for this to occur depends on the amount of the metal salt present. Silver nitrate and cupric sulfate, however, do not show this effect. These results are given in Table VI. Euler and Svanberg (6)noticed a similar effect of mercuric chloride on saccharase. These investigators also found that sucrose exercised a considerable protective action toward inactivation by mercuric ions, while no such action was observed toward the poisoning effect of silver ions on saccharase. I n the present experiments, sucrose, it is found, does not give such protective action to the urease toward similar inactivations. Effects of other salts on the urease reaction were tried in another series of experiments. Urea was completely decomposed by the urease when the stated amounts of each of the following salts were added to a 2-cc. sample containing 32.24 mg. of urea: 0.2 gram of magnesium sulfate heptahydrate, sodium chloride, potassium hydrogen phosphate, or melamine, and 0.1 gram of barium or calcium chloride.
Activity of Urease The activity of different batches of a commercial urease preparation from jack beans was found to vary greatly. AtTABLE111. INACTIVATION OF UREABEBY HEAVY METALS Weight of Metal Added Mg. 0.10 0.15 0.20 0.30 0.10 0.15 0.20 0.30 0.10 0.15
Heavy Metal Added Mercury (as HgClz)
Silver (as AgNOs)
Copper (as CuSOr)
n
20
0.30 0.50 0.70 1.00
Urea Decomposed
An. % 99.6 22.0 12.2 10.8 98.7 22.1 13.1 11.0 99.7 96.4 A.... I 1
73.6 52.4 42.9 33.6
TABLEIV. EFFECTOF UREASEON UREADECOMPOSITION IN PRESENCE OF HEAVY METALS Heavy Metal Added
Weight of Metal Added
Mg.
Ag (as AgNOs)
0.15
Cu (aa CuSOr)
1.0
Urease Used Uram
0.10 0.12 0.13 0.10 0.11 0.15 0.20 0.25 0.30
Urea Decomposed An. %
22.1 78.1 99.4 33.6 47.1 63.5 92.2 96.7 100.0
JULY 15, 1935
ANALYTICAL EDITION
TABLEV. EFFECTOF TIME OF UREASEREACTION ON UREA DECOMPOSITION IN PRESENCE OF HEAVYMETALS Heavy Metal Added
Weight of Metal Added M g
Hg
HgCld
0 15
Hg (ai3 HgCh)
0 20
(a3
Hg (an HgCld
0 30
Ag (aa AgNOd
0.15
cu
1.0
(a6
CUSO4)
Time of Urease Urea Reaction Decomposed Mzn. Av. % 15 22 0 30 99 7 15 12.2 30 14 1 45 51 0 60 99 7 90 100 0 90 18 7 180 100 0 15 22.1 30 57.9 45 77.1 60 90.5 70 98.5 90 100.0 15 33.6 30 36.3 60 43.0 180 51.8 240 55.5 504 96.8
TABLEVI. EFFECTOF TIME INTERVALS BETWEEN ADDITIONOF HEAVYMETALSAND UREASEON UREA DECOMPOSITION Heavy Metal Added
Weight of Metal Added
Ag (as AgNOd
0.15
Cu (as CuSOa)
1.0
TABLE
VII.
0 67 0 115
Urea Decomposed
20. 8 21. 1 33. 6
35. 7
ACTIVITYO F UREASE PREPARATIONS
VARIATIONS I N THE
Urease Mercury Urea Samples Added Decomposed Mg. Av.-% 3872 A 0 99.7
3872 A 3872
0.10 0.10
3965
0.10
9.3
4039
0.10
98.9
4039 4071
0.15
22.0 99.9
0.15 0.20
Time Interval between Addition of Metal and Urease
7.7 98.7
Remarks Bottle of urease tablets prepared, Feb. 15, 1932,a in use for about 9 months. Last 5 tablets, dark brown, were powdered and used in these experiments, Jan. 8, 1935 Hg added as HgCh Like 3872 A, except bottle waB just opened. Light greenish yellow Bottle of urease powder prepared, Apr. 3, 1933,O in use about 4 months , Bottle of urease iowder, prepared Jan. 22, 1934,a opene and used Dec. 11,1934
Bottle of urease ‘powder, prepared Oct. 15, 1934,a opened and used Jan. 2, 1935 4071 12.2 ..... 4039 0.20 12.1 ..... The dates of preparation were kindly given by the Arlington Chemical Co., Yonkers, N. Y.
mospheric moisture seems to have a deleterious effect on such preparations. The activity of urease from bottles that have been opened and in use for some time was found to have decreased considerably, while a sample of the same batch kept in a dark brown bottle and well sealed for 3 years maintained its activity much better. Results given in Table VI1 demonstrate these variations in activity of several urease samples.
Discussion This method has been in use for over a year in this laboratory and has been employed by other members of this bureau for the determination of ammoniacal and urea nitrogen in fertilizer samples. Urea solutions varying in concentration from 1 to 100 mg. of urea per 5-cc. sample have been successfully analyzed.
261
Van Slyke and Zacharias (13) have shown that phosphate buffers of proper concentration can maintain the enzyme action a t its maximum, but have also pointed out that the phosphates themselves exert a retarding effect on the action, especially when the urea concentration is low. Kay and Reid (8) demonstrated, with buffer solutions of pH values varying from 6.2 to 8.4, that 94 to 96 per cent of the urea can be hydrolyzed, with the maximum a t about pH 7 . In the presence of small amounts of mercuric and silver salts, according to Jacoby ( 7 ) ,urease is more active a t values higher than pH 7 . In the absence of buffers, the pH of a urea solution after the addition of urease will soon become and remain a t about 8.6, which is that of an ammonium carbonate solution. Since with excess of urease, complete decomposition of urea in concentrations as high as 2 per cent can be accomplished under the conditions of the present method, and in this way many unfavorable conditions for the enzyme action may be avoided, such as the presence of small amounts of heavy metal salts, etc., it is convenient to eliminate the use of buffers in routine analysis. Urease powder is now readily obtainable a t a reasonable cost and can be kept for a long time under proper conditions. Mercuric and silver ions have a very similar effect on the activity of urease. Cupric ions, however, seem to react quite differently, although their toxic effect is not as great. It is apparently more permanent. I n the presence of cupric salts, a much greater excess of urease is required to counteract the toxic effect, and complete decomposition of urea cannot be brought about readily by prolonging the time of reaction between the enzyme and the urea. I n the presence of either mercuric or silver salts, however, this can be accomplished easily. These results are in agreement with those of Hellerman and his co-workers (6), who found that urease was not readily poisoned by cupric ions and that hydrogen sulfide could not restore fully the activity of a solution of crystalline urease, which had been partially “extinguished” by cupric sulfate. On the other hand, they showed that the activity of urease, completely “extinguished” by organic mercurials, could be completely or almost completely restored by the action of hydrogen sulfide or potassium cyanide.
Summary The urease method has been modified so that it can be used to determine urea even in highly colored solution. The accuracy and precision of this modified method are well within the limits required for chemical analysis. A rapid method for the determination of ammoniacal nitrogen has been developed, whereby ammonia is liberated by distillation a t 40” C. with an alkaline alcoholic solution under reduced pressure together with aeration. Substances interfering with the urease reaction are discussed and means of overcoming their toxic effects are suggested.
Literature Cited (1) (2) (3) (4) (5) (6) (7) (8)
(9) (10) (11) (12) (13)
Dalman, L. H., J . Am. Chem. Soc., 56,549 (1934). Euler, H., and Svanberg, O., Fermentforschung,3,330 (1920). Fiske, C. H., J . Bid. Chem., 23, 455 (1915). Fosse, R., Ann. inst. Pasteur, 30,525 (1916). Fox. E. J.. and Geldard. W. J.. IND. ENG.CHEM..15. 743 119231. Hellerman, L., Perkins, M. E., and Clark, M., Proc’. Nat: Acad. Sci., 19,855 (1933). Jacoby, M., Biochem. Z.,262, 185 (1933). Kay, W. W., and Reid, M. A. H., Biochem. J . , 28, 1798 (1934). Kay, W. W., and Sheehan, H. L., Ihid.,28, 1784 (1934). Marshall, E. K., J . Bzol. Chem., 14,283 (1913); 15,487 (1914). Sumner, J. B., and Myrback, K., 2. physiol. Chem., 189, 218 (1930). Van Slyke, D. D., and Cullen, J. E., J . Biol. Chewa., 19, 141 (1914). Van Slyke, D. D., and Zacharias, G., Ibid., 19,181 (1914).
RXJCEWED May 1, 1935.
