A N A L Y T I C A L EDITION
March 15, 1941
TABLE I. TESTDATA FOR ACCURACY OF ANALYSIS OF BLOOD FOR MAGNESIVM Found er 100 M1. of riginal Blood
Found per, 100 M1. of Original Blood 1 Mg. of Mg per 100 hll.
Me.
MQ.
8
Errora
Found per 100 MI. of Original Blood 2 Mg. of Mg per 100 M1.
Errora
%
MQ.
%
+
+
3.10b 3.10
4.00
4.10
2.44
5.00 1.96 5.05 0.98 5.20 1.96 5.60 1.63 5.50 0.18 5.60 1.63 magnesium present in mg. per
0.00 2.44 4.48 0.66 4.40 2.44 4.60 1.99 5 Per cent error based upon total amount of 100 ml. b 3.10 is average value of three determinations of original blood sample 1. c 3.51 is average value of three determinations of original blood sample 2.
3.10 3.51C 3.51 3.51
4.20
187
of the disease which would be of diagnostic importance. A similar study on a variety of diseases was undertaken by Zimmer (16), in which no correlation was obtained. However, this lack of correlation may be due to the fact that consistently high results with an average precision of *13 per cent were obtained by the spectrographic method which she used.
Acknowledgment The author wishes to express her appreciation for the very fine cooperation and assistance given by the members of the X-Ray Department a t Harper Hospital, and to the General Motors Corporation for free license to Patent 1,979,964.
Literature Cited TABLE 11. MAGKESIUM DETERMINATION IN KORMAL BLOOD Sample v -
1
2 3 4 5 6
7 8 9 10 11 12 13
14 15 16
2.90 3.20 3.15
2.80
3.25 3.55 3.65 4.25
4.55
3.30 4.80 4.15
3.10 3.10 3.50 3.25
M g . per 2.75 3.20 3.20 2.85 3.35 3.65 3.90 4.30 4.65 3.15 4.40 3.75 3.00 3.40 3.60 3.40
100 m
2.85 3.35 2.95 3135 3.75 3.80
..
..
4:55 3.90 3.25 3.40 3.55
3.10
1
Av. . F 2.83 3.25 3.10 2.83 3.32 3.65 3.78 4.28 4.60 3.23
4.58 3.93 3.12 3.30 3.55 3.25
Deviation
%
%
+2.4 $3.1 +3.2 +0.7
-2.9
$0.9 $2.7
$3.2 i-0.5 $1.1 +2.2 +4.8 f5.5 +4.2 +3.0 +1.4 +4.6
-1.5
-4.8 -1.0 -2.2
-2.7 -3.4
-0.7 -1.1 -2.5
-4.0 -4.6 -3.8 -6.1 -1.4 -4.6
Table I1 presents the individual values, average values, and per cent deviation obtained on 16 normal blood samples run in regular routine practice. The values range from 2.83 to 4.60 mg. of magnesium per 100 ml. of whole blood, which agrees very well with the most reliable values reported in the literature. Work is now in progress on the analysis of a large number of blood samples from patients having malignant diseases to determine whether or not there is a correlation between the amount of magnesium present in the blood and the progress
(1) Cassen, B., J. Lab. Clin. Med., 25, 411-13 (1940). (2) Duffendack, 0. S., Wolfe, R. A., and Smith, R. W., IND. ENO. CREM.,Anal. Ed., 5, 226-9 (1933). (3) Ewing, D. T., Wilson, M. F., and Hibbard, R. P.. Zbid., 9, 410-14 (1937). (4) Gerlach. W.. Z. anoro. allaem. Chem.. 142. 383-98 (1925). (5j Greenberg. D. M., Luka, S . P., Mackey, M‘. A,, and Tufts; E. V., J . Biol. Chem., 100, 139-48 (1933). (6) Hald, P. M., and Eisenman, A. J., Ibid., 118, 275-88 (1937). (7) Hess, T. M., Owens, J. S.. and Reinhardt, L. G., IND.ENO. CHEM.,Anal. Ed., 11, 646-9 (1939). (8) Keirs, R. J., and Englis, D. T , Ibid., 12, 275-6 (1940). (9) Lundeghrdh, H., “Quantitative Spektralanalyse der Elemente”, Teil 2, Jena, G., Fischer, 1934. (10) Myers. V. C., and Muntwyler, E., Phys. Reo., 20, 1-37 (1940). (11) Sawyer, R. A., and Vincent, H. B., Spectrochimica Acta. 1, 131-6 (1939). (12) Thomson, K. B., and Lee, W. C., J. Biol. Chem., 118, 711-21 (1937). (13) Twyman, F., and Hitchen, C. S., Proc. Roy. SOC. (London), A133, 72-92 (1931). (14) Walker. B. S., and Walker, E. W., J. Lab. CEin. Med., 21, 713-20 (1936). (15) Watchorn, E., and McCance, R. A., B i o c h a . J., 26, 54-64 (1932). (16) Zimmer, E., Spectrochimica Acta, 1, 93-106 (1939). PRESEINTBD before the Division of Industrial and Engineering Chemistry at the 100th Meeting of t h e American Chemical Society, Detroit, Mich. T h e work was made possible through the generosity of Miss Florence Stroh in memory of her mother and father.
Determination of Ammoniacal and Nitrate Nitrogen in Decomposed Plant Material J. G. SHRIKHANDEI, Rothamsted Experimental Station, Harpenden, England
I
N STUDIES on the preferential utilization of different
forms of nitrogen during the decomposition of plant materials (5) the inadequacy of existing methods was apparent. This aspect of the problem of decomposition arises in connection with the practice of incorporating into the soil fresh undecomposed farmyard manure saturated with urine, or, less frequently, straw supplemented by a dressing of ammonium sulfate. I n such circumstances, in addition to the ammonia, nitrogen may also be available in the form of soil nitrate. Subsequent changes in the amounts of the various forms of nitrogen mere hard to follow, especially when the quantities involved were small, because of inaccuracies in existing procedures when applied to decomposing or decomposed residues and manures. I
Present address, Tea Research Institute of Ceylon, Talawakelle. Ceylon.
The common method of estimating ammoniacal and nitrate nitrogen in decomposed vegetable material is that of distillation in presence of magnesium oxide for ammonia, and subsequent reduction of the residue with Devarda’s alloy for nitrate. The conditions under which such estimations are made are drastic and some of the ammonia, which is liberated a t the abnormally high pH of 10 to 11 produced by the use of magnesia, comes from the plant amides. The results thua obtained are apparently higher than the true ammonia or nitrate content of the samples under examination. Another method which is sometimes employed is the extraction of the residues or manure with sodium chloride solution, thus liberating the ammonium ion by the process of base exchange, and subsequent distillation of this extract with magnesia. This method, being laborious and time-consuming, is still unsatisfactory, as some of the organic nitrogen may be dissolved out
INDUSTRIAL AND ENGINEERING CHEMISTRY
188
by the salt solution and hydrolyzed to ammonia a t the high pH, giving higher quantities of ammonia and nitrate than are actually present. It thus is evident that the chief defect of the distillation method lies essentially in the use of magnesium oxide, which produces a highly alkaline reaction conducive to the hydrolysis of some of the organic nitrogen. The ammonia so liberated increases the apparent ammonia content of the sample.
Experimental
AMMONIA.Nichols and Foote (3) showed that ammonia determinations in sewage and nitrogenous trade wastes were unsatisfactory if carried out by distillation with magnesia or 10 per cent sodium carbonate, because of the high alkalinity, and proposed the use of a phosphate buffer of pH 7.4 which causes no liberation of ammonia from such organic compounds as asparagine, acetamide, g!ycine, and arginine. Decomposing straw was accordingly distilled in the presence of amounts of phosphate buffers, giving four different reactions. The straw had been allowed to decompose without additional O F AMhfOh’IA ADDEDTO DECOMPOSED TABLE I. RECOVERY STRAW
(10 mg. supplied in each case) PH 7.0 7.4 S .O 8.5 10
NHa M Q. 9.50 10.11 10.84 11.65 11.96
(phosphate buffer) (phosphate buffer) (uhosuhate buffer) (phosphate buffer) (magnesia)
OF DISTILLING RETABLE 11. EFFECTOF COSCENTRATIOX AGEKTS ox HYDROLYSIS OF NITROGENOUS CONSTITUENTS OF AIR-DRIED PADDY STRAW
Phosphate Buffer Cc. 40 50
60 70
NHI Mg./6 0.
0 00 0.00 0.00 0 00
XlgO Grams 4 6
8
10
N H3 M0./6 g.
0 70 0.84 0.98 1 01
nitrogen having been supplied, and the high positive “nitrogen factor” of the straw (4)therefore precluded any possibility of ammonia accumulation in the decomposing residue. To this were added in each case 10 mg. of ammonia just prior to distillation (Table I). These results leave no doubt that the high alkalinity of the magnesia causes high ammonia figures to be obtained, and that by the use of a phosphate buffer at p H 7.4 this disturbing factor can be largely eliminated. Additional proof was provided by examining the effect of distilling 5 grams of undecomposed paddy straw (0.78 per cent nitrogen) with similar amounts of phosphate buffer and magnesia. Progressively increasing amounts of ammonia were obtained as the alkalinity was increased, no doubt as a result of hydrolytic deamination of organic nitrogenous constituents (Table 11). The procedure finally adopted consists of distillin about 10 grams of moist residue or manure with 30 ml. of phospfmte buffer in 300 ml. of distilled water. The buffer solution is prepared as described by Kichols and Foote (3) by dissolving 14.3 grams of KHtPO, and 91 grams of KzHPOa in 1 liter of distilled water. In this way distlllation is accomplished at pH 7.4 and the only precautions necessary are those to prevent undue frothing.
XITRATE.The cause of a n apparently high yield of ammonia by the magnesia method is also operative in the nitrate determination, and accordingly a modification of the Bengtsson (1) procedure was employed.
Vol. 13, No. 3
TABLE 111. RECOVERY OF NITRATE NITROGEN ADDEDTO MANURE Nitrate Nitrogen Added
Nitrate Nitrogen Recovered
Ma.
M Q.
5 10 15 20
5.01 10,06 15.15
20.25
Reoovery
% 100.2
100.6 101.0 101.3
TABLE Iv. RECOVERY O F AMMONIA AND NITRATE NITROGEN FROhf DECOMPOSING STRAW (Comparison of old and new methods. Grams of nitrogen per 100 grams of original straw) -Nitrate Nitrogen0 Id Modified Ammoniacal Nitrogen Days MgO Buffer method method N Added as Ammonium Nitrate
N Added as Ammonium Carbonate and Sodium Nitrate 0 0.57 0 57 0.57 0.57 3 0 45 0 36 0.48 0.48 7 0.25 0.04 0.35 0.12 0 28 0 03 0.30 0.03 15 30 0 21 0 02 0.22 0.01 0.044 0.17 0.02 56 0.14 0 Increase due to ammonification of fungal protein which sets in after about 6 weeks’ decomposition.
Ten grams of the fresh sample are extracted with 300 ml. of distilled water in 50-ml. portions. Each fraction remains in contact with the sample for about 10 minutes prior t o filtration through a cotton plug. Extraction is continued until no blue coloration is obtained with diphenyl benzidine, a test sensitive to one part in a million ( 2 ) . Suspended colloids are then precipitated by the addition of a few drops of sulfuric acid (20 ml. to 100 ml. of water). The solution is warmed and coagulated matter is removed by suction. By addition of caustic soda the filtrate is made alkaline, and boiled down to 30 ml. A little more caustic soda is added and the volume is made up to 200 ml. before concentrating again to 30 ml. In this way ammonia initially present as such, or capable of being liberated from the small amount of organic nitrogenous material present, is removed. The residue is finally distilled with Devarda’s alloy after dilution to 300 ml. The effect of this improved procedure on the recovery of nitrate nitrogen added to manure is shown in Tables I11 and IV .
Summary The magnesia distillation method is unsuitable for the d e termination of ammonia in decomposed plant residues or manure, because high result’s are obt’ained through the concurrent liberation of ammonia from organic nitrogenous substances. The substitution of a phosphate buffer giving a reaction of p H 7.4 is recommended. Xitrate nitrogen can be determined on the aqueous extract of such materials by the Devarda reduction method after removing free or liberated ammonia by boiling under alkaline conditions.
Literature Cited (1) Bengtsson, N., Medd. 415, Centralanstalt. fors6ksuueend.d. jordbruks. Bakt. avdel., No.58 (1932). (2) Letts, E. A., and Rea, F. W., J. Chem. SOC.,105, 1157 (1914). (3) Nichols, M. S., and Foote, M . E., IND.E m . CHEM..Anal. Ed., 3, 311 (1931). (4) Richards, E. H., and Norman, A. G., Biochem. J.,25,1769 (1931). (5) Richards, E.H., and Shrikhande, J. G., Soil Sci., 39, 1 (1936).
THE work described in this paper was commenced at the Rothamsted Experimental Station, Harpenden, Herts, England, under the direction of E. H. Richards, late head of the Fermentation Department, and completed a t the Tea Research Institute, Talawakelle, Ceylon.
