ANALYTICAL EDITION
March 15, 1941
Literature Cited (1) Baldeschwieler, E. L., and Wilcox, L. Z., IND.ENG. CHEM., Anal. Ed., 11, 525-6 (1939). (2) Ringham, E. C., “Fluidity and Plasticity”, 1st ed., p. 225, New York, MoGraw-Hill Book Co., 1922. (3) Claivoe, G. W., PTOC. Am. SOC.Testing Materials, 32,II, 689-704 (1 093) \AVV-,.
(4) Combes, K. C . , Ford, C. S., and Schaer, W. S., IND.ENQ. CHEM.,Anal. Ed., 12, 285-7 (1940). ( 5 ) Cornthwaite, C. R., and Scofield, F., Sci. Sect., Natl. Paint, Varnish Lacquer Assoc., Circ. 547 (1938). (6) Gardner, H. A., and Parks, H. C., Paint Mfrs. Assoc. U. S., C~TC . 414-28 (1926). 265,
185
(7) Gardner, H. A,, and Van Heuckeroth, A. W., IND. ENQ.CHEM., 19, 724-6 (1927). (8) Gray, D. M., and Southwick. C. A., Jr., GEass Packer, 2, 17, 77 (1929). (9) Hall, F. P., Bur. Standards, Tech Paper 234, 345-66 (1923). I:lo) J.. “GraDhical and Mechanical Computation”. New . , LiDka. York, John Wiley & Sons, 1918. (11) McIntyre, G. H., and Irwin, J. T., J. Am. Ceram. SOC.,15,433-8 (1932). (12) Sward, G. G., and Stewart, J. R., Am. Paint Varnish Mfrs. ASSOC., C~TC 394, . 317-22 (1931). (13) Turnbull, E. D., Ceram. Age, 15,330-1 (1930).
Determination of Blood Magnesium Quantitative Spectrochemical Method FRANCES W. LARZB’, Harper Hospital, Detroit, Rlich.
A spectrochemical method for the determination of magnesium in blood which is accurate, rapid, and simple to perform and requires only 1 ml. of sample is described. The blood samples are first diluted with a potassium alum solution which acts both as a spectroscopic buffer and internal standard and then atomized into a spark between graphite electrodes. Analysis is made by measurement of relative intensities. The average error obtained by this method is less than 3 per cent.
I
N GENERAL clinical practice biological fluids are only
occasionally analyzed for magnesium. However, during recent years i t has become desirable to be able to determine accurately the magnesium content of blood as well as of other biological fluids in order to ascertain its possible relationship to certain pathological conditions. A comprehensive review of the work reported in this field, including the most reliable values for blood magnesium, is given by Myers and Muntwyler (10). As stated in the original articles (6, 6, 14, 15), the magnesium determinations were made by chemical methods of analysis. However, because of the limited amount of sample available, it is necessary to have a method requiring only a few milliliters for the analysis. For this reason the possibilities of a spectrochemical method embodying speed, accuracy, and minimum sample requirement have been investigated. A number of methods for the spectrochemical analysis of biological fluids for various elements have been reported in the literature. Among these are the methods described by Hess, Owens, and Reinhardt (7) and by Thomson and Lee ( I d ) , both of which make use of the well-known internal standard method of Gerlach (4),intensity calibration of each plate, and photometric measurement of the relative intensities of spectral lines as described by Duffendack, Wolfe, and Smith (9). Both methods require wet-ashing of the sample, a time-consuming step which the method here described successfully eliminates. The method used is that of atomizing a solution into a 1
Present address. Bohn Aluminum & Brass Corporation, Detroit, Mich.
spark between graphite electrodes, the atomizer being similar to the one employed by LundegHrdh (9) in his flame method. Several other methods have been described for introducing solutions into a spark: the Hitchen (13) sparking tube for solutions, the source used by Thomson and Lee ( I d ) in which the spark passes between two horizontal quartz jets from which the solution drips, and the more simple method employed by Keirs and Englis (8) in which the spark is formed between carbon electrodes, the upper one being hollow to permit inserting a glass capillary from which the solution drips. While each of these methods has a number of good points, the manipulations required to clean and refill the apparatus are more involved than is desirable for a method which is to be used for the routine analysis of a large number of samples. The preliminary work on this method has been reported by Cassen (1). However, a number of changes and refinements in technique have been introduced in order to obtain the necessary accuracy. The three features which combine t o make the method accurate, rapid, and simple to perform are use of a spectroscopic buffer which also acts as an internal standard, use of electrodes of special design, and photometric measurement of relative intensities of spectral lines. The blood is diluted with a solution of potassium aluminum sulfate whicli serves both as a spectroscopic buffer and as an internal standard. It has been esta,blished by experiment that when aluminum is present in a concentration correspondper liter of soluing to 47 grams of A12(S04)a.K2S04.24H20 tion (using the conditions of exposure standardized upon and described under procedye for analysis) the densities of tke aluminum line a t 2816 A. and the magnesium line a t 2795 A. are equal for a solution coiitaining 0.35mg. of magnesium per 100 ml. and also fall near the center of the straight-line portion of the characteristic curve of the photographic emulsion. Both conditions are ideal for obtaining reproducible results, and are especially desirable since this figure corresponds to 3.50 mg. of magnesium per 100 ml. of blood, which is about the average value reported for normal whole blood, the range being 2.75 to 5.00 mg. of magnesium per 100 ml. (6). The compound containing this internal standard, in the concentrat’ion used, is also a very effective spectroscopic buffer. The advantages of the use of spectroscopic buffers in analyzing biological materials are fully discussed elsewhere (3, 7 ) . Briefly, in this case, the problem is reduced t o the determinat’ion of small amounts of magnesium in a potassium aluminum sulfate base solution; and any effects which might be introduced by the comparatively small amount of organic material present in blood or other biological fluids have been
186
INDUSTRIAL AND ENGINEERING CHEMISTRY
found by experiment to be negligible. Proof of this statement is shown by the experimental results given in Table I. Thus it is possible to analyze the samples directly after proper dilution with the internal standard stock solution. This results in a marked saving of time as compared with the time required for wet-ashing of samples and also makes it possible to adapt the method easily to the analysis of other biological fluids.
CAQsON ELECTRODES w \THSWRPEO ENDS
h
FIGVRE 1. APPARATUS
The electrodes used are shaped as shown in Figure 1. These were found greatly to increase the uniformity of exposure since, as the electrodes burn away, the cross-sectional area (6.25 X 2.5 mm., 0.25 X 0.10 inch) remains constant, making i t possible to use the same pair of electrodes for a large number of samples. This narrow rectangular electrode also prevents wandering of the spark, for the source of illumination is at all times in optical alignment with the slit of the spectrograph. Further, it has been established experimentally that by sparking the electrodes in a spray of distilled water for 60 seconds after each sample all traces of aluminum and magnesium are removed. This fact, together with the shape of the electrode, makes it possible to use one pair of electrodes for all the exposures on one plate. This greatly reduces the number of manipulations required and is a great saving in time. The method of measuring relative intensities of spectral lines ( 2 ) has been used in order to obtain more reproducible results and to eliminate the necessity of repeating standard solutions on each plate. While it is necessary to place a calibration pattern on each plate, which is accomplished by nieans of a step sector, yet it is possible to run sixteen separate or eight samples in duplicate on a single plate.
Vol. 13, No. 3
the charateristic curve drawn for each plate, were plotted against the logarithms of the corresponding concentrations of magnesium. Figure 2 gives the resulting analytical curve for the determination of magnesium in a potassium aluminum sulfate base solution. PROCEDURE FOR ANALYSIS. The atomizer is adjusted to a rate of 15 ml. per 120 seconds. Before the first exposure and after each succeeding exposure the electrodes are cleaned by a preliminary sparking for 1 minute in a spray of distilled water. The distance between the electrodes is set a t 2.35 mm. (0.094 inch) by a spacer. The blood sample is diluted by pipetting 1 ml. of blood into 9 ml. of the internal standard stock solution, and then carefully mixed until a stable uniform suspension results. The diluted blood sample or a standard magnesium solution, as the case may be, is placed in the atomizer, the stop watch being started a t once; after the solution has atomized for a period of 30 seconds, the spark is started and a 30-second exposure made. The graphite electrodes are then cleaned by sparking in distilled water for 60 seconds and the procedure is repeated for a series of samples. For producing a calibration pattern on each plate (Eastman process) the step sector is rotated in front of the slit and a solution of ferrous sulfate is sprayed into the spark in order t o produce lines of suitable blackening for determining the characteristic curve of the late. The plates are rapidly rocessed by the procedure descriied by Sawyer (11) except that Eastman developer D-19 is substituted for D-8. The blackening of the spectral lines is then measured and the ratios of the relative log intensities of the selected pair of aluminum and magnesium lines are determined. By referring t o the analytical curve the amount of magnesium present is quickly obtained.
Results and Discussion I n order to determine the accuracy of the method a number of magnesium determinations mere made on whole blood, followed by redeterminations after known amounts of magnesium were added. Two different samples of blood were run in triplicate. Sample 1 was found to contain 3.10 mg. of magnesium per 100 ml. and sample 2, 3.51 mg. of magnesium per 100 ml. of whole blood. To each sample 1 mg. of magnesium as magnesium sulfate heptahydrate was added per 100
Apparatus For this work a Zeiss quartz spectrograph (chemist's model) was used. A rotating step sector (ratio 1.5) was placed directly in front of the slit for obtaining the plate calibration patterns. The atomizing unit (Figure 1) was placed 1 meter from the slit with no intervening lens system. A Hilger nonrecording microphotometer was used for making the photometric measurements. The spark source used was a condensed spark with synchronous interrupter.
Method of Analysis PREPARATION OF SOLUTIONS.Standard solutions containing 47 grams of AlZ(SO~)s.K2S04.24H~O per liter and 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0 mg. of magnesium as magnesium sulfate he tahydrate per liter are prepared. An internal standard stock sogtion for dilution of blood samples containing 52.22 grams of potassium aluminum sulfate per liter is prepared. DETERhfINATION OF ANALYTICAL CURVE. A number Of plates were made from the standard solutions using the technique described below. The blackenings of the aluminum line at 2816 -4. and the magnesium line at 2795 A. were measured pliotometrically. The logarithms of the ratios of the intensities of these two lines, obtained by application of the measured blackenings t o
CURVEFOR DETERMINAFIGURE2. ANALYTICAL TION OF MAGNESIUM ml. of blood. These additions were made on three separate portions of each sample, and the total magnesiums redetermined spectrographically. I n a similar manner 2 nig. of magnesium were added per 100 ml. to three separate portions of each sample and again the total magnesium contents redetermined. The results of these determinations are given in Table I. It is noted that the maximum error in these twelve determinations is 2.44 per cent, with an average error of 1.5 per cent.
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 per 100 MI. of Original Blood 2 Mg. of Mg per Errora 100 M1. Errora Me. MQ. % MQ. % 4.00 2.44 5.00 3.10b 1.96 3.10 4.10 0.00 5.05 0.98 4.20 2.44 5.20 3.10 1.96 3.51C 4.48 0.66 5.60 1.63 3.51 4.40 2.44 5.50 0.18 3.51 4.60 1.99 5.60 1.63 5 Per cent error based upon total amount of magnesium present in mg. per 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. Found er 100 M1. of riginal Blood
8
Found per, 100 M1. of Original Blood 1 Mg. of Mg per 100 hll.
+
+
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
1
Av. . F 2.83 3.25
3.10 2.83 3.32 3.65
..
3.78
4:55 3.90 3.25 3.40 3.55
4.58 3.93 3.12 3.30 3.55 3.25
..
3.10
4.28 4.60 3.23
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
