Organic Microanalysis I.
Nitrogen by Dumas Method
R. T. MILNER
AND
MILDRED S. SHERMAN
Bureau of Chemistry and Soils, U. S. Department of Agriculture, Washington, D. C.
D
URIXG the last four years over a thousand organic
Supremax glass, bas been used for several hundred analyses with no sign of d e t e r i o r a t i o n ; in fact, mechanical fracture has been responsible for the loss of all tubes since using the electric furnace. The movable Bunsen burner is e q u i p p e d with a wing tip and mounted on a heavy base plate by a rough rack and pinion, paralleled by a scale ruled on the base. This enables the burning to be accomplished very smoothly and regularlv. The burner is advanced 1 em." per minute until decomposition starts and, after that, 1 or 2 mm. per minute until the desired bubble rate is reached. Since the burner is never advanced too far by a slip of the hand, very little attention is required for the combustion. With the azotometer now in use, the analysis is finished 25 minutes after the end of the first burning. On the average 1.25 hours are required for an analysis, allowing 30 minutes for the first combustion. The azotometer is made of Pyrex glass designed without a stopcock a t the top. Difficulty was always experienced with the stopcock of the old type of azotometer. Either grease would contaminate the potassium hydroxide or the stopcock would freeze on standing idle for short intervals. The present azotometer has a cup opening a t the top, closed by a short glass rod ground into the azotometer tube. There is no constriction of the graduated portion of the tube, such as there was in the old azotometer. Smaller volumes of nitrogen can be read and the use of a trace of ceresin on the ground joint has eliminated sticking. The plug is sealed by the alkali itself and can be held firmly seated by a rubber band stretched over it. The graduated portion with groundin plug can be made a t moderate cost by any apparatus maker and, after calibrating with mercury, can be seated to the wide lower portion of the azotometer. Pyrex resists the action of alkali very well, since recalibration after one year's use showed no deviation greater than 0.001 cc. from the original Bureau of Standards calibration. The potassium hydroxide solution is made of equal weights of c. P. stick potassium hydroxide and distilled water without any additional treatment. The potassium hydroxide is renewed after approximately ten analyses and sticking of bubbles at the mercury surface is rarely encountered.
A n all-glass apparatus, which is easily handled and includes an improved azotometer without stopcock, has been designed and used in many hundred analyses. All arbitrary procedure in correcting for errors has been eliminated and the corrections have been experimentally determined. More accurate results are thus obtained without excessive time or attention.
compounds have been analyzed in this laboratory. These compounds have been of the most varied types, and the original methods of Pregl (IO) as well as m a n y modifications suggested later have been checked carefully. A brief description of the amaratus and Drocedure now employed should be of assistance to all microanalysts. The analysis is easier, quicker, and somewhat more accurate than when using Pregl's directions. Carbon dioxide generators of the Kipp type, using the procedure recommended by Pregl, were found unsatisfactory because of the short life after the exhaustive purification required. Various modifications with provision for the use of carbon dioxide above the acid (3, 6, 13) were tried and discarded because they were cumbersome or difficult to manipulate. Generators using a solid carbonate with heating were unsatisfactory because of the troublesome heating required before use (4, 8). The generator described by Poth (9) in its original form (no advantages were found for the modified generator, 7 ) has proved the best. Such generators after careful filling deliver to a small mercury-filled gasometer carbon dioxide which contains less than 0.001 per cent impurity (not condensed by liquid air). One filling will deliver about 100 liters of carbon dioxide and the gas is uniformly pure and available on turning a stopcock. The most careful recent study of the Dumas method has been made by Trautz ( l a ) ,and the combustion tube filling and the combustion procedure follow his directions. The points discussed below, however, differ from Trautz.
Apparatus
The size of the opening through which the bubbles enter An electric furnace, split type, was constructed cheaply by the azotometer is of importance, but cannot be regulated boring a 1.1-cm. (0.44-inch) hole lengthwise through an Armstrong easily, since in sealing on the capillary inlet tube (approlrirefractory brick, cutting the brick in halves, and then cutting eight slots around the periphery of this central hole with a hackmately 0.8 mm. inside diameter) it is impossible to avoid saw blade. Nichrome wire, No. 26, was laid in these slots, hairwidening it slightly. The volume of one bubble can, however, pin fashion, and held in place by a light coating of alundum cebe easily ascertained. I n the azotometer now in use 1 bubble ment. The two halves were mounted in sheet-metal holders equals 0.0167 cc. Weygand (IS), who discusses this point at hinged together and held on a rod. Such a furnace, with the two halves connected in series, has a resistance of about 10 ohms and, when carrying a current of approximately 4 amperes regulated by a General Radio Co. Variac, is easily maintained at 700' C. A thermocouple buried in the a l u n d u m lining indicates the temperature on a c a l i b r a t e d millivoltmeter. The replacement of the long burner with this electric furnace has proved very advantageous. The furnace can be opened, slid back, and the tube cooled with an air blast in 10 minutes, so that there is no time lost before the next analysis. Likewise the heating of the room is greatly reduced. FIGURE 1. DIAGRAM OF APPARATUS The t u b e now in use, of Jena
A
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INDUSTRIAL AND ENGINEERING CHEMISTRY
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some length, has an obvious misprint (p. 29) in giving the volume of 20 bubbles as 4.0 cu. mm. or less. Friedrich probably gives the best criterion for true microbubbles-namely, their uncertain, slow (40 to 90 seconds) rise in the graduated part of the azotometer. Naturally, each azotometer is different with respect to the volume of the single bubbles so that the rate of burning employed and the size of the final microbubbles will vary, depending on the diameter of the azotometer inlet tube. The gas collected in the azotometer is subject to three corrections: (1) the wall error of the azotometer, (2) the vapor pressure of the alkali solution, and (3) the nitrogen present in the carbon dioxide or in the interstices of the tube filling or leaking in through the rubber connections. All the above corrections were considered by Pregl to be satisfactorily taken into account by the deduction of 2 per cent of the gas volume as read. Of this, 1.5 per cent was supposedly due to the wetting of the walls and 0.5 per cent was due to the vapor pressure of the potassium hydroxide solution. Trautz later pointed out after careful experiments that the wall error was really only 0.5 per cent. This has been confirmed by measurements made in this laboratory. Trautz also assumed (p. 308) that the vapor pressure correction was only 0.3 per cent. However, measurements made by Clark ( 1 ) as well as some made in this laboratory show this to be too low. The method of Smith and Menzies (11) was used and both fresh and used (10 analyses) potassium hydroxide solutions were measured. The results are shown in Table I. For comparison the values found by Clark and data interpolated from International Critical Tables (6) are given. The agreement among the results is not very good, but it is sufficient for the purposes of the correction, and the values from International Critical Tables have been used as the best basis for the correction. As can be seen, the variation with temperature is such that no simple correction for all temperatures can be made. Accordingly the values from the International Critical Tables have been used to construct a table of corrections to be applied to the barometric reading. HYDROXIDE TABLE I. VAPORPRESSUREOF POTASSIUM (Vapor pressure, in mm. of mercury, of,solution containing approximately 71.5 grams of potasslum hydroxlde per 100 grams of water) Fresh Used Temperature Solution Solution Clark I. C. T.
c.
The value of the last correction, for air leakage or adsorption, depends on both the manner in which the apparatus is assembled and the pressure rela tionships within the combustion tube. Using the azotometer in its holder, as usually purchased, with the combustion tube about 22.5 em. (9 inches) above the desk top it is found that, even with the alkali leveling bulb lying on the desk top, a pressure greater than atmospheric exists within the combustion tube. With rubber connections in the train, this may well lead to low nitrogen values. If, however, a 0.5 or 0.3 per cent correction has been made instead of the true, higher correction for vapor pressure, this error may be compensated and approximately correct nitrogen values found. Unless the rubber is exceptionally tight, it is almost impossible to guard against leaks either into or out of the tube because, during a combustion, the pressure within the tube will vary appreciably as the gas collects in the azotometer. The all-glass apparatus now in use in this laboratory has a combustion tube with two ground-glass joints with the final stopcock sealed directly to the azotometer. Since all regulation of the gas flow is accomplished by the cock on the carbon
VOL. 8, NO. 3
dioxide gasometer, this final cock is a three-way one, so that the tube can be evacuated before starting the combustion. I n this way the measured error due to air in the carbon dioxide and adsorbed on the copper oxide has been reduced to 0.002 cc. On compounds of known purity, values are consistently obtained within less than *0.1 per cent of theory. TABLE 11. NITROGEN CONTENT OF SOMEREPRESENTATIVE COMPOUNDS Aaobenaene, CizHioNz Ammonium sulfate, (NHa)zS04 Urea, CH4ONz 1,2-Dihydro-2-keto-4-ethoxypyrimidine, CeHsOaNz Parabanic acid CaHzOaNn 6-Methyluracil: C1He0pNa l-Tetraacetylglucosido-7-acetylcytosine, CZOHZLO~~NS 1-Glucosidocytosine nitrate monohydrate, CioHi~08Na~HNOaHsO 1,2-Dihydro-2-keto-l-ethyl-4-ethoxypyrimidine, CsHizOzNz Cytosine, C4HsONa 1-Methylcytosine, CaHrONs 5-Bromouracil, CaHaOnNaBr
2,4-Dimethoxy-5-bromopyrimidine, CeH7OnNzBr
Found Fpund Dumas’ Calculated XJeldakh 15.44 15.39 21.10 21.20 ... 46.61 46.66 , , .
...
20.11 24.56 22.22
20.01 24.57 22.22
8.86 15.59 15.60 16.47 16.39 32.13 35.05 31.72 32.52 14.04 14.17 10.71 11.33
8.70 15.82
...
16.66
16.71 16.77 37.58 37.63 33.58 33.63 14.55 14.70 12.89 13.02
37.84 33.60 14.67 12.79
,
, ,
...
,
...
Some years ago it was found that, contrary to the statements in many standard texts, some compounds cannot be analyzed correctly by this method even when potassium chlorate is used. This has recently been pointed out by Friedrich (3, p. 74). The last half of Table I1 gives some examples that have been found in this laboratory. The Kjeldahl method used, which does not involve any preliminary reduction or treatment, will be described in a forthcoming note. X o generalizations can be made because, while bromine appears to be a disturbing factor, it is not present in the cytosines which also give low values. Likewise, in this apparatus, compounds such as urea, containing a high percentage of nitrogen, are analyzed with success. In the analysis of pyrimidines and possibly other cyclic ureides, imidazoles, and purines which are often obtained from natural products, it would seem best to check the Dumas nitrogen values by the Kjeldah1 method. Many compounds have been encountered which stick so obstinately to the sides of the mixing tube, especially in winter when the relative humidity is low, that now, as a matter of routine, all weighings are made in porcelain boats and mixed with fine copper oxide after introduction into the combustion tube. This has also been experienced by others (2).
Literature Cited Clark, J . Assoc. Oficial Agr. Chem., 16,575 (1933). Drake, N. L., University of Maryland, private communication. Friedrich, A., “Die Praxis der quantitativen organischen Mikroanalyse,” Leipsig and Vienna, Frans Deuticke, 1933. Govaert, Mikrochemie, 9,338 (1931). Hein, F., 2. angew. Chem., 40, 864 (1927). International Critical Tables, Vol. 3, p. 373. Lowe and Guthmann. IND. EXG.CHEM,.Anal. Ed., 4,440(1932). Poth, I b i d . , 2,250 (1930). Ibid., 3,202 (1931). Pregl, “Quantitative Organic Microanalysis,” 2nd ed., London, J. and A. Churchill, 1930. Smith and Menzies, J . A m . Chem. Sac., 32, 897 (1910). Trauts, Mikrochemie, 9,300 (1931). Weygand, C., “Quantitative analytische Mikromethoden der oraanischen Chemie,” D. 36, Leipzig, Akademische Verlagsgeiellschaft, 1931. RECEIVXD May 19, 1936. Presented before the Microchemical Section a t the 9 l s t Meeting of the American Chemical Society, Kansas City, Mo., April 13 to 17, 1936.
