V O L U M E 2 4 , NO. 5, M A Y 1 9 5 2 7 years without significant changes. An analysis of a sample in duplicate requires 40 minutes, including the time required to weigh the samples and the mass spectrometric analysis. The authors have successfully analyzed with this apparatus the following types of compounds: fatty acids, amino acids, sugars, porphyrins, sterols, halogen, nitro and thio compounds, and salts of organic acids. In the analysis of sulfur-containing compounds care must be exercised that the oxides of sulfur do not reach trap C. ACKNOWLEDGMENT
The authors are deeply indebted to Konrad Bloch, Herbert
881 Anker, and Henry Hoberman for their advice a t various times and to Irving Sucher for the mass spectrometric analysis. LITERATURE CITED (1) Farkas, A , , and Farkas, L., Proc. Roy. SOC. (London), A144, 567 (1934). (2) Horiuchi, J., and Polanyi, M.,Nature, 132, 819 (1933). (3) Hughes, E. D., Ingold, C. K., and X'ilson, C. L., J . Chem. S O C . . 1934,493. (4) Keston, A. S., Rittenberg, D., and Schoenhelmer, R., J. Biol. Chem., 122,227 (1937). ( 5 ) Libby, W. F., J . Chem. Phys., 11, 101 (1943). ( 6 ) Rittenberg, D., and Urey, H. C., Ibid., 1, 137 11934). RECEIVED for review July 9 , 1951. Accepted February 6, 1952.
Microdetermination of Nitrogen in Organic Compounds W. C . ALFORD National I n s t i t u t e of Arthritis a n d Metabolic Diseases, ,Vational I n s t i t u t e s of Health, Bethesda, M d . The need for a reliable method for the microdetermination of nitrogen in organic compounds and the frequent failure of previous methods on refractory materials prompted development of a new procedure. The standard Dumas combustion tube is modified to permit combination of the combustion technique of the carbon and hydrogen train with the nitrogen isolation principle of the Dumas method. The sample is burned in a stream of pure oxygen, the combustion products are passed over copper oxide and platinum at 723" C., the oxygen is remoi-ed
T
HE necessity for nitrogen determinations in the characterization of many organic compounds emphasizes the need for a universally applicable method. Experience in this and other laboratories has shown that neither the Kjeldahl method, with its various modifications, nor the Dumas method can be relied upon to give correct results on all classes of compounds, particularly those containing heterocyclic nitrogen. This is borne out by the large number of papers dealing with attempts to modify both methods so as to make them applicable to a wider variety of materials. The most significant of these have been covered in recent reviews by Willits and Ogg (9,16,17). The same authors (18) have published a series of papers describing the development of a modified Kjeldahl procedure, which permits more accurate determination of nitrogen in heterocyclic compounds, such as tryptophan and nicotinic acid. However, no claim is made that the method can be applied to all types of organic compounds and, in fact, certain exceptions are noted. The widely used Friedrich (3) hydriodic acid reduction method for compounds containing oxidized nitrogen was criticized as being unreliable and it was concluded that optimum conditions for the Kjeldahl digestion are as yet unknown. The Dumas method, as adapted to microanalysis by Pregl (11), was originally thought t o be a universal method, but its failure on many compounds is now generally recognized. Such failure is usually attributed to incomplete combustion of refractory materials in the presence of hot copper oxide. Several attempts have been made to overcome this difficulty. Hayman and Adler ( 4 ) achieved some success by adding powdered copper acetate to the temporary filling to catalyze combustion, but others (1, 12, 1 3 ) found the method unsatisfactory. Spies and Harris ( 1 3 )and Ronzio (12) suggested the use of potassium chlorate, or other strong oxidants, to promote complete combustion of the
by hot copper, and the nitrogen is swept into potassium hydroxide with carbon dioxide. The time required for a complete analysis is about 40 minutes, about one half that needed for a standard microDumas analysis. Virtually all classes of nitrogen compounds have been analyzed successfully with no analytical failures on compounds of established purity. The method is especially useful for refractory heterocyclic compounds, but its speed, simplicity, accuracy, precision, and reliability have led to its use for all classes of Compounds.
sample. Although this method gives good results in some cases, it has not been found generally applicable. Its weakness may be that the temperature a t which the oxidant becomes active is either too low to effect combustion of the sample or so high that the sample boils or sublimes away from the oxidant. Recently, Sternglanz et al. (14) modified the Zimmermann (19) method and suggested the use of cobaltic oxide in lieu of previously used oxidants. Excellent results were reported for a few known refractory compounds, but the method was apparently not tested on other difficultly combustible materials such as phenazines, pyrazines, triazolee, benzimidazoles, and pyridine derivatives. Kirsten's (6) high-temperature, nickel-nickel oxide permanent filling is a radical departure from the standard Dumas method. However, the sample is mixed with powdered copper oxide in the usual temporary filling and no improvement in results can be expected where analytical failure is due to the formation of tars or charred masses which resist complete burning by hot copper oxide. Few results are reported for compounds that might ordinarily be expected to give low results by the regular Dumas procedure. Unterzaucher ( 1 5 ) described a method in Tvhich the sample is burned in a stream of moist carbon dioxide and oxygen. The oxygen is obtained by bubbling carbon dioxide through 307, hydrogen peroxide solution containing finely divided platinum. It appears that the carbon dioxide-oxygen ratio would be difficult to control. This method cannot be evaluated from the results reported because all the compounds listed are readily analyzed by conventional methods. I n this laboratory, the necessity for handling a wide variety of nitrogenous compounds, including various heterocyclic systems, and the frequent failure of previously used techniques on such
882
ANALYTICAL CHEMISTRY
materials, prompted the development of a new method which has proved effective on all classes of compounds encountered thus far. The method combines the combustion technique or^ the usual carbon and hydrogen microcombustion train with the nitrogen isolation principle of the Dumas method-Le., the sample is burned in a stream of pure oxygen, the combustion products are passed over hot copper oxide and platinum, the oxygen is removed by hot copper, and the nitrogen is swept into a nitrometer with carbon dioxide. I n principle the method is based upon the theory that failure of the Dumas method is due to incomplete combustion, and the fact that those compounds which give low nitrogen results offer no difficulty in the carbon and hydrogen determination, where complete combustion is, of course, a prerequisite for accurate results. After more than 6 months of routine use, no failure has been observed on any sample that has been judged to be pure on the basis of physical constants and elemental analyses. The procedure requires about 40 minutes, which is much less than the time required for the standard Dumas method. The only disadvantage is the requirement of oxygen completely free of inert gsses. APPARATUS
The entire apparatus (Figure 1) is assembled from corniercially available equipment, with the exception of the gasometer, G, and the bubble counter, C, which were constructed of borosilicate glass as shown. The essential components of the apparatus are described briefly as follows:
Platinized Asbestos. Thirty per cent platinized asbestos is prepared by dissolving a weighed amount of platinum in aqua regia, after which the calculated amount of micro grade asbestos is added. The mixture is well stirred, evaporated to dryness, and finally ignited a t red heat for 10 to 15 hours. Platinum Gauze. The gauze is formed into a roll which fits the combustion tube snugly. Hydrogen. Commercial tank hydrogen. Oxygen. Because the oxygen must be free of inert gases and because none of the commercial oxygen that was tested (electrolytic and from liquid air) met this requirement, it was prepared cheniically as follows: A 2-foot length of stainless steel tubing (1/4 X 3/38 inch) iyith a "tee" and needle valve in the center, was brazed to the outlet of a needle valve which fitted a "20-cubic-foot" oxygen cylindei . The other end of the tube was connected to a 7OO-ni1. highpressure hydrogenation bomb. The bomb was charged with ti40 grams of C . P . potassium permangante, over which a thick layer of borosilicate glass wool was packed firmly. The syateni was alternately evacuated (10 minutes) with a Cenco Hyvac pump and charged with commercial oxygen (100 pounds per square inch) through the valve on the tee. -4fter three such operations the vacuum pump was run for 1 hour to remove all residual air. The valve on the tee was closed and the bomb was slowly heated to 250' C. in a thermostatically controlled jacket heater. This temperature was maintained for 10 minutes and the main valve on the oxygen cylinder was closed. At 240" C. potassium permanganate decomposes according to the follo~vingequation: 2KMn04
K&lnO,
+Ah02 +
01
The theoretical yield is about 45 liters of oxygen; which, with allowance made for normal losses, is sufficient for a t least 500 analyses. Once the atmaratus is assembled. about 4 houriire required for the prep: aration of the oxygen, which has been found to be completely free of inert gases. PREPARATlON OF APPARATUS
The combustion tube is cleaned and dried as usual. A 1-cm. plug of glass wool is placed in the tube a t the capillary outlet and is followed by a 32-cni. F F layer of copper oxide which is tapped down firmly to eliminate air spaces. A 5-mm. layer of 30% platinized asbestos is packed into place with a glass rod and the tube filling completed, as shown in Figure 1. The tube is COPPER OXIDE R E D U C E D COPPER PtGAUZE CuO Pi. flushed with coniniercial hydrogen at a rate of 100 to 150 ml. per minute for 5 BCM J G b 3 0 C M 12 CM. 2 0 CM. w Iminutes. Then a micro combustion furnace, F , heated to 700" C., is Figure 1. Diagram of Apparatus for Microdetermination of Sitrogen closed around the tube so that the section marked '*reduced copper" (Figure 1) is heated. Hydrogen flow continuously. The reduction proceeds smoothly and requires about The carbon dioxide genrrator (CO,) is a 1-liter Dewar flash. 1 hour a t the flow rate suggested. If the rate of flow is faster than fitted with a rubber stopper, a delivery tube and a Hershberg ( 5 , indicated, the temperature rises to a point where the reduced mercury safety valve. A medium-sized cylinder ( 0 2 ) , (20 cubic copper melts, thus destroying its porous surface. The hydrogen feet), as commonly used for medical grade oxygen, holds the should flow from the open end of the tube ton-ard the capillary specially prepared oxygen. Gasometer G is made from a 25-m1n. t o avoid staining the empty portion of the tube with powdered test tube and has a capacity of 50 ml. I t is sealed to a bubble copper oxide which is carried along by the water formed in the counter, C, similar to those used on a microcombustion train reaction. Khen the reduction is complete, the tube is cooled Butyl phthalate is used in the bubble counter. The combustion arid then flushed with carbon dioxide to remove hydrogen. The tube is made of Vycor glass (No. 790) and is identical with Corntube is conditioned by placing it in both furnaces (Figure 1) ing item 18660 except that a 25-cm. length of similar tubing is and heating at 500" C. for 3 or 4 hours (or overnight if desired), sealed on to give a total length of 75 cm., exclusive of capillary during which time a slow stream of carbon dioxide is passed tip. (The longer tube is available from Corning Glass Co. on through the tube. It is then ready for use. special order.) Two microcombustion furnaces, F (Fisher SciThe nitrometer is cleaned and filled with mercury and 50% entific Co., Item 20-286), are used. The nitrometer, AT,conforms potassium hydroxide solution according to Niederl (8). After t o the specifications given by Niederl (8). Butyl rubber tubing, the nitrometer has been filled, the surface of the mercury is lubricated with glycerol, is used for making glass to glass joints. ovidized electrolytically to prevent sticking of bubbles. Two All connecting glass tubing has a 2-mm. capillary bore. dry-cell batteries are connected in series and, by means of fine copper m r e , the positive pole is connected to the mercury REAGENTS through the inlet tube of the nitrometer, \vhile the negative pole is connected to the alkali solution in the leveling bulb. Twenty Carbon Dioxide. Commercial dry ice is crushed to a fine powder. to 30 minutes are required to produce a coating of finely divided oxidized mercury, which has proved more effective than the Copper Oxide. Wire form copper oxide of "micro" grade. materials commonly used to prevent sticking of bubbles. Potassium Hydroxide, 50%. Five hundred grams of C.P. potassium hydroxide are dissolved in 500 ml. of distilled water, The carbon dioxide generator is charged with crushed dry ice the day before it is to be used, so as to permit sweeping of all 1 ml. of isoamyl alcohol is added t o prevent frothing, and the residual air from the flask. solution is stored in a rubber-stoppered bottIe.
,*,
--+
V O L U M E 2 4 , NO. 5, M A Y 1 9 5 2 With the aid of a thermocouple, voltmeter, and empty combustion tube, the rheostats of both furnaces are adjusted so that a temperature of 700’ to 725” C. is attained inside the combustion tube. This rheostat setting is then maintained during future analyses. ANALYTICAL METHOD
Both furnaces are preheated to 700” C. while a slow stream of carbon dioxide (two to three bubbles per second) is passed through the combustion tube. A sample, estimated to contain from 0.35 to 0.55 mg. of nitrogen, is w-eighed in a platinum boat on the microbalance. The 2 tube is disconnected from the combustion tube and the boat and sample are placed a t a point about 5 cni. from the furnace. The 2 tube is replaced and the system is swept with a rapid stream of carbon dioxide (6 to 10 bubbles per second) for 10 minutes to remove all air. Then, with the leveling bulb, L1, of the nitrometer lying on the table and stopcock S 1 open, the nitrometer is connected to the capillary tip of the combustion tube. After a few seconds LI is raised and the nitrometer is filled with the 50% potassium hydroxide solution. S1 is closed when the funnel on top of the nitrometer is about one half filled. The bulb is placed in a split-ring holder which is adjusted so that the level in the bulb corresponds to about the 0.3-ml. mark on the nitrometer. This position of the nitrometer is different from that ordinarily used. The usual test for “micro bubbles” is made-e.g., the bubbles should require 30 seconds or more to reach the top of the nitrometer. Stopcocks Sa and S3 are closed, the main valve on the oxygen cylinder is opened, and by means of the needle valve on the cylinder (or diaphragm regulator valve) a slow stream of oxygen is allowed to escape through the mercury safety valve, V . The mercury-filled leveling bulb, L,, connected to gasometer G, is lowered, and stopcocks S4and SSare opened to allow oxygen to displace the mercury in the gasometer. When the gasometer is filled, S4 is closed, L? is raised, and 85 is opened to the air to discharge the oxygen from the gasometer. (This operation is omitted on subsequent analyses, but is practiced on the first analysis of each day as a safeguard against possible air contamination.) Stopcock Sbis closed while oxygen is still escaping. S, is opened, and the gasometer is again filled with oxygen by lowering La. S4 is closed and the valve on the oxygen cylinder is turned off. The leveling bulb, Lz, is raised so that it is above the gasometer, and by means of Sz about 20 ml. of oxygen are passed a t a fairly rapid rate into the combustion tube. Then L1 is raised and any gas or debris is allowed to escape from the nitrometer. From now until the end of the analysis, LI is lowered only enough to permit the desired flow of gas through the system. This is a variation of the usual technique. The flow rate is reduced to two or three bubbles per second with S2 and the sample is burned with the full flame of a Bunsen burner in a manner exactly like that described by Niederl (8) for burning samples in a micro carbon and hydrogen train. A Nichrome wire gauze, S,shields the tube from the direct flame. The first burning requires about 7 minutes, while the second is completed in 5 minutes, a t which time all the oxygen should have been forced from the gasometer. The times given for the combustion periods will, of course, vary with the nature of the sample. Refractory materials require a longer heating period. The progress of the combustion may be followed visually as in the micro carbon and hydrogen determination. Stopcock Ss is closed and S a is opened to sweep the system with carbon dioxide. The bubble rate is adjusted with Sa.The tube is swept a t a fairly rapid rate ( 4 to 6 bubbles per second) so that 30 to 40 ml. of carbon dioxide are passed through the tube in about 8 to 10 minutes. Sweeping is continued for no longer than 10 minutes more, when microbubbles, as described by Pregl (11), are certain to have appeared. The nitrometer is then disconnecfed from the combustion tube. The empty boat is removed while the carbon dioxide is still flowing and the furnaces heating. Another sample is placed in the tube and sweeping yith carbon dioxide is commenced, preparatory to another analysis. After 10 minutes the volume of gas in the nitrometer is read. Two per cent of the volume is subtracted, according to Pregl(11). and any predetermined “air blank.” The per cent nitrogen in the sample is calculated as usual. Two samples are weighed while the furnaces are heating and thereafter one sample is weighed during the 10-minute interval allowed for the nitrogen in the nitrometer to equilibrate. The total time for an analysis is 40 to 45 minutes and ten samples can be run in an 8-hour day without difficulty. DISCUSSION
The copper in the combustion tube filling is sufficient for about 75 analysee, after which it may be reduced again in less than 2
883 hours. The useful life of the tube is not known, as it has been the practice to preoare a new tube after about 200 analyses because of the discoloration which occurs a t the point where samples are burned. SJveepingis carried out a t a much faster rate than in the standard Dumas procedure and no particular care is necessary in regulating the flow to a specified rate, Complete reduction of nitrogen oxides and oxygen is accomplished by the unusually large amount of hot metallic copper. This is in conformity with the findings of Colson ( 3 ) that, despite a large amount of reduced copper in the combustion tube, no carbon monoxide is formed. At first, the carbon dioxide was metered with the gasometer according to Niederl (8), but later it was found that the simpler expedient of sweeping to the reappearance of microbubbles, according to Pregl i l l ) , gave equally good results. Sweeping with 50 ml. of carbon dioxide a t a rate of approximately 2 ml. per minute gave the same air blank as that observed when sweeping was a t the rate of 10 ml. per minute for 5 minutes. I t was decided to sweep a t a rate approximating 5 ml. per minute, which is sufficient to complete the process in 8 to 10 minutes. .4lthough it has been reported that the so-called air blank can be reduced to negligible proportions by painstaking preparation of carbon dioxide (8, I O ) , the use of dry ice as a source seems preferable because of its simplicity and convenience. I t has been found that with dry ice a small air blank is invariably observed on a “blank” run or when a standard sample is analyzed. However, for any given lot of dry ice the blank is constant and has in general been found to be about 0.010 nil. The blank is determined by analyzing Bureau of Standards acetanilide and backcalculating to determine the excess gas collected. The use of a small but constant blank introduces no appreciable error into the analysis. Several tanks of commercial oxygen tested were found to contain from 0.2 to 0.5% of inert gases, which was sufficient to make the oxygen unsatisfactory for use. The method described above for preparing pure oxygen is satisfactory and not too timeconsuming, provided the necessary equipment is available, but a commercial source would, of course, be desirable. Other chemical means would doubtless be equally satisfactory. The usual three-way precision stopcock between the combustion tube and nitrometer is not mentioned in the section “Analytical LIethod.” It was not used during the course of the work reported here, because it is not necessary for the regulation of flon- rates. Recently, however, it has been added to the apparatus to eliminate the necessity of disconnecting the nitrometer after each analysis. .hother variation in the usual technique is the position of the nitrometer leveling bulb during the analysis. It is lowered only enough t o permit the desired flow of gas through the system. By so doing the troublesome tendency of the potassium hydroxide solution to leak past the stopcock of the nitrometer is largely eliminated. The horizontal position of the gasometer tube is desirable because it decreases the rate of change of pressure as the mercury displaces the gas in the gasometer. Thus, if the leveling bulb, Lz, is raised a fen- inches above the top of the gasometer, the flow rate can be kept reasonably constant nith only two or three adjustments of Sp in a 10-minute period. Advantages of Present Method. Of prime importance is the fact that it has proved entirely satisfactory on a wide variety of nitrogen-containing compounds, including most of the common heterocyclic systems. Compounds such as pyrimidines, pteridines, triazoles, benzimidazoles, imidazoles, pyridinee, indoles, and phenazines are analyzed with the same ease, speed, accuracv, and precision as are simple amino and nitro compounds. The only variation in technique required for refractory materials is a more vigorous (longer) heating of the sample in cases where combustion proceeds s l o ~ ~ l yThe . fact that the progrees of the combustion can be followed visually, as in a carbon and hydrogen
ANALYTICAL CHEMISTRY analysis, is in itself an advantage over methods which call for mixing the sample with copper oxide. It seems probable that nitrogen can be determined successfully in any organic compound which can be analyzed for carbon and hydrogen content by conventional methods. By eliminating the temporary filling entirely, the method is made cleaner and possible errors due to adsorption of nitrogen on the fine copper oxide are avoided. There is no necessity for cooling the combustion tube between analyses; the next determination can be commenced a t once. The time required for complete analysis (40 t o 45 minutes) compares favorably with any previous method and is much less than that of the conventional Pregl method (90 t o 105 minutes). The time used in the reduction of copper oxide after 75 analyses and the preparation of oxygen after about 500 analyses is negligible, when compared to the time saved.
Table I. Comparative Results of Pregl-Dumas, Ma and Zuazaga Kjeldahl (7), and Present Methods Compound D-Glucose phenylosotriazole
Nitrogen Found, % Nitrogen Present Calcd., % Dumas Kjeldahl method
15.84
15.17
2.59 8.52
15.77 16.03 15.73 15.83 6.97
lO-Chloro-pyrid0[3,2-a]phenazine 15.82
14.67
2-Chloro-8-ethoxy-5-methyl-phena-6.97 zinium methyl sulfate monohydrate 2-Bromo-7-ethoxy-5-methyl-phena- 6.27 zinium methyl sulfate A’-Methyl nicotinamide chloride 16.23
6.19
4-~-gZycero-2-Hydroxymethyl-2,4-16.66 cis-di(Z-benzimidazoIyl)-l,3-dioxolane Codeinone oxime hemimethanolate 8.53 Codeinone picrate 10.50
8.59 14.93
a,e-Di(g- henanthry1)biguanide h y drochoride
13.02 13.82 9.37
13.89
...
10.05
55.27 54.69 56.34
55.72
56.97
14.29
N,Nf-Di(9-phenanthryl)guanidine 10.21 1 -Methyl-3-cyanoguanidine
57.11 *
a
... ... 6.63 8.21
... 5.55
6.34
8.94 11.47“ 14.98 16.75a
16.12
.. ... ,
16.59 16.43 8.41 10.30 10.27 14.28
Table 11. Typical Results Obtained on Research Samples of Known Purity
Nitrogen, % Compound Calcd. Found D-Iodoheptose phenylosotriazole 14.22 14.19 L-a-Rhamnohexose phenylosotriazole 15.04 14.85 L-a-Rhamnohexose phenylosazone 15.04 15.01 Thiamine hydrochloride 16.61 16.49 N-(4-Chloro-2-nitrophenyl)xenylaminehemihydrate 8.41 8.47 2-Chloro-6,lO-dimethylphenaziniummethyl sulfate 7.90 8.05 7-Desoxy-~-gala-~-manno-heptobenzimidazole 9.94 9.99 D-Altro-D-gluco-heptobenzimidazole 9.40 9.45 4-~-gZycero-2-hydroxymethyl-2,4-cie-di(2-benzimidazolyl)-l,3-dioxolane 16.66 16.59 L-a-Rhamnohexobenzimidazole 9.90 9.88 L-B-Rhamnohexobenzimidazole 9.33 9.26 Benzedrine dinitrobensoate 13.95 13.87 Glycerol tri(p-nitrobenzoate) 7.79 7.65 Cystine5 11.66 11.58 2-Carboxymethyl-4-benzoxycyclohexanone semicarba- 14.70 Codeinone-2,4-dinitrophenylhydracone 14.70 zone Imidazol aldehyde-thiosemicarbazone hydrochloride monohydrate Folic acid” Tryptophan0 a Commercial samples of proved purity.
12.61
12.53
31.31 22.22 13.72
31.39 22.15 13.64
The method was then adopted for routine use on all organic compounds received by the laboratory and, over the course of many months, has been applied to hundreds of ssmples representing virtually all the common classes of nitrogenous compounds. Typical results obtained on research samples of established purity are shown in Table 11. As Tables I and I1 give results obtained from a series of compounds of established purity and known nitrogen content, it is possible to combine these data and compute the standard deviation of the differences between the theoretical and observed values. This was also found to be 0.112%. With a standard deviation of this size, and a normal distribution of errors, we could expect, on the average, that 99.3% of all determinations would fall within the conventional &0.3%. The close agreement between this standard deviation and that for the acetanilide series is, of course, largely coincidental.
4-hour digestion period.
ACKNOWLEDGMENT
The summation of the advantages of the method may be concluded by the statement that since its adoption it has been more reliable and trouble-free than any other routinely performed analysis in this laboratory. RESULTS
The method described was applied first to simple nitrogen compounds such as acetanilide to establish ita accuracy and precision on tractable materials. For example, over a period of several weeks the air blank was determined 26 times, using 3- to 5 m g . samples of Bureau of Standards acetanilide. The average blank was found to be 0.013 ml. This value was then applied to each of the 26 analyses. Under these adverse circumstances (constant purity of carbon dioxide falsely assumed) the maximum error was O.26Ljb. Eighteen of the calculated results varied from theory by less than 0.1% while only two exceeded 0.29”. In fairness, it may be stated that, for a given lot of dry ice, results on acetanilide are consistently reproducible with less than O.l$Zo variation from theory. The standard deviation of the differences between the theoretically computed values and the per cent nitrogen found by this method was 0.112%. Having established its reliability on normally amenable compounds, the method was next applied to a series of more refractory substances which, although thought to be pure on the basis of other analyses, had failed to give good nitrogen determinations by the Kjeldahl and Dumas methods. In every case, acceptable nitrogen values were obtained without difficulty. Duplicate analyses were run when sufficient sample was available. The results of these analyses are shown in Table I.
The author is grateful to Margaret M. Ledyard for her helpful suggestions and performance of many of the analyses. LITERATURE CITED
(1) Brancone, L. M., and Fulmor, W.,ANAL.CHEM.,21, 1147 (1949). (2) Colson, A. F., Analyst, 75, 264 (1950). (3) Friedrich, A., 2.physiol. Chem., 216, 68 (1933). (4) Hayman, D. F., and Adler, S., IND.ENG.CHEM.,ANAL.ED.,9, 197 (1937). (5) Hershberg, E. B., and Wellwood, G. W., Ibid., 9, 303 (1937). (6) Kirsten, ANAL. CHEM.,19, 925 (1947); 22, 358 (1950). (7) Ma, T.S.,and Zuazaga, G., IND. ENG.CHEM.,ANAL.ED.,14, 280 (1942). (8) Kiederl, J. B., and Nederl, V., “Micromethods of Quantitative Organic Analysis,” 2nd ed., pp. 79-94, New York, John U‘iley & Sons, 1946. (9) Ogg, C. L., and Willits, C. O., ANAL.CHEM.,23, 47 (1951). (10) Pagel, H. A., IND.ENG.CHEM.,ANAL. ED.,16, 344 (1944). (11) Pregl-Grant, “Quantitative Organic Microanalysis,” pp. 63-85, Philadelphia, Blakiston Go., 1946. (12) Ronzio, A. R., IND.ENQ.CHEM.,ANAL.ED., 8, 122 (1936); 12, 303 (1940). (13) Spies, J. R., AND Harris, T. H., Ibid., 9, 304 (1937). (14) Sternglanz, P. D., Thompson, R. C., and Savell, W. L., ANAL. CHEM.,23, 1027 (1951). (15) Unterzaucher, J., Chem. Ing. Technik, 22, 128 (1950). (16) Willits, C. O., ANAL.CHEM.,21, 132 (1949). (17) Willits, C. O., and Ogg, C. L., Ibid., 22, 268 (1950). (18) Willits, C. O., and Ogg, C. L., J . Assoc. OBc. Agr. Chemists, 31, 565 (1948); 32, 118, 561 (1949): 33, 100, 179 (1950). (19) Zimmermann, W.,Mikrochemie ser. Mikrochim. Acta, 31, 42 (1943).
w.,
RECEIVEDfor review October 24, 1951. Accepted January 25, 1952. From a thesis submitted in partial fulfillment of the requirements for the degree of doctor of philosophy, Georgetown University, June 1951.
