am
A N A L Y T I C A L CHEMISTRY
action is kinetically complex (in part heterogeneous), no kinetic treatment of the data is made. The results of this fractionation study, presented in Table 111, show conclusively that serious errors are made if the reaction is not driven to completion. LITERATURE CITED
Alfin-Slater, R. B., Rock, S. M., and Swislocki, hl., ANAL. CHEM.,22, 421 (1950). Orchin, M., ]Tender, J., and Friedel, R. A., Ibid.,21, 1072 (1949).
(3) Rittenberg, D., ”Preparation and Measurement of Isotopic Tracers,” p. 31, 4nn Arbor, Mich., Edwards Bros., 1946.
(4) Stevenson, D. P., and Wagner, C. D., J . Chem. Ph~js.,19, 12 (1981). (5) Thomas, B. W., i l . v . 4 ~ . CHEY.,22, 1476 (1950). (6) Turkevich, J., Friedman, L., Solomon, E., and Wrightson, F. AI , J . Am. Chem. SOC.,70, 2638 (1945). (7) Zeremitinoff, Bet-., 40, 2023 (1907). RECEIVED for review August 8, 1951. Accepted February 2 5 , 1952. Research carried out under the auspices of the U. s. Atomic Energy Commission.
Microdetermination of Deuterium in Organic Compounds JACK GRAFF’ .4ND DAVID RITTENBERG Department of Biochemistry, College of Physicians and Surgeons, Columbia University, Y e w Y o r k , IV. Y .
The usual procedures for determination of the deuterium concentration in the hydrogen of organic compounds require samples of about 100 mg. A study of various procedures in which the final analytical determination would depend on a mass spectrometric determination of hydrogen gas derived from the hydrogen of the organic sample has resulted in a micromethod requiring samples of from
T
HE common procedures for determining the deuterium
concentration of the hydrogen of an organic compound require the oxidation of the compound to yield water. The water is then purified and some physical quantity which is a function of the concentration of deuterium is precisely determined. The most widely used methods determine the density of the purified water. The densities of deuterium oxide and water differ by more than 105 p.p.m. and the density can be determined conveniently t o 2 p,p.m. by the falling drop procedure ( 4 ) . A sample sufficiently large to yield about 100 mg. of water is required for routine determinations. The procedure is long and requires careful attention t o details in order to obtain reliable results. The mass spectrometer can easily determine the deuterium concentration in as little as 0.1 cc. of hydrogen gas (4 micromoles). The analysis of water directly in a mass spectrometer is not feasible. If pure deuterium oxide vapor is admitted to a mass spectrometer, constructed of glass and Nichrome V, which has been baked until the water peak a t mass 18has a low intensity and no peaks are observable a t either mass 19 (HDO) or 20 (D20),a spectrum shown in Table I results. It is clear that the sample of deuterium oxide admitted into the mass spectrometer has become diluted with normal hydrogen atoms of compounds present in the instrument. Hydrogen atoms are present either as adsorbed water or as OH groups of the silicates of the glass vacuum chamber. Experience has shown that molecular hydrogen is the most suitable compound for mass spectrometric determination of deuterium. It is easily introduced and pumped out of the mass spectrometer, and impurities present either in the sample or in the mass spectrometer produce ion peaks far removed from those formed by hydrogen itself. Were it possible to liberate all the hydrogen from the water formed in the complete oxidation of an organic compound, it vould be possible to carry out an isotope determination on as little as 0.1 or 0.2 mg. of compound. While the oxidation of such quantities of organic compounds involves no technical difficulties, the conversion of this quantity of water 1 Present address. Department of Physiological Chemistry, Yale University. New Haven, Conn.
3 to 5 mg. The sample is burned in a stream of dry oxygen and the water formed is reduced by hot zinc to yield hydrogen. The accuracy is equal to that of previous macromethods and the time required for an analysis is considerably shortened. This method is especially useful in the study of the metabolism of natural compounds, where often i t is not feasible to obtain 100-mg. samples for analysis.
to hydrogen is not simple. The obvious methods such as reaction of water with sodium are not suitable, because these reactions do not completely convert all the water to hydrogen. Because of kinetic factors the isotope concentration of the deuterium in the hydrogen is from one third to one fourth that of the starting water ( 3 ) . The same fractionation exists in all reactions which do not go to completion.
Table I. Relative Intensities of Ion Beams at Masses 16, 17, 18, 19, and 20 after Introduction of Deuterium Oxide Vapor
Mass 16 17
18 19
20
(Intensity of mass 20 taken as 1000) Ion Intensities Immediately after ‘ admission of DzO Background into mass spectrometer 0.3 94 0.8 43 2.8 702 0.0 777 0.0 1000
ANALYSIS OF WATER BY EQUILIBRATION WITH HYDROGEN GAS
In an attempt to circumvent this difficulty, the authors studied the feasibility of equilibrating the water sample with a known amount of normal hydrogen. The reaction
HDO
+ Hz
HzO
+ HD
(1)
can be catalyzed in several ways ( 2 ) . As the equilibrium constant is known ( 5 ) , knowledge of the quantities of water and hydrogen employed and the isotope Concentration of the equilibrated hydrogen permits the calculation of the isotope concentration of the ifiitial water sample. Water formed by the combustion of a known amount of organic compound (about 5 mg.) was transferred to an evacuated vessel (about 300-ml. volume) and mixed with 1 ml. of normal hydrogen. The equilibration of hydrogen and water (Reaction 1) was carried out by a platinum
V O L U M E 2 4 , NO. 5, M A Y 1 9 5 2
879
wire a t white heat (- 1700" Iheequilibrated hydrogen is lower than that of t,he water, the analysis of samples containing nearly the normal abundance becomes difficult. (3) This apparatus has a large "niemorj- c,ffect," which arises from the large glass surface of the fa,& in rr-hich the water and hydrogen were equilibrated. PREPARATION O F HlDROGEN FROM XIILLIGRAM SAMPLES O F WATER
In principle the methods involve the oxidation of the organic compound in a stream of dry oxygen and the subsequent reduction of the xater formed by a hot metal. The initial step, the oxidation of the organic compound, involves no technical difficulties. The reduction of the water formed can theoretically be carried out by any metal above hydrogen in the electromotive force series. In practice, not all metals are equally suitable, for one ie required which Till completely convert all the hydrogen of the water t o elementary hydrogen. If the conversion is incomplete, as is the case Kith iron, or if the metal oxide forms a very stable
880
ANALYTICAL CHEMISTRY
vessel, F. (The volume of this vessel is 15 mi. I t may be obtained from Eck and Krebs, New York, N. Y.) If the tempera: ture of B is not sufficiently high the reduction of water will not be complete, By turning stopcock 2 to connect the Toeppler pump to trap C and transferring the dry ice from D back to C, the unreacted water can be sent back through the hot zinc and the hydrogen formed can be pumped by the Toeppler pump into F . When the apparatus is first assembled a test run should be made with increasing temperature of the furnace surrounding B, starting with 360 . Conversion starts slightly above this temperature and in a new converter is complete a t about 384 . With progressive use, higher temperatures (up to 415 ”) are reuired. One filling of tube B suffices for 200 samples or more. ‘%he complete reduction of water to hydrogen takes from 5 to 8 minutes, depending on the age of the converter tube. If conversion is incomplete in one pass even when the zinc is kept a t 415 “C., a new zinc filling is required. Since the reaction between zinc and water is quantitative, no fractionation occurs. The deuterium concentrations in the first and second halves of the hydrogen produced are the same within the experimental error. When all the hydrogen has been transferred to tube F , the mercury is brought below the sample tube, stopcock 4 is closed, and the sample tube is removed from the system. The cooling trap is removed from trap D (or C) and the entire system to the left of the Toeppler pump is evacuated. The system is now ready for the next sample. Care should be exercised to manipulate the stopcocks so that air never enters converter tube B while it is hot. In most cases water samples even though impure-e.g., blood or urine-need not be passed through the combustion tube, A . A 5-mg. sample can be placed in tube G and directly transferred to trap C by distillation in vacuo. Great care must be exercised to prevent dilution of these small water samples by atmospheric moisture. The simplest way to clean tube B before filling it with fresh zinc is to crack the system a t point a, blow a hole a t one end of B , and dissolve the filling with dilute acid. The tube should be loosely filled with the zinc granules. All the apparatus described suffer from a common defect. If a normal compound is analyzed after one that had a high isotope concentration, the isotope concentration of the normal compound xi11 be above normal. When, for example, a large sample of deuterium oxide containing 86 atom % excess deuterium was passed through the system and followed by a 10-mg. sample of normal palmitic acid, the hydrogen produced from the latter contained 0.53 atom % deuterium excess. There had apparently been a hold-up of 62 micrograms of water in the apparatus. This holdup is not localized in any specific part of the apparatus. The authors have been unable to eliminate this holdup. The tvater (or OH groups of the silicates) is very firmly adsorbed on the glass.
Table 11. Analyses of Palmitic Acid (Apparatus in Figure 1 saturated with DiO,then 5-mg. samples of normal palmitic acid analyzed) Sample Atom yo Excess 86 Dz0 1st sample 0.82 2nd sample 0.27 3rd sample 0.053 4th sample 0.029
The extremely low vapor pressure of the compound (ROH) responsible for the “memory” is illustrated by the following experiment. A borosilicate glass tube (25 X 200 mm.) containing a loop of platinum wire (0.05 mm. in diameter, 25 cm. long) connected to the external source of current by tungsten presses and sealed to a vacuum line was saturated with deuterium oxide by distilling in 0.5 gram of deuterium oxide. The deuterium oxide was removed by pumping. The tube was then heated t o incipient fusion under high vacuum to remove adsorbed water. After cooling, 1 cc. of dry hydrogen was admitted and the filament was ignited for 3 minutes. On analysis the hydrogen then had a deuterium concentration of 0.06 atom % excess. Another 1-cc. sample of normal hydrogen and 0.5 mg. of water were now introduced. The platinum filament was again heated for 3 minutes. The hydrogen gas on analysis now was found to contain 5.1 atom % deuterium.
Table 111. Analysis of Water Samples of Known Concentrations Sample A, 11.01 atom % excess. Sample B, 0.237 a t o m % excess. .\lass spectrometer employed is a r type single collector slit instrument. No correction factors were employed in calculation of deuterium concentrations. Bample Atom 70 D Excess 10.8 11.06 11.06 0.260 0.240 0.240
AI Az A8 Bi
Bi Ba
Table IV.
Representative Analytical Values of Organic Compounds
(Each sample analyzed in duplicate. D 174 A and D 174 B are separate determinations of same compound) Sample D 174 A D 174 B D 175 A D 176 B D 176 A D 176 B D 177 A D 177 B D 178 A D 178 B D 183 A D 183 B
Atom % Excess D 0.106 0.099 0.383 0.372 1.41 1.39 1.75 1.72 1.98 2.11 0.017 0.014
Sample D 184 A D 184 B D 185 A D 185 B D 186 A D 187 186 A B
D 187 B D 188 A D 188 B D 189 A
D 189 B
Atom % Excess D 0.002 0.002 0.004 0.004 0.008 0.004 0.104 0.121 0.196 0.195 0.115 0.109
This experiment demonstrates that there exists on the glass surface a deuterio compound which has such a low vapor pressure that it does not enter the gas phase and so cannot undergo the exchange reaction with hydrogen on the hot platinum surface. If, however, water vapor is added to the system, the water can exchange with the deuterio compound on the glass surface, come back into the gas phase, and exchange with the hydrogen on the hot platinum wire. It appears plausible that a t least part of the deuterium on the surface is there in the form of OD groups of the silicates. The exchange between water and the deuterium bound to the glass surface is not an instantaneous reaction. For example, when the apparatus in Figure 1 is saturated with deuterium by successive additions of 20-mg. samples of 86% deuterium oxide, which are then followed by the analysis of 5-mg. samples of normal palmitic acid, the data given in Table I1 were obtained. I t is clear that we are not dealing with a simple dilution of the adsorbed deuterium with the normal water of the stearic acid combustions. It may be that there exist crevices in glass into which the water molecules can only slowly diffuse in and out. In any case, from the data obtained from the first combustion of palmitic acid, there appears to be a holdup of the order of 54 micrograms of water in the entire apparatus. For this reason 2 to 5 mg. of water are employed for analysis. With a 5-mg. sample the holdup is but 1%. If the deuterium concentrations of two successive samples are not too widely different, the holdup will have little effect on the final results. Analysis is always done in duplicate. The results obtained on the analysis in triplicate of known water samples are given in Table 111. Samples A and B were water samples prepared by careful dilution of pure deuterium oxide (99.98% deuterium) and contained 11.01 and 0.237 atom % deuterium excess, respectively. The sample analyzed before A contained 1.7 atom yo excess deuterium. Sample B was analyzed after sample A. The effect of holdup is seen in the low value of analysis A1 and the high value of 131. Samples of cholesterol containing 0.286 and 0.142 atom ”,, excess, respectively 0.005 and (by the falling drop method) gave values of 0.291 0.144 i~ 0.05 atom Yo excess (average of three determinations). Analyses of normal organic compounds give zero values =k0.003%. A random selection of data obtained in this laboratory is given in Table IV. Each sample was analyzed in duplicate. This apparatus has been in use in this laboratory for the past
+
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
