927
N O V E M B E R 1947 closed by turning m, si turned in such a way that ci is closed, and st and sg so that the combustion tube communicates with one of
the nitrometers. The T e c h burners are placed one a t each end of the capsule and lighted. When the ends of the capsule have become red hot the burner nearest T is moved towards the sample. The other burner remains stationary during the combustion. Ordinary microsamples not containing long carbon chains can be burned off instantly. Large samples and samples containing very long carbon chains must be burned more slowly (at a rate of about 1 my. per minute) to secure complete oxidation. When the reaction is finished c1 is opened and the tube is washed with carbon dioxide. A flat chimney is put onto one of the T e c h burners and the whole capsule is heated t o red heat (750 O to 800 C.) during the washing. With the present apparatus the necessary washing time is 8 minutes. During washing the next sample is weighed in. When microbubbles are obtained the burners are turned off, the liquid level in the nitrometer is adjusted, and the capsule is taken out. A new capsule map now be put in and 11-ashed free from air. The sample is burned, the nitrogen blown into the other nitrometer, etc. Automatic combustion of the sample may be easily ariangcd with the present apparatus. One tube filling can be used for about 30 analvses of 15-mg. samples with very long carbon chains khich might correspond to 150 to 200 ordinary microsamples. The results of the analysis of a number of nitrogen-containing compounds of xvidely different types are shoxm in Table I. hnalyses 5 to 9 were carried out t o show the reproducibility of the method. Pregl-Roth (6, p. 88) stat:s that the ordinary Pregl method is able to give results which differ less than 0.1% from the calculated. The author has not been able t o get so good
results with the Pregl method, and in his experience the precision of the new method is better-but not so good that the differences from the theory are less than 0.1%. ACKNOWLEDGMENT
The author is indebted to E. Stenhagen for his interest in the work and t,o L. Finn for preparing the figures. The work was made possible by a grant from the Rockefeller Foundation to Dr. Stenhagen. LITERATURE CITED
(1) Clark, E. P., J.Assoc. Oficial Agr. Chem., 16, 575-80 (1933). (2) Hallett, L’, T., IND.ENG.CHEM.,ANAL.ED., 14, 975 (1942). (3) Lamb, A . B., Bray, W. C., and Frazer. J. C. W., Znd. Eng. Chenz., 12, 213-21 (1920). (4) Lindner, J., “Mikro-massanalytische Bestimmung des Kohlenstoffes und TVasserstoffes mit grundlegender Behandlung der Fehlerquellen in der Elementaranalyse,” p. 44, Berlin, Verlag Cheniie, 1935. ( 5 ) Miher, 11. T., and Sherman, Af. S., IND.ENG.CHEX., AXAL. ED.,8, 331 (1936). (6) Pregl-Roth, “Quantitative organigche Mikroanalyse,” pp. 84105, Berlin, Verlag Julius Springer, 1935. (7) Stallberg-Stenhagen, S.,Arkiv Kemi, Mineral. Geol., 23A, No. 15 (1946). (8) Stenhagen, E., and Tagtstrom-Eketorp, B., unpublished. (9) Unterzaucher, J., Ber., 7 3 B , 391-404 (1940). (10) Zimmermann, Cl., Ann. Chem. Pharm., 232, 342 (1886). RECEIVED September 17, 1946.
Estimation of Ultramicroquantities of Urea and Kjeldahl and Amino Acid Nitrogen Improvements in Microaeration Technique ALBERT E. SOBEL, ALBERT HIRSCHMAN, ~ R LOTTIE D BESIIAN Depurtment of Biochemistry and Pediatric Research Laboratory, Jeuish Hospital of Brooklyn, Brooklyn, .V. Y . Two nelc types of microaeration tubes are described. One, w i t h a sealed-in bubbling tube, simplifies aeration of from 10 to 200 micrograms of ammonia nitrogen. The other type utilizes conical centrifuge tubes which make feasible the determination of frpm 1 to 10 micrograms of aeration and direct titration.
T
HIS paper presents t\vo improvements in the determination of ultramicro quantities of urea and Kjeldahl and amino acid nitrogen by aeration (1,2). A new aeration tube has been developed t o eliminate cumbersome featureq of the microaeration tube previously proposed ( 2 ) ,and another tvpe of aeration tube is suggeqted for handling less than 10 micrograms of nitrogen. METHOD FOR 10 TO 200 MICROGRAVS O F NITROGEN
A new design of aeration tube is used which has the essential features of the microaeration tube of Sobel, lIaver, and Gottfried (2) arid eliminates many of its disadvantages The new tubes are Pyrex test tubes with side armand bubbling tube sealed on. For aeration, they arc set up as shown in Figure 1, the outlet of one tube being connected to the inlet of the next, etc. Over 100 tubes may be connected in se5ies to the same pump. These tubes eliminate the necessity of individually adjusting each bubbling tube so that the tip reacheq close to but does not touch the bottom of the aeration tube; this is a nuisance, espcciillv when many determinations are done simultaneouslv. The outlet of one tube can be connected to the inlet of the next tube bv means of a short piece of rubber tubing, s? that there is p r x t i -
cally a glass-to-glass connection, or by a ground-glass joint. There are no cumbersome, long rubber connections. The sealed-in bubbling tube does not have to be washed out prior to titration, because it is used as a stirring device. I n the tube previously proposed (21, dilution of the boric acid solution due t o washing of the bdbbling tube tended t o reduce the sharpness of the end point, unless very small amounts of water were used in the washing. The stopper is easier to handle, as it does not carry a bubbling tube connected to rubber tubing. B closer and quicker fit can be made with solid rubber stoppers. I n addition, the stoppers carrying bubbling tubes sometimes developed cracks around the hole, Tvhieh caused leaks in the system. Reagents. The same reagents are uqed as in previous work ( 1 , a ) . [There is an error in the directions given previously (2) for making the stock standard ammonia solution; 4.7186 grams of ammonium sulfate are dissolved in distilled water to a final volume of 100 ml. to make a solution in which 1 ml. equals 10 mg. of nitrogen.] Apparatus. Pyrex test tube., 125 mm. long and 1.5 mm. \vide, with a side arm and bubbling tube sealed on about 35 mm. from the top of the tube (Figure 1). The bubbling tube is 3 t o 4 mm. in outside diameter and is constricted to a 1-mm. bore at the tip. It is kept close to the side of the tube in order hot t o interfere
-
V O L U M E 19, NO. 1 1
928 with the titration a t the end of the determination. Tubes may be obtained from Emil Greiner Co., New York, N. Y. No. 0 solid rubber stoppers. Rubber tubing, 40 to 50 mm. long, 10 mm. in outside diameter, and 4 mm. in inside diameter. A wooden test tube rack with holes 18 mm. in diameter, the centers spaced 63 mm. from each other. A capillary microburet. An electrically operated oil pump or a good water pump.
C0,-FRE A\R
Figure 2.
Figure 1. Schematic Diagram of Microaeration Apparatus for 10 to 100 Micrograms of Nitrogen
Procedure for Aeration and Titration. The preliminary steps, such as Kjeldahl digestion, incubation with urease, or reaction with ninhydrin may be carried out in the aeration tubes according t o the original directions ( I , 2 ) . Should any of the liquid get into the bubbling tube before all the reagents (except the alkali) are added, it should be blown out gently to ensure mixing; a small nipple bulb is convenient for this purpose. When ready for aeration, the tubes are set up in series (Figure 1) with short pieces of rubber tubing. For ease in handling, the ends of the rubber tubing may be moistened in a mixture of glycerol and water. The aeration is conveniently set up in special test tube racks, described above. After 1.5 ml. of the boric acid-indicator mixture are added to the receiving tubes, these tubes are stoppered; 0.5 ml. of the appropriate alkali is added to the specimen tubes, and each tube is immediately stoppered before proceeding to the next. The first tube in the series is connected to a suction pump. The suction is started gently and is increased until all tubes are aerating at a moderate rate. After the required period of aeration, depending on the method used (1, b ) , usually 20 to 40 minutes, the stoppers are removed, starting with the tube farthest from the suction, and gradually cutting down the suction as more stoppers are removed. There will be no trouble from li uid sucking back, as is the case if the suction is suddenly cut The boric acid solutions in the receiving tubes are titrated with 0.0714 N acid to their original pH by means of a capillary microburet in the manner illustrated in Figure 2. Stirring is effected by blowing a stream of air (previously passed through soda lime,
08
Table I.
Determination of -4mmonia Nitrogen Ammonia Nitrogen Found
49.7 49.8 50 0 50 2 50 0 49 5 50 0 49 8 50 2 50.0
Av.
49.93
f
99.8 a9.5 99.98
0.17
if desired) through the bubbling tube. The tip of the microburet is kept beneath the surface of the liquid. The titration is finished when the color matches the original color of the borie acid indicator solution. Results. These tubes were carefully tested by aerating ammonium sulfatestandards. The results are shown in Table I. The average deviation for !O micrograms of nitrogen was * 0.17 microgram, and the average deviation for 100 was * 0.22 microgram. These tubes have been in routine use in this laboratory for the determination of urea for over a year. The analysts showed an unqualified preference for these tubes over the previous tubes used. A summary of their results, obtained as routine analytical controls, is shown in Table IT. METHOD FOR 1 TO 10 MICROGRAMS OF NITROGEN
In work with 1 t o 10 micrograms of nitrogen, the end point is not sharp enough in a volume of 1.5 ml. to give accurate results. For this purpose, a 5-ml. conical centrifuge tube is fitted with a two-hole rubber stopper carrying a bubbling tube ending in a capillary and an outlet tube. The upper portions of the bubbling and outlet tubes are bent horizontally, so that there is almost a glass-to-glass connection and short rubber connnections may be used. The tubes are set up for aeration as shown in Figure 3. The narrow conical bottom of these tubes allows the circulating gases to bubble through a greater depth of solution for a given volume than would a round-bottomed tube. The tubes are not large enough to seal in a bubbling tube and still have ample room for the tip of ft microburet. For this reason, they are not SO convenient as the first tube described in this paper. However, complete absorption of ammonia takes place with but a.1 ml. of 2% boric acid in the receiving tube. Apparatus. Conical centrifuge tubes, 5 ml. No. 00 twohole rubber stoppers. Rubber tubing, 40. to 50 mm. long, 3 mm. in inside diameter, 7 mm. in outside diameter. Glass tub-
100 y present 99.7 99.8 100.0 100.0 100.0 100.0 100.0 101.0
50 7 present
Schematic Diagram of Titration Assembly
TcAIR
* 0.22
Table 11. Recovery of Urea Nitrogen
Sample
0.1 ml. of standard solu-
tion 0.1ml. of standard solution added to 0.1 ml. of blood
NO. of Determinations
Added
34
10.0
29
50.0
N
Y
N Recovered
Average Deviation
Standard Deviation
Y
10.2
% 0.22
% 0.27
50.1
0.76
0.88
TRAP FOR
Figure 3. Schematic Diagram of Microaeration Apparatus for 1 to 10 Micrograms of Nitrogen
929
N O V E M B E R 1947 Table 111. Determination of Ammonia Nitrogen i n 0.1 A l l . of Solution 5.0~ Present
10.07
Present Y
10 10 10 10 9 10
10 10 10 10
9Y
3 0 2 0 9 0 0 0 2 2
10.1
2.07 Present
Y
Y
4.94 5.02
2.04 2.06 2.00 2.04 2.00 2.00 1.96
5.04
5.10
5 00 5.06
4.92 4.88 5.10 5.10 5.00 i :0 . 1
5.01
t
0.05
2.01
* 0.03
ing, 4 mni. in outside diameter, 3 mm. in inside diameter. Capillary microburet. Electrically operated oil pump or a good water pump. Procedure for Aeration and Titration. The tubes arc set up as shown in Figure 3. The bubbling tube ends in a capillary which reaches almost to the bottom of the tube. It may be more convcnient to set up the tubes in a zigzag formation, so that tubes containing boric acid are in one row and those containing the specimen are in another. Boric acid-indicator solution, 0.2 ml., is added to each receiving tube. After the appropriate preliminary steps, 0.1 ml. of
alkali is added to each specimen tube, which should not contain more than 0.2 ml. of solution. More alkali should be added if the volume is larger. Each tube is promptly stoppered as won as the alkali has been added. Suction is started gently and the tubes are aerated for 30 minutes. After the aeration, the tubes are disconnected and the bubbling tubes are washed by forcing a small amount of water through them (about 0.05 ml.) by means of a small capillary pipet with a nipple bulb. The outsides of the bubbling tubes are rinsed off with 2 drops of water. The solutions are titrated with 0.00714 A; acid until their color matches that of an equal amount of boric acid-indicator solution plus the amount of water used in the washing. These tubes were tested by aerating known amounts of ammonium sulfate standards (Table 111). The average deviation for 2 micrograms of nitrogen is h0.03 microgram, for 5 i t is *0.05 microgram, and for 10 it is *0.1 microgram. LlTERATURE CITED
Sobel, -4. E., Hirschman A ., and Besman, L., J . B i d . Chew%., 161, 99 (1945). Sobel, A. E., Mayer, A . M., and Gottfried, S. P., Ibid., 156, 355 (1944). RECEIVEDFebruary 3, 1947. Presented before the Division of Analytical and ZIicro Chemistry at the 109th hleeting of ' e AVERICASC K E M I C A L SOCIETY, Atlantic City, N. J.
Microdetermination of Alkaline Earths as Normal MoIyb.dates ROBIN RIOSER' AND REX J. ROBINSON, D e p a r t m e n t of C h e m i s t r y , University of Washington, Seuttle 5 , Wccuh.
T
HE alkaline earths are usually determined on the micro basis by modification of the well-known macromethods. This adaptation is accompanied by accentuation of the various errors, so that the micromethods leave much to be desired. The present work summarizes the results of an investigation of the microdetermination of calcium, strontium, and barium as the molybdates. By this method the influence of weighing errors is lessened, as the gravimetric factors are very favorable and the molybdates are not hygroscopic. An effective separation may also be made from magnesium, since magnesium molybdate is soluble. The alkaline earths have been determined as the molybdates only on the macro basis. Wiley (8) reported satisfactory results for calcium when calcium molybdate was precipitated even in the presence of magnesium. Searing (8) made a detailed study of the factors involved in the determination. Results varying from the calculated by as much as 1% have been obtained by Tanii and eo-workers (6). Similar quantitative methods have not been reported for the determination of strontium or barium, although extensive studies of the molybdates of the alkaline earths have been made by Smith and Bradbury (4) and Cllik ( 7 ) . SOLUTIONS
Standard Solutions. Solutions of calcium chloride, barium chloride, and strontium chloride (0.2 iV) were prepared from C.P. chemicals and carefully standardized by standard macromethods. Ammonium Molybdate Reagent. Three and a half grams of ammonium paramolybdate and 9.6 grams of ammonium nitrate were dissolved in 4 ml. of 6 N ammonium hydroxide and diluted to 100 ml. Sodium Molybdate. This solution was prepared by dissolving 5 grams of sodium molybdate (Na2M00,) in 100 ml. of water. In the later work, both sodium and ammonium molybdate I
Present address, Joseph E. Seagram and Sons, Louisville, Ky.
solutions were acidified to p H 4 by addition of hydrochloric acid, in order to reduce the absorption of carbon dioxide from the atmosphere. EQUIPMENT
The equipment used in this work was simple. The precipitate was formed, filtered, ignited, and weighed in a platinum dish of about 10-ml. capacity and 5-gram weight. The supernatant li uid was drawn off through a Pyrex glass filter stick which ha% been constructed according to Benedetti-Pichler (1). The blank with this filter amounted to about 0.005 mg. A calibrated micro muffle furnace was used for ignition of the precipitate. Weighings were made on a Kuhlman microbalance. FINAL PROCEDURE
The alkaline earth solution, containing about 0.2 to 1.0 mg. of the element, was pipetted into the platinum dish, the volume was adjusted to about 6 ml., and 0.8 ml. of sodium molybdate solution was added. The pH of the solution was adjusted to about 6 or 7 with filtered ammonium hydroxide as indicated by methyl red indicator. The mixture was boiled gently for 2 or 3 minutes and cooled for 15 minutes or longer. The supernatant liquid was filtered off and the precipitate washed with a minimum of hot water. The dish and filter stick were then ignited to constant weight at 700" C. Five minutes' ignition a t this temperature was normally sufficient. Effect of Temperature upon Precipitation. Boiling of the solution during precipitation is necessary to precipitate the alkaline earth molybdates completely. I n the earlier part of the work, the precipitation mixture was heated only to the temperature attained on the steam bath, which was usually about 80" C. Invariably low results were obtained. Stability of Reagent. The reagents as first prepared were not acidified. It w&s observed that the reagents, either ammonium or sodium molybdate, precipitated the calcium completely when first prepared, but after a few days became acid and gave erratic and incomplete precipitation. Any attempt to restore the efficacy of the reagents by making the solution alkaline made
