358
ANALYTICAL CHEMISTRY
and i t is probably one of the first that will be extended into the range of cellular studies because of its tremendous importance in biological investigation. SUMMARY
The present status of the Kjeldahl method leaves much to be desired in spite of its wide use and tremendous value t o industry, nutrition, physiology, biochemistry, and medicine. Fundamental research is still needed to elucidate and improve the digestion process for use with different forms of nitrogen. The question of reducing and oxidizing agents other than sulfuric acid, and the matter of the proper catalyst, particularly require further fundamental investigation. Most significant is the rapid development and wide adoption of the micro and ultramicro forms of the Kjeldahl analysis. It is here that many of the most striking and significant developments are still to be expected, particularly in lowering the range of application and applying it to the exploration of minute biological systems, perhaps the living cell itself. LITERATURE CITED
Borsook, H., J . BioZ. Chem., 110, 451 (1935). Bradstreet, R. B., Chem. Rev., 27, 331 (1940).
ENG.CHEN.,ANAL.ED., 12, 657 (1940). Bradstreet, R. B., IND. Brael, D., Holter, H., Linderstr$m-Lang, K., and Rbzits, K., Compt. rend. trav. lab. CarZsherg, Ser. chim., 25, No. 13, 289 (1946). Chibnall, A. C., Rees, M.W., and Williams, E. F., Biochem. J . , 37, 354 (1943). Conway, E. J., and Byrne, A, Ibid., 27, 419 (1933). and King, G. B., ISD.ESG.CHEM.,- ~ N . L L . ED., Dalrymple, R. S., 17, 403 (1945). Friediich, A , , .Uikrochem., 13, 114 (1933).
Friedrich, A . , Kuhaas, E., and Schnurch, R., 2. physiol. Chem., 216, 68 (1933). Gunning, J. W., 2. anal. Chem., 28, 185 (1599). Jonnard, R., IXD.ENG.CHEM.,ANAL.ED., 17, 246 (1945). Kaye, I. A., and Weiner, N.,Ibid., 17, 397 (1945). Kirk, P. L., “Chemical Determination of Proteins,”in “Advances in Protein Chemistry,” Vol. 111, New York, Academic Press, 1947. Kirk, P. L., IND. ENG.CHEM.,ANAL.ED.,8, 223 (1936). Kirk, P. L., Mikrochem., 16, 13 (1934). Kirk,P. L., Rosenfels, R. S., and Hanahan, D. J., ANAL.CHEM., 19, 355 (1947). Koch, F. C., and Meekin, T. L., J . Am. Chem. Soc., 46, 2066 (1924). Lundin, H., Ellburg, J., and Riehm, H., 2. anal. Chem., 102, 161 (1935). Margosches, B. M.,and Vogel, E., Ber., 55B, 1350 (1922). Milbauer, J., 2. anaZ. Chem., 111, 397 (1935). Miller, G. L., and Miller, E. E., ANAL.CHEM.,20, 481 (1948). Needham, J., and Boell, E. J., Bwchem. J . , 33, 149 (1939). Osborn, R. A,, and Krasnitr, A., J . Assoc. Ofic.Agr. Chembte, 16,107 (1933) ; 17,339 (1934). Osborn, R. A,, and Wilkie, J. B., I b i d . , 18, 604 (1935). Patel, S.M., and Sreenivasan, A., ANAL.CHEM.,20, 63 (1948). Poe, C. F., and Schafer, R., J . Dairv Sci., 18, 733 (1936). Schwoegler, E. J., Babler, B. J., and Hurd, L. C., J . BwZ. Chem., 113,749 (1936). Self, P. A. W., Pharm. J . , 88,384 (1920). EXG.CHEX.,ANAL.ED., Shirley, R. L.. and Becker, W.W., IND. 17, 437 (1945). Sreenivasan, R., and Sadisivan, V., Ibid., 11, 314 (1939). Tompkins, E. R., and Kirk, P. L., J . BioZ. Chem., 142, 477 (1942). Van Slyke, D. D., and Hiller, A., Ibid., 102, 499 (1933). Wicks, L. E., and Firminger, H. I., ISD. EKG.CHEM.,AXAL. ED., 14,760 (1942). RECEIVEDMay 16, 1949. Presented before the Division of Analytical and I I i c r o Chemistry, Symposium on Determination of Sitrogen in Organic Compounds, a t the 114th Meeting of the A?IIERICASCHEMICAL SOCIETY St. Louis, 110.
Dumas Nitrogen Determination Using Nickel Oxide
‘
WOLFGAYG KIRSTEK, Institute of Medical Chemistry, University of Lippsala, Sweden
HE Dumas nitrogen determination apparatus previously deTscribed by the present author ( 4 ) has now been used in this laboratory and in several industrial and scientific laboratories during a longer period, and experience has indicated improvements in certain constructional details, which lower the risk of breakage and add to the convenience in using the apparatus. A schematic view of the apparatus is shown in Figure 1. I n order to avoid the risk of breakage, most of the tapered ground joints have been replaced by ball and socket ground joints, or flat ground joints, held together by clamps. The ball and socket ground joints were satisfactory when kept in a fixed position during use, as is the case with the joints on the combustion tube. The joints between the four-way stopcock and the nitrometers are sealed together with Kronigs glass cement. tl and tP are flexible metal tubes, connected with the stopcocks through flat ground joints of metal and glass. t B and tr are glass tubes shaped in a U to provide flexibility. sz, a special four-way stopcock, replaces two three-way stopcocks in the old apparatus.
A laboratory for mineral oil research, which had t o determine small quantities of nitrogen in oil products, wanted a tube filling with a greater oxidation capacity. The part of the tube which is situated in the hot furnace, k , was therefore made wider. As this seemed t o have no bad influence on the microanalyses, every spparatus is now provided with this wider tube, which makes it possible t o carry out more analyses with one tube filling and gives
more assurance of complete combustion. Samples of 50 mg. of motor oil are burned with good results, using a combustion time of about 20 minutes Although the suitability of the nickel-nickel oxide tube filling may be doubted because of the experiments of Bell (Z), this tube filling has always worked well, in good agreement with the experiments performed by Kurtenacker (6). The part of the quartz tube situated within furnace k must have a wall thickness of at least 1.5 mm. During use a thin layer of nickel silicate is formed on the inside of the tube, which seems to protect the quartz from further chemical attack. Jf the quartz walls are too thin this layer will cause cracking of the tube because of the difference in thermal expansion. The score in the ground joint of the capsule can be dispensed with, as it has no perceptible influence on the blanks and the necessary washing times, and it is unnecessary t o have a plug in the capsule during the combustion. Instead of the plug rod 1 is now used, which is taken out of the tube after the capsule is put in place. The capsule and the rod are provided with a better supporting device, the use of which IS shown in Figure 1. Outside the apparatus the capsule is handled with the tweezers, p and I, and the stand, d. The stopper, m, for closing the combustion tube has been redesigned (Figure 1); it should be lubricated with silicone grease. The nitrometers, n1 and w,are more rigidly constructed than formerly. The capillaries leading doR-n into the
359
V O L U M E 2 2 , NO. 2, F E B R U A R Y 1 9 5 0
hopcalite will give off gas for a long time, and i t will be imWeight Tempossible to get microbubbles. x x of Pres- peraSubstance Sample sure ture Nz Found Calcd. The nickel oxide used in the KO. Mm. combustion furnace should be Mg. Hp C. Ml. 70 % free from metals which reduce 0.643 6.52 6.52 11.27 1 Glucosamine hydrochloride 0.347 3.73 3.73 10.59 Carbobenzoxyglycine benzhydryl ester 2 carbon dioxide. A specimen 0.810 7.19 7.14 12.85 N-Acet 1-p-aminosalicylic acid 3 0.675 7.03 6.99 11.05 D ( + ) 3 - H ethylhendecanamide (7) of nickel oxide (Baker's ana? 3.50 3.54 0.272 8.943 24-Methylpentacosanamide ( 1 ) 3.56 3.54 0.413 (+)-2 (~),g(~)-Dimethyltetracosanamide( 7 ) 1 3 , 1 2 4 lyzed), which according t o 6 3.99 4.01 Az1:zz-Tricosvnsmide (8) 0.497 11.524 7 the manufacturers contained 4.18 4.13 0.352 9.50 Diphen).l-p-ioluidinophosphonare 8 3.79 3.82 0.581 17.22 Dibenzyl-p-toluidinophosphonate 9 0.35% of cobalt and 0.02% of 3.63 3.67 0,305 9.61 Dibenaylphenylethylaminopho~pti- i.,tte 10 5.64 5.62 0.543 10.98 Diphenylaminophosphonate 11 iron, was found unsatisfactory 8.89 0.567 7.43 8.88 12 m-Chloronitrobensene 3.42 0.351 3.41 11.53 D-2-Methglhexacosanamide ( 8 , 13 for the present method. 1,049 11.20 11.20 10.68 Taurine 14 If mainly samples containing 11.23 0,336 11.20 3.434 Taurine 15 0.490 36.74 36.81 1 525 Thiourea 16 nitrogen not combined with 1 335 46.61 rrea 0 546 46.65 . . ~ 17 33.44 33.33 Cy&acetamide 1.036 i5l 24 0.305 18 oxygen are analyzed, the use 8.24 15 16 763 25 1.086 8.25 Silver nitrate 19 42.7 ,.. .. 0.00'24 ... 0.007 Motor oil A 20 of metallic nickel in the tube 0.006 45.9 ... .. 0.0025 ... 21 Motor oil A is superfluous. When the tube 0 029 4 0 , .5 ,., . . 0.0100 ... Motor oil B 22 ... 0.029 Motor oil B 43.5 ,.. .. 0.0110 23 filling is exhausted it may be 0.007 40 2 ,.. .. 0.0025 ... Motor oil C 24 0,008 44.3 Motor oil C ... . . 0.0030 ... 25 reoxidized by leading a stream .inalyses 20 t o 25 were carried o u t i n a n industrial laboratory, using a special grade of copper oxide and a of oxygen through the hot apspecial type of nitrometer, t h e construction of which will be subject of a later publication. paratus for 1 or 2 hours. hfter a few analyses have been performed, so much nickel has been reduced that even nitro compounds can be analyzed mercury are ground oblique at the end to give small bubbles. with good results. The author has used his apparatus in this The funnels of the nitrometers can be closed with rubber stoppers manner without refilling during a whole year. The filling is still when the apparatus is standing idle. When the tube is being filled the hopcalite must not be situated in use. t o o near k . which might cause undue heating. The overheated When the capsule has been put into the apparatus for combus-
Table I.
Analysis of Nitrogen-Containing Compounds
~
Figure 1. b . Furnace a t 100°,C. Cz, CI, Ca. Capillaries e. E n d of capsule fi. Quartz wool fz. Hopcalite fa. Nickel oxide f4. Kickel f6. Coarse copper oxide
Schematic Diagram of Apparatus Sample mixed with fine and coarso copper oxide g,h. Holders for stopcocks Capsule SI. Three-way stopcock sc. Score in stopper m st. Side tube of combustion tube t h . Thermos flask
f6.
3.
360
ANALYTICAL CHEMISTRY
tion according to the procedure dchcribed (4),the backstream is admitted in such a manner that the carbon d i o d e passes through both c4 and st. On a few occasions the fine copper oxide clogged the capsule, almost preventing the carbon dioxide from passing through. Atmospheric nitrogen may then remain in the capsule and cause high results. I n order to avoid this the procedure has been changed. When the backstream is turned on, stopcock SI is turned in such a manner that the stream passes only through ca, and no gas passes through st. By putting the tongue near c 2 , or by putting a rubber tube on cp and allowing the gas to bubble through water, it is easy to ascertain that a good stream is passing through the capsule. If this is the case, sLis turned so that the stream also passes through st. If too little carbon dioxide passes through the capsule, the apparatus is allowed to stand in this position until i t can be expected that the atmospheric nitrogen has been swept out of the capsule. The analysis is then continued as described (4), although it may be necessary to extend the sweeping time after the combustion. I n the previous paper (/t) i t is stated that the method is not so good that the results differ by less than 0.1% from theory. The author had calculated the difference according to the formula I theory - TOfound x 100 crror = ';io theory 1
-,
ruidcr the mibtaken impres3ion that the accuracy claimcd by
Iloth (6) was calculated in the Same manner. However, 110th I the~ more general practice of calculating thc error according to the formula , % error = 19; theory found1
L
When calculated in this manner the results given in ( 4 ) differ by less than 0.1% from theory and only one (No. 18) of the results givcn in the present paper differs by 0.11% from theory. In the same paper Unterzaucher's work in 1940 (9) is referred to a b the origin of the backway streaming system used in the present apparatus. The author did not know then that Gysel (3)had *hortly before (in 1938) used an apparatuc: with backway streaniing For the determination of nitrogen according to Dumas. 4 C K Y O I LEDGMENT
The author is indebted to Einar Stenhagen for his interest in the work arid for reviewing the manuscript; t o Norstedt & Soner, Stockholm, manufacturer of the apparatus, for technical antl financial assistance; and t o Lars Finn for drawing the figure. 1.IlER.ATCRE CITED
.\rosenius, K. E., Stallberg, G., Stenhagen, E., and TiigtstrijnlEketorp, Arkiu Kemi, .lIineral. Geol., 26A, No. 19 (1948). ( 2 ) Bell, Chem. S e w s , 23, 253 (1571). (3) Gysel, H., Helv. Chim. Acta. 22, 10% (1939). (4) Kirsten, Wolfgang, ASAL.CHEM.,19, 925 (1947). 15) Kurtenscker, 2. anal. Chem.. 50, 548-65 (1911). ((i) Pregl-Itoth, "Quantitative organiache hfikroanalyse," p. 8 8 , Berlin, Julius Springer, 1935. l i ) Stallberg-Stenhagen, Y.,Arkiu Kemi, .1fineraZ. Geol., 26A, No.12 (1948); A r k i v Kemi, 1 , Nos. 18, 21 (1949). (SI Stenhagen, E., Ibid.,1, S o . 13, 99-104 (1949). ($1) Untersaucher, J., Ber., 73B,3'31-404 (1940). (1)
ItECEIVED
I'cbl'Uary 1, 1049
Separation of Cotton and Rayon or Cotton and Acetate for Analytical Purposes OSKAK IIEIM 5943 48th Ave., Foodside, .V. Y .
THE method given in this paper effects a clean separation of
1 cotton and rayon (regenerated cellulose) or cotton
and acetate a t room temperature, using a solution of sodium zincate antl :rinmonium thiocyanate. The solutions are readily filterable :itid no cutting up of the sample is required. T h e method offers ronsiderable advantages over the one in general use a t present (.I.S.T.PII. D 629-46T, using calcium thiocyanate); the gelatinous condition of rayon solution in calcium thiocyanatc ('ituses great difficulties in the separation of the two fibers arid gives uncertain results. Pure undegraded cotton may bc completely insoluble in thc: sodium zincate-ammonium thiocyanate solvcnt, but 1xc:iuw rotton textiles have various histories of kiering, bleaching, long storage, exposure t o light,, etc., amount of degradation can bci cspected to be reilected in solubility. The variation, however, is ordinarily small-on the order of 0.5 to 1% in the limited number of samples tested. .Z correction of 0.7yois made-added when the result is i n tcJlms of per cent cotton and subtracted nhen i n terms of per cent t,uyoti-for kiered cotton fabric, and a correction of 0.5% i.: madr for the same fabric n-hen kiered and partly mercerizcd. This is the amount lost during treatment with the solvent. Th(9 0.7% correction was also found to apply to one case of cottoti I'utj1,ic after a U.F. resin finish had bccn removcd by 0.5 -I'hyclrovhloric arid a t 60" C. in 1 hour. PROCEDURE
Make a stock solution from 20 parts by weight of sodium hytiroxide, 9 of zinc oside, and .51 of water by adding an amount of
water equal to the sodium hydroxide, stirring (applying further heat if necessary) until all the zinc oxide is dissolved, then cooling and adding balance of water. This solution (double strength) is stable and zincate crystals do not separate from it. The fin:il percentage composition of the solvent is: Sodium hydroxidc Zinc oxide Aininonium thiocyanate
10 4.5 10
For samples up to about 1 gr:~nis (dorwined, desized, Ate., and tli.ird) use 100 grams of solvent made by niising 40 grams of the double-strength stock n-it,h 50 nil. of cold water, and press the sample in the solution for :t I ' mminutes by means of a rubber stopper attached to a rod. This will sn.ell and disperbe most of the r:iyon. Add 10 grams of ammonium thiocyanate and contittue pressing n.it>hthe rubber stopper for about a minute. Pour the liquid through an unp:ttldcd Gooch filter, using nonv or vci'y little suction. By i n g with the rubber stopper, reniovc :ts much liquid as 1 If,. (.\ bag made of fine-mesh tiylon mny he used instcwl of thcx Gooch.) .igsin add 40 grams of stock :tiit1 50 nil. of cold water, work the namplc, add 10 grams of aniiiioiiiuni thiocyanate, and press thoroughly for a few niinutv. Itcniove the liquid by the Gooch as hci'ort,.
.\(Id 40 nil. of natcr and 10 nil. of strong ammonia :ind work the snniple with the ruiher. lteriiove liquid by Gooch. Itepeat with hot r a t e r (70" to 90" C.), then with hot 5% acetic acid, and finally with hot water. This removes the zinc, considerablcl :mounts of which otherwise: remain. A final treatment with alcohol may be given t o shorten the clryirig time. The zinc content of the insoluble material (cotton) is ncgligihle-0.190 total : ~ , 5 1 1 .
