ANALYTICAL CHEMISTRY
760 There was no loss when a-tocopherol was added to duplicate samples of spleen or of fat. In three oils and four cuts of meats analyzed in duplicate (Table 111), the amounts of tocopherol found were similar but usually less than those reported by others
( 4 , 9J
ll).
Because so many substances other than tocopherol reduce the Emmerie-Engel reagent, it might be argued that the method giving the lower values for tocopherol is the more accurate. This is almost certainly the case for brewers' yeast, which contains so little tocopherol that it has been used routinely in basal diets for the bioassay of vitamin E. According to the saponificationchromatography method, this material contained 1.03 micrograms of tocopherol per gram, whereas by the distillationhydrogenation method ( I S ) the value was 4.17 micrograms per gram (I?'). Until there is an absolute standard against which the accuracy of tocopherol determinations can be evaluated, Stern's rule of thumb (16) may be helpful. If the apparent tocopherol content of the sample does not increase more than 10% during the interval from 2 to 10 minutes folloiving the addition of ferric chloride to the tubes under his conditions, he assumes the color mearured is due to tocopherols. LITERATURE CITED (1) Binnington, D. S., quoted by Chipault, J. R., Lundberg, It-.O., and Burr, G. O., A d i . Uzochem.. 8,321 (1946).
( 2 ) Dunford, R. A , , Can. Chem. Process Inds., 35,47 (1951). (3) Farber, hl., hlilhorat, A. T., and Rosenkrantz, H., Federation Proc., 10, 294 (1951). (4) Harris. P. L.. Quaife. M. L.. and Swanson. W.J.. J . 9uLrilion. 40, 367 (1950). ' Hines, L. R., and Mattill, H. A,, J . Biol. Chem., 149,549 (1943). Kachmar, J. F., Boyer, P. D., Gullickson, T. V., Liehe, E., and Porter, R. M., J . Xutrition, 42,319 (1950). Kidlhede, K. T., quoted by Kanntorp, H., and Xordlnind. G., Ark'iv Kemi Mineral. Geol., 25, 1 (1947). Kj$lhede, K. T., Z.Vitaminforsch., 12, 138 (194%). Kofler, M,, Hela. Chim. Acta, 28, 26 (1945). Parker, IT. E., and McFarlane, W. D., Can. J . Rescarch, B18, 405 (1940). Quaife, hl. L., J . Biol. Chem., 175,605 (1948). Quaife. hI. L.. and Biehler, R., Ibid., 159,663 (1945). Uuaife, &I. L., and Dju, bII. Y., Ibid., 180,263 (1949). Quaife, M. L., and Harris, P. L., IND.ENG.CHEM.,ANAL.ED., 18,707 (1946). Quaife, &I. L., Swanson, W.J., Dju, A l . Y., and Harris, P. L., Ann. S . Y . Acad. Sci., 52, 300 (1949). Stern, b l . H., quoted by Baxter, J. G., Biol. Symposia, 12,484 (1947). Swi'ck, R. IT.,Ph.D. thesis, University of Wisconsin, 1951. Tosic, J., and Moore, T., Biochem. J., 39,498 (1945). Wanntorp, H., and Nordlund, G., Arkia K e m i Mineral. Geol., 25, 1 (1947).
RECEIVED for review August 24, 1951. Accepted October 25, 1951. Published with the approval of the director of the Wisconsin Agricultural Experiment Station. Work supported in part h y the Wisconsin hlumni Research Foiindation.
Hydrazones, Semicarbazones, and Other Nitrogenous Substances Requiring a Reductive Pretreatment '4 Semimicro-Kjeldahl Procedure \ E L M E R B. FISH W m . H . Chandler Chemistry Laboratory, Lehigh University, Bethlehem, Pa.
CRING the past few years the micro- and semimicroKjeldahl determination of nitrogen has become increasingly popular in research and control laborat'ories. The Kjeldahl method presents certain advantages over the Dumas method. The time required per analysis is less, because several samples may be analyzed simultaneously. Recent developments in the adaptation of the Kjeldahl method to the micro or semimicro scale and the discovery of very efficient Kjeldahl catalysts have greatly reduced the time required for conversion of organic nitrogen to ammonium salts. There are, however, certain recognized limitations in applicability of the Kjeldahl method which require various modifications of the usual procedure. If the nitrogen atom is attached to either oxygen or another nitrogen atom a special reductive pretreatment is essential before the usual digestion with sulfuric acid in the presence of a catalytic mixture. Certain nitrogen heterocyclic compounds are very resistant to the usual method of acid digestion and require an extended afterboil. Ogg and Willits ( 7 , 1 4 ) and Kirk (6) have brought together a great deal of information regarding the conditions that are most favorable to the application of the Kjeldahl method to refractory compounds. Many of the recommendations cited by these authors are applied in the method of Cole and Parks (3). The catalyst used by Cole and Parks consisted of a mixture of mercuric oxide, selenium, and potassium sulfate, which was satisfactory for use with certain refractory nitrogen compounds if an afterboil of 1 hour was used. The 1-hour afterboil is much less than that recommended bj. Shirley and Becker ( 1 2 ) and others (6, 7 ) . Pat.el and Sreenivasan (8) found that if the afterboil were extended to 6 hours considerable nit>rogen was lost. They found a mercuric oxide-selenium mixture t'o be a more
effective catalyst than either alone m-hen used on refractory nitrogen compounds. A search of the literature for a satisfactory method for use with compounds containing N-S and YO2 groups disclosed that the Friedrich ( 4 ) method, which is highly recommended by Clark ( 2 ) ,is probably the most widely accepted for compounds of this type. Several methods have been used for reducing the nitro group, such as the use of sodium hydrosulfite ( I I ) , zinc dust and acid ( I S ) , sodium thiosulfate (1, 5, 9, IS), and potassium iodide with sulfuric acid ( I O ) . The Friedrich method of reduction with hydriodic acid is satisfactory when correctly applied to derivatives of hydrazine, semicarbaeones, or azo, nitro, and nitroso compounds. However, the elimination of the iodine by steam distillation following the addition of sulfuric acid and water is slow and tedious. The methods described in this paper are designed to eliminate the need for hydriodic acid treatment in the analysis of derivatives of aldehydes, ketones, and other substances requiring a special reduction treatment. The pretreatment is simple and the extra time required is about 30 minutes. The material is dissolved in acetic acid and methanol, reduced by the action of zinc and hydrochloric acid, and digested, and the nitrogen is determined as described by Cole and Parks ( 3 ) . APPARATUS
.i microbalance was used to weigh samples containing 20% or more of nitrogen and others were weighed on a balance adjusted to a precision of 0.03 mg. The samples were weighed in tin-foil cups made by cutting out circles of pure tin foil with a S o . 15 cork borer. The circles nere formed into cups by shaping them over the end of a 0.16-inch plastic rod. The average weight of the sample cups thus prepared x a s about 140 mg.
V O L U M E 24, NO. 4, A P R I L 1 9 5 2
761
T h e samples were digested in semimicro-Kjeldahl flasks using gas heaters. Distillations were made in the improved PreglParnas-Wagner apparatus.
Table 11. Determination of Nitrogen b y Kjeldahl Method Preceded by Reduction % Nitrogen
REAGENTS
Compound Found 2,4-Dinitrophenylhydrazones 2-Renzy1oxy.3-niettiox~t~~nzal~l~hpde 13,15 13.10
The reagents wed ~ c r eprepared, without modification, according to the direcations of Cole and Parks (3). Catalyst. Grind and mi.: thoroughly together 150 grams of anhydrous potassium sulfate, 10 grams of mercuric oxide, and 5 grams of selenium metal. Mixed Indicator, Mix 10 ml. of a 0.1% solution of bromocresol reen in 95% alcohol with 2 ml. of a 0.1 % alcoholic solution of m e k y l red. Boric Acid, 4%. Dissolve 20 grams of boric acid crystals in 500 ml. of boiling distilled xater. Sodium Hydroxide, 48%. Dissolve 480 grams of sodium hydroxide in 520 ml. of distilled aater. .411ow to settle and use the clear supernatant solution. Sodium Thiosulfate, 44%. Dissolve 88 grams of sodium thiosulfate pentahydrate in 112 ml. of distilled Jsater. Standard Hydrochloric Acid. Prepare a 0.015 S solution and standardize against a suitable primary standard such as sodium carbonate using methyl red as an indicator. The additional reagents used include zinc dust, glacial acetic acid, concentrated hydrochloric acid, and methanol. All were analytical grade reagents.
18.60 18.08
18.66
Pro pior h e n o n e
17.88 17.73
17.80
as-Diphenylacetonr
14 28 14 40
14.30
Cyclopentanone
21.30 21.20
21.15
14.22 14.15
14.35
23.70 23 5 5
23.70
17.51 17.70
17.60
7 68 7.64
7.72
8.40 8.35
8.38
Benzilidine azine
13.41 13.38
13.45
2-hlethylpyridine pirratea
17.40 17.50
17.37
Semicarbazones Acetophenone Benzophenone Oximes 2,3-Dimethoxyhenzaldehydr 2-Hydroxy-3-met hoxyhrnzaldeliyd~
a
13.35
Acrtophenon~
Benzil diphenylhydrazone
PROCEDURE
Weigh a sample which requires a reductive pretreatment, and containing nitrogen equivalent to approximately 7 nil. of 0.015 il; hydrochloric acid, in a tin foil cup and place in the digestion flask. D o not close the sample cup in any way, as it is essential that the sample be dissolved in acetic abid. To dissolve the sample add 1 ml. of glacial acetic acid and warm the mixture gently if neceesary. Allow t o cool slightly and add 200 mg. of powdered zinc and 1.5 ml. of methanol. Add 4 drops of concentrated hydrochloric acid and warm over a small flame t o promote the reduction. After 2 to 3 minutes add a second 4-drop portion of hydrochloric acid and continue to warm the mixture. Continue the addition of 4-drop portions of hydrochloric acid until a total of 16 drops have been added. Increase the heat slightly and boil the mixture to remove part of the volatile solvents. Evaporation to near dryness must be avoided or poor recovery of nitrogen is likely to result.
Theory
8-Hydroxyquinoline picratea 15.07 Time of afterhoil for these compoiinds was 1.5 hours.
14.97
receiver until the delivery tube is entirely out of the distillate and rinse the outside of the tube with a little water. Titrate the blue solution with standard 0.0150 N hydrochloric acid until the blue color has just changed to a pink tinge. Run a blank uaing all the reagents used in the determination.
A few refrartory nitrogen compounds were analyzed and the period of afterboil necessary was found to be a t least 1.5 hours. Results of these experiments are shown in Table I. I n Table I1 are shown the results obtained by this modification of the Kjeldahl method.
Table 1. Determination of Nitrogen in Refractory Compounds
Compound
(Results expressed Theoretical N, 70
Nicotinic acid 2-Xethyl pyridine picrate Tryptophan
11.38 17.37 13.72
9s
% nitrogen) Time of Afterboil, Hours 0.75 1.0 1.5 2.0 19:98 ,
, .
10.90 16.90 12.30
11.37 17.40 13.72
l7:42 13.70
Allow the solution to cool and add 3 ml. of concentrated sulfuric acid. Heat to boiling and continue heating until the water is driven off and the solution darkens. Again allow to cool for a few minutes; then add 1.5 grams of the catalyst and 1 ml. of sulfuric acid. Continue boiling until colorless, then keep the mixture boiling for 30 to 45 minutes longer. A white precipitate remains throughout the entire digestion treatment, but very rarely causes bumping if a moderate rate of boiling is maintained during the afterboil. Rlaterial which does not require a reduct,ive pretreatment may be x-eighed in the tin foil cup and placed in the digestion flask, and 1.5 grams of catalyst may be added, folloxed by 4 ml. of concentrated sulfuric acid. Digest the material until colorless and then for 30 to 45 minutes longer. In either case, ext,end the period of afterboil to 2 hours if refractory nitrogen compounds are being analyzed. Allow the digestion mixture to cool and then rinse into t'he distillation apparatus using a total of about 25 ml. of water. Use a 125-nil. Erlenmeyer flask containing 10 ml. of 4y0boric acid and 6 drops of mixed indicator to receive the distillate. Add about 12.5 ml. of 48% sodium hydroxide slowly to the diluted digestion mixture in such a way that it reacts with the sulfuric acid during the addition and does not, form a layer. Add approximately 4 ml. of 44% sodium thiosulfate and rinse into the apparatus with about 2 nil. of distilled water. Then pass steam rapidly through the misture until about 35 ml. of distillate are collected. Lower the
Table 111.
Effect of Solvent for Prereduction upon Per Cent of Nitrogen Found Solvent
-
Theoretical Compound
1L'. YO
Acetlc acld
Xethanol
Acetic acid and methanol
Benzophenone sernicarbazone
17 60
13.60 14.75
17.55
...
17.51 17 70
36. $50 13.4.5
27.20 10 72
36.20 13.45
36.20 13.41
18.66
18.62
16.45
18.60
Acetone semioarbaaone Benzilidine azine Acetophenone, 2 , 4 dinitrophenylhydrazone
ils shown by Table 111, neither acetic acid nor methanol alone is an entirely satisfactory reduction medium. Acetophenone 2,4-dinitrophenylhydrazoneis insoluble in methanol, hence is incompletely reduced. The higher alcohols are unsatisfactory, because they form carbonaceous material and so deplete the sulfuric acid when it is added to the reduced sample. Acetic acid is a satisfactory reduction medium for the dinitrophenylhydrazones but is unsatisfactory for semicarbazones and azines. The primary purpose of the reductive pretreatment is to rupture the N--S and i'-0 linkagesin such a way as to convert the nitrogen to either ammonia or amino compounds which are easily digested in the regular Kjeldahl procedure. The conditions in this method are not satisfactory for the determination of nitrogen in reitain heterocyclic compounds in which the ring
ANALYTICAL CHEMISTRY
?62 contains a N-N linkage. Hydriodic acid was also found to be unsatisfactory on certain compounds of this type. CONCLUSIONS
The adaptations of the Kjeldahl method of nitrogen determination herein described make it possible to determine nitrogen in such compounds as azines, hydraaones, oximes, and semicarhasones. The method is also satisfactory for use with other types of compounds containing nitrogen to oxygen linkages and for refractory nitrogen compounds such as pyridine and quinoline derivatives. ACKNOWLEDGMENT
It is a pleasure to acknowledge the help and encouragement received from E. D. Amstute and N. R. Easton during the progress of this work. The author is also grateful to the members of the organic research staff who have tested these methods.
LITERATURE CITED
Bradatreot. R. B..ANAL C a z ~ .21, . 1012 (1949). (2) Clark, E. P., J . Assoc. Osc. Agr. Chernista, 24, 641 (1941). (3) Cole, J. O., and Parks, C. R., IND. ENG.CKEM.,ANAL.ED.. 18, (1)
G1-2 (1946).
Friedrich. A.. Kuhaas.. E... and Schnuvoh. R.. Z. ohu~iol.Ciiern.. 216, 60 (1933). ( 5 ) Grillemord. H.. BuU. doc. chim.. 1, 1 9 6 2 0 0 (1907) ( 6 ) Kirk, P. L.. ANAL,Cmnr.. 22, 354-8 (1915n) ..,. (7) Ogg, C . L., and Willits. C. 0.. Ihid., 23, 47-51 (19511. (8) Patel. S. M.. and Sreenivasan.A.. 16id..20. 63 (i948): KAI.. NO. (9) Pepkbwitu, L. P.,and Shive. J. W., IND.'E Eo., 14, 914 (1942). 110) Rose, E. D.. and Ziliotto. H., Ibid.. 17, 211 (1945). Shaefer. W. E., and Beoker. W. W.. Ibqid, 19, 307 (1947). Shirley, R. L.. and Beeker, W. W., 1bir1., 17, 437 (1945). Shuey. P.MoG.. Ibid.. 19.882 (1947). WillitB, C. 0.. and Ogg, C. L.. J . Assoc. OBc. A0 ', 33, (4) . .
.-n 0 0 ,.nzn> 1,v-00 ,'VU",.
RzcErvEDforreview June20, 1951. Accepted Ortnber 31, 1911.
Contrihuted b y MAX R. WILLIAMS A N D W. P. VAN M ETER, D e p a r t m e n t of Chemistry, Oregon S t a t e College, Colvallif i, Ore. 7.r
11:-C-N-N-0 11 11 H I1 0 Structural Formula for 8-Acetylphenylhydrazine XCELLENT crystals of p-acetylphenylhydraaine I can be obtained from acetone, ethyl alcohol, and thymol. Acetone and thymol solutions give tablets or plates, while alcohol solutions tend t o give a needle habit. Figure 1shows crystals from thymol. Figure 2 is an orthographic projection of a typical tablet from solution.
E
Formula Weights per Cell. 8 (8.06 calculated fmm x-ray data). Formula Weight. 150.18. Density. 1.280 (Rotation and specific gravity balance); 1.286 (x-ray). Prinoipsl Lines d 10.88 5.18 4.74 4.64 4.35 4.01 3.96 3.73 3.45 .%,,lllrjr .L---
---
I/Iz 1.00
0.61 0.39 0.30 0.09 0.30 0.14
0.28 0.30
io.77(ozo) 5.22(121) 4.78(200) 4.e5(210) 4,371220) 4.oo(141) 3.98(211) 3.7~(012) 3 . 5 0 112)
3.49h51) -.LA-
yl/ln
d
I/J
Indexn
3.34
0.20
3.37(032)
0.08
a.05(320) 2.92(311) Z.S~(ZZZ) 2.64(212)
Index-
L:.L ,"yLIy
3.06 2.93 2.88 2.66 2.57 2.51 2.43 1:ifi
-:^l.
llllbjll
0.58 0.18 0.14 0.10 0.08 0.05
2.65(003) 2.53 (013) ~.44(302)
2.44(113) i.xe(511)
_^_._ .^ "" alii
:L...^ u"IIIII"""~
,:..""
"1151s L.llrv
"-2
the indicated indexes are only suggested as wssible contributing renections.
Figure 1. B-Aoetylphenylhydrazine I Crom Solution
CRYSTAL MORPHOLOGY Crystal System. Orthorhombic. Form and Habit. Plates and tablets !ying on mnrropinncoid, 100, and needles elongated along e showing prierns, { I I O I , pyramids, 11111, and brachydomes, I O l l l . Axid Ratio. a:b:e: = 0.444:1:0.3_5. Int_erfacial Angles (Polar). 12OA120 = 57" (83.2", x-ray); 101A101 = 76" (77.2', x-ray). X-RAYDIFFRACTION DATA Cell Dimensions. a = 9.55 A,; h = 21.54 A,; e = 7.04 A
