235
V O L U M E 2 6 , N O . 1, J A N U A R Y 1 9 5 4 Table 111. Effect of Total Activity S-35-Labeled Methionine, Micromole 0.0176 0.00878 0.00351 0.00176
Observed Specific Activity Counts/ Min./Mioromole 166 142 165 156
Counts/Min. 2925 1243 580 275
I
1
I
>
t 2 0 4
#
MILLIGRAMS
cUC12 2H20
Figure 5. Effect of Copper on Counting Rate of Samples Prepared by Method B
on planchets. The standard error for a mean calculated from triplicate determinations is less than 4% by either method. This compares favorably with the precision obtained after the more tedious procedure involving conversion to barium or benzidine sulfate ( S ) , particularly as such methods require at least two additional transfers of small amounts of materials. The less desirable features of this method of sample preparation include the absorption of radiation by the paper. The counting rate of samples prepared by Method B was approximately 20% of that observed with identical samples prepared by Method .4. The availability of compounds of high specific activity makes this loss permissible in many procedures. It is also obvious that the independence of counting rate and specific activity can introduce errors if large amounts of the labeled species are present and the total weight of sample is not suitably corrected. Finally, any factor, such as complex formation, which alters the movement of the labeled species through the paper, can lead to erroneous results. I n practice, these sources of error can be generally avoided in designing the experiment. The chief advantage is the relatively small and constant alteration in counting rate introduced by other components of the mixture in which the radioactivity is to be determined. The changes in absorbers tested represent more drastic changes than would normally occur in practice. This would seem to make this a satisfactory method for the direct comparison of radioactivity in heterogeneous mixtures.
Each point is average of 10 samples
after drying in the usual manner. In this case the counting rate for the upper side of the sheets, stdrting with the top one, was 7 4 i , 192, 121, 131, and 380 counts per minute, respectively. Although this procedure introduced the additional variable of a series of interfaces between the sheets, the results serve to indicate that the active species moves both up and down through the paper as evaporation proceeds. I t aould seem that the equations derived by Henriques et al. for solid precipitates do not apply to this process (S). Presumably the actual rate of movement depends on a variety of factors, including temperature. I t is significant that when water was added to samples and permitted to re-evaporate under the same conditions, no marked effect on the counting rate was observed. I n spite of the complexity of the process leading to the distribution of the labeled substance in the paper, the precision obtained is a t least as great as that observed with direct evaporation
LITERATURE CITED
(1) Calvin, &I., Heidelberger, C., Ried, J., Tolbert, B., and Yankwich, P., “Isotopic Carbon,” New York, John Wiley & Sons,
1949. (2) Greenberg, D. ll., “Amino .kcids and Proteins,” Springfield,
Charles C Thomas, 1951. (3) Henriques, F. C., Kistiakowsky, G. B., Nargoretti, C., and Schneider, W. G., IND.ESG. CHEM.,ANAL.ED.,18, 349 (1946). (4) Hogness, J. R., Roth, L. J., Leifer, E., and Langham, W. H., J . Ant. Chem. S ~ C 70, . , 3840 (1948). (5) Le Page, G. A., and Heidelberger, C., J . Bid. Chern., 188, 593 (1951). (6) Ruben, S., Hassid, W. Z., and Kamen, 31.D., J . Am. Chena. SOC. 61, 661 (1939). ( 7 ) Yankwich, P., Sorris, T., and Huston, J., -4v.k~.CHEM.,19, 439 (1947). RECEIVED for review M a y 15, 1953. .4ccepted August 17, 1953. Supported by a grant-in-aid from The American Cancer Society upon recommendation of the Committee on Growth of the National Research Council.
Determination of Nitro Nitrogen by the Kjeldahl Method R. B. BRADSTREET The Bradstreet ~!aboratorks, lnc., 7356 North Broad St., Hillside,
T
H E Kjeldahl method ( 4 , is not applicable to every form of organic nitrogen. However, owing to the comparative ease of operation, many attempts have been made to generalize the method. Treatment of nitro conipound.5 with phenols to convert them into a more easily reducible form was first suggested by Jodlbauer ( 3 ) . Later, Cope ( 2 ) substituted salicylic acid for phenol. Complete recovery of the nitrogen in nitro and other so-called refractory compounds is not always accomplished by the use of these, even in conjunction with sodium thiosulfate. The generally accepted Procedure for nitro compounds uses 5 grams of sodium thiosulfate and 10 grams of potassium sulfate a boiling point raiser. The severity of this reaction is increased by using larger amounts of potassium sulfate, which has been shown by other investigators (5,6). Using p-dinitrobenzene as a typical compound whose nitrogen is not readily available, it
N. 1.
was determined that 18 grams of potassium sulfate was the optimum (see Table I). EFFECT O F INCREASED DIGESTION TERIPER.4TURES
T~ demonstrate the difference between the use of the conventional 10 grams of potassium sulfate and 18 grams, a number of compounds were using the follon+ng procedure: Weigh into a Kjeldahl flask 0.1 to 0.15 gram of sample and 35 ml. of concentrated sulfuric acid containing 1 gram of salicylic acid. Let stand for 0.5 hour on the steam bath, or until the sample has completely dissolved. Transfer to the digestion rack and add 5 grams of sodium thiosulfate. Let stand for 0.5 hour and then heat gently until the mixture carbonizes. Cool and add potassium (I), and 0.25 gram Of mixed (FeSOa.iHzO-Se). Heat strongly until the mixture clears, and boil gently for 1 hour, Coo], dilute wit,h distilled water, determine the nitrogen in the usual manner.
236
ANALYTICAL CHEMISTRY Table I. p-Dinitrobenzene
Table 111. Effect of .4romatic Hydroxy Compounds on oand p-Dinitrobenzene
(Calculated 16.68%)
KzSOI
Added, Grams 2
4 6 8 10 12 a
Pasty
Nitrogen,
%
15.01 15.04 15.19 15.23 15.18 15,47 digest on cooling.
Kif301
Added, Grams 14 16 18 20 22 24
%
l5,49 15.30 16.27 15.1Iy l5.20Q 15.48'
~ _ _
Table 11.
i
Effect of Increasing Potassium Sulfate to 18 Grams K,SOr,
Sitrogen, V0b KzSOi, 18 grams 14.48 16.67
Compounda 10 grams o-Dinitrobenzene 12.98 m-Dinitrobenzene 16.66 15.15 p-Dinitrobenzene 15.49 6.17 6.76 m-Nitrochlorohenzene p-Nitrochlorobenzene 6.61 8.07 2 .5-Dichloronitrobenzne 2.40 4.58 3,4- Dichloronitrobenzene 3.82 5.57 22.01 2,4-Dinitroaniline 14.00 13:io 2,4-Dinitrophenol 14.70 15 46 2 4-Dinitroresorcinol 3:5-Dinitrobenzoic acid 12.28 12.62 6.89 7.07 3-Nitrosalicylic acid 7.06 7.31 5-Sitrosalicylic acid Eastman Kodak Co. highest purity or equivalent. b Average of t w o determinations.
I
':'
16.68 16.68 16 68
13.38 13 21 7 6.5 7.65
'
"
Calcd.
8.89 8.89 7.30 7.30 22.95 15.22
o-Dinitrobenzene, 16.68% N 14.48 15.09 14.95 14.80 14.00 15.63 15.12 13.17 15.65 l3,89
Concd. HnSOr, 1 Gram/35 Ml. Salicylic acid I-Naphthol 2-Naphthol Catechol Resorcinol Pyrogallol Hydroquinone Phloroglucinol Gallic acid Salicylic acid : Pyrogallol Salicylic acid Hydroquinone Gallic acid ,I : Pyrogallol Gallic acid 1-Xaphthol Pyrogallol I-Naphthol Pyrogallol 2-Xaphthol
Nitrogen,
p-Dinitrobenzene, 16.68% .V 15.49 16.03 15,88 15.23 15.49 16.00 14.77 15.72 15.76 16.13
14.88
15.09
15.90
15.89
15.69
15.79
lL95
16.28
15.77
15.38
Comparison of Results Using Salicylic .4cid and 1-Naphthol-Pyrogallol
Table IV.
Salir>lic acid 1 4 48
Compounda o-Dinitrobenzene
Nitrogenb. % I-Naphtholpyrogallol 16 15 16 02
Calcd. 16 68
I~. 5 9 .5 .~
From Table 11, it will be seen that while the results do not reach the calculated percentage, in most cases there has been an appreciable gain owing to the larger amount of sulfate used and the consequent increase of digestion temperature. E F F E C T OF HYDROXY COMPOUNDS O N CONYERSIO\ OF l I T R O GROUPS
Because phenol and salicylic acid, both hydroxy compounds, apparently help in the conversion of nitro groups, it was believed that other aromatic hydrovy compounds might have a similar effect. The following procedure was used, the salicylic acid being replaced in the same amount by the variouc: hydro\\- compounds. 0- and p-Dinitrobenzene, typical refractory coml)ounds, ncre used. Weigh into a Kjeldahl flask 0.10 t o 0.15 gram PROCEDURE.
of sample and add 35 ml. of concentrated sulfuric acid containing 1 gram of the hydroxy compound (or combination). Let stand for 0.5 hour on the steam bath, or until the sample has completely dissolved. Transfer to the digestion rack and add 5 grams of sodium thiosulfate. Let stand for 0.5 hour, then heat gently until the mixture carbonizes. Cool, and add 18 grams of otassium sulfate and 0.25 gram of mixed catalyst (FeSO1.7Hdbk). Heat strongly until the mixture clears, and boil gently for 1 hour. Cool, dilute with distilled 11-ater, and determine nitrogen in the usual manner. The results (Table 111)show that the most suitable combmation is a mixture of equal parts of 1-naphthol and pyrogallol. Further determinations were made on a number of compounds using the above procedure with 1-naphthol-pyrogallol. A comparison of results is shown in Table 11'. In some cases, the calculated amount of nitrogen has been reached; in every case an increase is noted over the results found by the use of the conventional salicylic acid. In a recent paper, 1lcCutchan et al. ( 7 ) have suggested the use of thiosalicylic acid. Comparative results using this and 1naphthor-pyrogallol are shown in Table V. For the compounds analyzed, the method using 1-naphthol-pyrogallol, in moqt cases, gives somewhat higher results. CONCLL-SIONS
The data presented indicate that the use of l-naphtholpyrogallol tende to give better results than salicylic acid on the
m-Xiti ochlorobenzene p-Kitrochlorobenzene 2 5-Dichloronitrobenzene 3:4-Dichloronitrohenzene 2 4-Dinitroaniline 3'5-Dinitrobenzoic acid 3:Xitrosalicylic acid 5-Kitrosalicylic acid 2,6-Dichloro-4-nitrophenol b
4.58 5.57 22,Ol 12.62 7.07 7.31 6.35
8.89 8.89 7.30 7.30 22.9.5 13.21 7 .65 7.65 6.74
Comparison of Results Using 1-NaphtholPyrogallol and Thiosalicylic Acid Xitrogenb, ?& l-Saphtholpyrogallol
Compound' p-Nitrochlorobenzene 2,4-Dinitrochlorobenzenr m-Dinitrobenzene o-Kitrotoluene Picrolonic acid Azobenzenc Pyrrole Piperinr Piperidine b
6.i6 8.07
16.68
Eastman Kodak Co. higheht purity or eqiliralent. Average of two determination.-.
Table V.
a
16.33 16.28 16.62 16.22 16.47 7.84 8.80 6.64 6.81 22,42 13,26 7.69 7.65 6.53
15.49
v-Dinitrobenzene
8.80 13.84 16.66 10 60 17.23
ThioEalicylic acid 8.42
13.36 20 64 4 63 12.09
13.88
16.72 8.73 12.39 15.33 19.76 4.30 10.56
Calcd. 8.89 13.82 16.68 10.22 21.21 15.38 20.87 4.69 14.43
Eastman Kodak Co. h i g h e ~ tpririty or equivalent. .4vrrage of two determinations.
compounds analyzed. h comparison of this procedure and the procedure using thioealicylic acid shows some improvement over the latter. LITER4TURE CITED
(1) Bradstreet, R. B., ISD. ENG.CHEM.,ANAL.ED., 10, 696 (193s). (2) Cope, W. C., J . Ind. Eng. Chem., 8, 592-3 (1916). (3) Jodlbauer, &I., Chem. Zentr., 57, 433 (1886). (4) Kjeldahl, J., Medd. Carkberg Lab., 2 (1883); Z.mud. Chem.. 22, 336 (1883). (5) Lake, G. R., RIcCutchan, Philip, Van Meter, Robin, and ?;eel, J. C., ANAL.CHEM.,23, 1634 (1951). (6) Ogg, C. L., and Willits, C. C., J. Assoc. Ofi. Agr. Chemists, 33, 100-3, 179-88 (1950). (7) LIcCutchan, Philip, and Roth, W. F., ANAL.CHEM.,24, 369-70 (1952). RECEIVED for review June 6, 1952. .4ccepted September 22. 19.53
