ANALYTICAL CHEMISTRY
722 will serve to determine safrol or other similar compounds and formaldehyde in the presence of each other. Speck (10)has pointed out that formaldehyde will interfere with the determination of diacetyl by his procedure. Samples of diacet yl when carried through the recommended procedure produce no color. Furthermore, diacetyl, at least in small concentrations, does not interfere with the determination of any free formaldehyde that may be present. Therefore, a combination of these procedures will serve to determine formaldehyde and diacetyl in the presence of each other. Compounds which are not readily volatile a t 170" C. may still interfere with the formaldehyde reaction. However, the usefulness of this reagent has been greatly extended with this modified procedure. This procedure gives a linear calibration line and thereby eliminates the necessity of using a calibration graph to determine the amount of'formaldehyde corresponding to a given optical density.
ACKNOWLEDGMENT
The authors gratefully acknowledge the financial assistance of the grant made by E. I. du Pont de Nemours & Company to sponsor fundamental chemical research at Princeton University. LITERATURE CITED
Boos, R. N., ANAL.CAEM.,20, 964 (1948). Boyd, 31. J., and Logan, M. A , , J . B i d . Chem.. 146, 279 (1942). Bricker, C. E., and Johnson, H. R., IXD.ESG.CAEM.,ANAL.ED., 1 7 , 4 0 0 (1945).
Bricker, C. E., and Roberts, K. H., ANAL.CHEM.,21, 1331 (1949). Eegriwe, E., Z. anal. Chem.. 110, 22 (1937). Huckabay, W. B., Newton, C . J., and Metler, A. V., ANAL. CHEM..19, 838 (1947). Kleinert, T.. and Srepel, E., Mikrochemie uer. Mikrochim. Arta. 33, 328 (1948).
MacFayden, D. A., J . Bid. Chem., 158, 107 (1945). Schryver, S. B., Proc. R o g . Soc. (London), 82B,226 (1909). Speck, J. C.. Jr., ANAL. C H E M .20, . 647 (1948). R E C E I V E DSovember 28. 1949.
Determination of Benzylpenicillin DOROTHY J. HISCOX Department of Wational Health and Welfare, Ottawa, Ontario, Canada
1946, Page and Robinson (6) published a colorimetric IN (N1-naphthyl) method for the determination of benzylpenicillin, using ethylenediamine dihydrochloride as a color reagent. By this method the benzene ring of the penicillin was nitrated, and the nitro compound was reduced, then diazotized and coupled with the color reagent in the presence of ethyl alcohol. A violet color showing maximum absorption a t a wave length of 560 mp was produced. This method did not give reproducible results when tested in this laboratory. During investigational work, two reasons for the failure of this method were found: An excess of sodium nitrite reacts with the color reagent, and under the conditions outlined by the arithors, nitration was not complete. This is also true of other methods for the determination of benzylpenicillin which are based upon the nitration of the benzene ring (f-S). Under none of the conditions used in these methods could reproducible results be obtained when tested colorimetrically with N( 1-naphthyl) ethylenediamine dihydrochloride. The error did not lie in the color development, as there was no significant variation in the color developed in numerous aliquots taken from single nitrations. Nitration was more complete with smaller esmples of penicillin. For routine work the simplest conditions for nitration would be to heat the sample with nitration mixture in a test tube in boiling water. It was decided to see if complete nitration could be effected under these conditions. Surprisingly, it was found that nitration was more complete when smaller aliquots of nitration mixtures were used. When the nitration mixture was broken down into its two components, it was shown that this result wa3 due to the decrease in the amount of sulfuric acid present with the smaller aliquots. Typical results are shown in Table I. With sample 1, as the amount of sulfuric acid increased, the color produced decreased. This could be due to the decrease in the concentration of potassium nitrate present in the mixture. However, samples 2 and 3 show that i t was due to the amount of sulfuric acid present. With these samples, different amounts of the same nitration
mixture were used, but the concentration of the potassium nitrate was unchanged. Bitration was more complete with the smaller amount of nitration mixture-that is, with less sulfuric arid present . It was found that, when a sample of 0.5 mg. of penicillin or less is heated in boiling water for 2 hours with 0.5 ml. of a nitration mixture containing 60 grams of potassium nitrate in 100 ml. of concentrated sulfuric acid, nitration is complete. Under these conditions, reproducible results are obtained. The density of the color from an amount of aniline equivalent to 0.5 mg. of benzylpenicillin, as determined in a Beckman quartz spectrophotometer a t 560 mfi, was 1.20 and 1.30. The density of the color from 0.5 mg. of benzylpenicillin was 1.19, 1.23, and 1.33. Although this cannot be taken as an absolute comparison, it is a good indication that nitration by the author's procedure is complete. When such a small amount of nitration mixture is used, it is necessary to add more sulfuric acid after nitration is completed before the color is developed to ensure its completp development. Heat from 0.1 to 0.25 nig. of penicillin with 0.5 ml. of nitration mixture (60 grams of potassium nitrate in 100 ml. of concentrated sulfuric acid) in a 25 X 100 mm. test tube in boiling water for 2 hours. Add 5 ml. of water and 0.2 gram of granular zinc and heat an additional 15 minutes. Transfer to a 25-ml. volunwtrib flask, rinsing the tube with two 3-ml. portions of water and decanting the liquid from the zinc residue. Add l ml. of ronren-
Table I.
Effect of Sulfuric Acid on Nitration of Benzylpenicillin
Sample
KSOr, G.
HzSO4. MI.
1
0 40
0 40 0 40
1 0 1 5 2 0
2
0 40 0 80
1 0 2 0
3
0 25 0 50
0 5 1 0
70Absorption 57 9 17 4
51 9 16
c(
3 2 74 7 27 3
73 8 24 2
89 9 75 4
'10 2 74 0
4 7
V O L U M E 22, N O . 5, M A Y 1 9 5 0
723
Table 11. Determination of Benzylpenicillin by Three Methods
b C
% Benzylpenicillin NEP" UVb Sample 89.4 89.0 41,369 41,518 90.5 87.1 93.1 94.8 42,522 94.3 98.7 43.678 89.4 90.0 46,385 88.6 91 . o 46,574 92.0 89.5 46,975 91.0 88.8 48.623 89.1 48,743 88.5 92.0 94.6 48,774 91.0 93.8 48,949 93.4 92.9 50,233 92.5 95.8 50,345 93.9 93.3 50,346 92.4 95.8 49,523 88.3 92.9 49,038 91.3 92.3 Mean N-ethylpipendine. Ultraviolet. N(1-naphthyl) ethylenediamine dihydrochloride.
NEDC 89.7 96.8 96.8 97.1 95.2 87.2 91.2 91.1 86.8 96.2 92.0 94.0 97.0 95.2 94.8 95.1 93.5
trated sulfuric acid, cool, and add 1 ml. of 0.1% sodium nitrite and 1 ml. of 0.25% ammonium sulfamate, shaking well after the addition of each. Add 10 ml.. of 95% alcohol. Add 1 ml. of 1.5% N ( 1-naphthyl) ethylenediamine dihydrochloride, prepared fresh daily. Make to volume. Let stand a t least 1 hour and determine the density of the solution a t 560 mp in a Beckman quartz spectrophotometer. Crystalline penicillin may be used, or the residue from a 1-ml. aliquot of a solution of penicillin in water or buffer which has been taken to dryness in boiling water. The procedure may be interrupted after nitration, but once reduction has taken place the color must be developed within 1 to 2 hours. With many samples of penicillin color development is complete within 15 minutes, but some samples require 1 hour for maximum color development. The color is stable for at least 3 hours after the maximum color is reached. Though different brands of N ( 1 -
naphthyl) ethylenediamine dihydrochloride possess different physical properties, under the above conditions the same results are obtained. For refereme the author established a regression line using the above procedure and amounts of sodium benzylpenicillin varying from 0.05 to 0.5 mg. The 67 points used were determined on 5 days during a period of 2 weeks. Two brands of N ( 1-naphthyl) ethylenediamine dihydrochloride and two standard benzylpenicillins were used. The equation of'the line was Y = 0.399032 X 0,0059, where Y represents milligrams of benzylpenicillin and X is the density. The correlation coefficient was +0.995 and the standard error of prediction +0.014 mg. In Table I1 results obtained by this method on a number of commercial samples of penicillin are compared with results by the gravimetric N-ethylpiperidine ( 5 ) and the ultraviolet absorption ( 4 ) methods. Although these methods are based upon three different principles, they show good agreement. They vary greatly in the amount of time required, the size of sample used, and the precautions that must be observed. The choice of a method will depend upon the conditions existing in individual laboratories. ACKNOWLEDGMENT
The author wishes to thank Merck & Co. and Ayerst, McKenna & Harrison, Ltd., both of Montreal, for gifts of crystalline benzylpenicillin. LITERATURE CITED
(1) Boxer, G. E., and Everett, P. M., ANAL.CHEM.,21, 670 (1949). (2) Canback, T.,Farm. Rea., 46,97 (1947). (3) Del Vecchio, G., and Argenziano, R., BUZZ. soc. ital. biol. 8pcc.. 22, 1190 (1946). (4) Levy, G.B.,et al., ANAL.CHEM.,20, 1159 11048). ( 5 ) Mader, W. J., and Buck, R. R., Ibid., 20,254 (1948). (6) Page, J. E., and Robinson, F. A , , Nature, 158,910 (1946).
RECEIVED July 19, 1949.
Identification and Microdetermination of Nickel In Presence of Iron by Means of 1,Z-Cyclohexanedione Dioxime H. I. FEINSTEIN National Bureau of Standards, Washington, D . C . DVANTAGES and disadvantages in the use of 1,2cyclodioxime (called nioxime) as compared with dimethylglyoxime in the analytical chemistry of nickel and palladium have been reported (2-4). Voter, Banks, and Diehl (3) devised a procedure using this reagent for the gravimetric determination of nickel in the presence of a number of elements, but were not successful in preventing interference by iron. The cause of this difficulty was not explained, but i t seems likely that the masking agents did not form sufficiently stable complexes a t a p H of 4 to 5 to prevent oxidation of the reagent by ferric iron. Accordingly, a change in the pH of the solution a t the time of precipitation might eliminate the interference from iron. Therefore, experiments were performed using neutral or slightly ammoniacal solution a t room temperature followed by digestion a t 60" C. Furthermore, nioxime wag added to the already neutralized solution, whereas Voter, Banks, and Diehl (S) adjusted the p H after the reagent was added. The use of acetate as a buffer was eliminated, because tartrate could function both as buffer and masking agent. I t is believed that tartrate has no appreciable reducing action on ferric ion under the conditions of the procedure. The procedure finally adopted was tested on three National Bureau of Standards standard samples of iron and steel. The
A hexanedione
results of nickel determinations employing single and double precipitation on these samples are given in Table I. GRAVIMETRIC PROCEDURE
A weighed sample containing 10 to 25 mg. of nickel is tranaferred to a 400-ml. beaker and decomposed by accepted methods Table I.
Values Obtained for Nickel by Single and Double Precipitations Nickel Found, % Single Double precipiprecipitation tation 3.32 3.27 3.32 3.26
Weight of Sample, 0. 0.5
Nickel Present,
82ab
1.0
1.07
1 12 1.12 1 12 1.11
lOlbC
0.1
8.99
9.17 9.13 9.18
S.B.S. Standard Sample 33ca
0.2
%
3.28
1.10 1.10
8.90 8.99 a Nickel steel (SAE 2335). M n 0.733; Cu 0.031; Cr 0.052; M o 0.032; AI 0.032. b Nickel-chromium cast iron. M n 0.65. Si 2.06. Cu 0.08; Cr 0.33. 0 18 Chromium-9 nickel steel (SAE '30905). 'Mn 0.597; Cu 0.168; Cr 18.49; V 0.049; Mo 0.078; Co 0.078; Cb 0.062; Sn 0.012.
