V O L U M E 2 1 , NO. 11, N O V E M B E R 1 9 4 9
1389
Anallsis of Synthetic Samples
hydrolyzed by dilute sulfuric acid. These esters were not run by the above method; however, the method should be readily applicable to their analysis and should yield good results.
Table U T .
Decyl Kitrate Added
Stabiliaer Added
110 0
113.7
114.2
+0..5
0 4
108.8
109.1
+0.4
0.5
105.5
104.2
-1 -1
Mg.
117.0
Diphenylamine Diphenylamine derivatives (mixture of 2-nitrodiphenylamine, 2,4-dinitrodiphenylamine, p-nitrosodiphenylamine) Diethyldiphenylurea
Decyl
Nitrate Found .ti@. 116.3 109.5
121.8
120.2
D*.viation
Deviation
Slg. -0.7 -0.5
0 6
0.5
3
1
4CKNOB LEDGXIEYT
‘70
The authors wish to express their sincere appreciation to Clayton Huggett and Franz Rathmann for their many helpful comments during the course of this work.
1 2 0 9
It is essential to have a shaking apparatus that trnds to homogenize the rciaction mixture as well as to shake it, and the shaking action should be vigorous. X shaker speed of 240 to 250 complete oscillations per minute was sufficient to effect the complete hydrolysis of the nitric acid esters. .4number of synthetic samples nere made up containing decvl nitrate together with roughlv equal weights of diphenylamine, or diphenylamine derivatives, or diethyldiphenvlurea. These were then analyzed. Table VI1 gives the results of these runs. h-0 interference was found from these compounds. Interferences. Organic nitrites cause serious errors in the determination and should be absent from the materials to be analyzed. Sitrites when hydrolyzed in acid solution give nitrous acid, which reacts with the 3,4-xylenol t o give a variety of steamdistillable colored products. Other Nitric Acid Esters. The application of this method to nitrocellulose mas attempted. Low results were obtained Kith nitrocellulose due to incomplete hydrolysis in 80% sulfuric acid. The hydrolysis was very slow (about 6 hours were required to reach a steady value) and only 87.5y0of the nitrocellulose was recovei ed. Ktroglycerin and diethylene glycol dinitrate are very easily
LlTER4TURE ClTED
(1) dlten. F.. Wandrowskj,, B., a n d Hille, E . , Bodenkunde zi. Pflanze,Lernlihr., 1, 340 (1936), (2) .\Iten, F.. and Weiland, H., Z Pflnntetwrndhr. Diingung u . Bodenk., 32A,337 (1933). (3) =iuwers, K. C., and Borsche, E., Ber., 48,1714 (1915). (4) .\uwers, K. C., and Michaelis, F., Ibid., 47,1289 (1914).
(5) Beilstein, “Handbuch der organischen Chemie,” 4th ed.. Vol. VI, p. 480, Berlin, Julius Springer, 1918. (6) Blom, J., and Treschow, C., Z . Pflanzenernahr. D i i i ~ g i ~ i i gu . Bodenk., 13A,159 (1929). (7) Diepolder, E.. Ber., 42,2916 (1909). (8) Holler, A . C., Huggett, C., and Rathmann, F. H., J . 9 m . Chem.
Soe., to be published. (9) McVey, IT. C., J . Assoc. Ofic. A g r . Chemists, 18,469 (1936). (10) Mueller, G. P., and Pelton, W.S., J . Am. Chem. SOC.,71, 1504 (1949). (11) Xoelting. E., and Pick, B.. Be?., 21,3169 (1888). (12) Treschow, C., and Gabrielsen, E. K., 2 . Ppanzenernlihr. Diinyiing t i . Bodenk., 32,357 (1933). (13) Wallach, O., and Blembel, A , , Ann. 319,99 (1901). (14) Welcher. F., “Organic Analytical Reagents,” Yol. I, p . 188. New York, D. Van Nostrand Co., 1947. (15) Werr, F.. Z . anal. Chem., 109, 81 (1937). (16) Wheland, G. W., Brownell, R. M.,and Mayo. E. C., J . A m . Chem. Soc., 70, 2494 (1948). (17) Wieland, H.. Ber.. 54,1780 (1921). (18) Yagoda, H., IND. ENG.CHEM.,A s a ~ED., . 15,77 (1943). RECEIVEDFebruary 12, 1949. Research supported by the C. S. S a v y , Bureau of Ordnance, through Contract XOrd 9680 with the University of Minnesota.
Spectrophotometric Determination of Uracil, Thiouracil, and Related Compounds WENDELL L. HOLT AND LELAND N. MATTSON, Cole Chemical Company, S t . Louis, ,Wo.
A
COLORIhIETRIC method for assaying barbiturates, introduced by Dille and Koppanyi ( 2 ) , is based on the color produced \)-hen a barbiturate reacts with a cobalt salt in an alkaline medium. Further investigations ( 1 , 3-5) describe optimum conditions and application of this reaction to the barbiturates. During a study of this reaction in the determination of phenobarbital it was noted that compounds with -COSHCOand -COXHCSgroups interfered in the reaction bj- giving similar color. This interference prompted investigation of the color reaction for possible use in assay of compounds containing these groups. dmong the compounds tested were uracil, thiouracil, and propylthiouracil and it was found that by using conditions similar t o those used for the barbiturates, reproducible quantitative results accurate to within about 1 or 2% could be obtained. These results are noted in Table I. REAGEATS REQUIRED
Chloroform, C.P. grade. Drv over anhydrous sodium sulfate. Methyl Alcohol Absolute, C.P. grade. D r y over anhydrous sodium sulfate. Cobalt Acetate Reagent. Dry cobalt acetate for 2 hours at 100’ C. Dissolve 0.25 gram of dried cobalt acetate in 200 ml. of dried ahsolute methyl alcohol, C.P. grade.
Isopropylamine Reagent (Eastman) to 200 ml. with Isopropylamine Reagent (Eastman) to 200 nil. with
A. Dilute 50 nil. of isopropylamine dried chloroform. B. Dilute 50 ml. of isopropylamine absolute methyl alcohol.
4S.4 LYTICAL PROCEDURE
Because optimum conditions for the determination of the and -CONHCSgroups vary, an example of each is presented.
-COXHCO-
Compounds with -CONHCSGroup. Accurately weigh a dry sample sufficient to give a concentration of approximately 1 mg. per ml. and transfer to a volumetric flask. Dissolve the sample in methanol and dilute t o volume. Transfer exactly 5 ml. to a dry 25-ml. volumetric flask and add reagents in the folloning order: 5 ml. of isopropylamine reagent .4,5 ml. of cobalt acetate reagent, and dilute to volume with chloroform. Mix thoroughly and take readings a t 530 niM. Run a blank on reagents consisting of 5 nil. of cobalt acetate reagent, 5 ml. of isopropylamine reagent A, and 5 ml. of methanol, diluting to volume with chloroform. Subtract the blank reading from that obtained for the sample. Prepare standard graphs, using the dried standard compound dissolved in methanol in suitable concentrations. Compounds with -CONHCOGroup. Accurately weigh a drv sample sufficient to give a concentration of 0 . i 5 mg. per ml.
ANALYTICAL CHEMISTRY
1390
Uracils, thiouracils, and related compounds react with cobalt salts in anhydrous alkaline medium to form stable color complexes. The complexes formed obey Beer’s law within limits and this reaction may be used for accurate quantitative measurements. Optimum conditions for color development and application of this reaction to analysis nf some pharmaceuticals and biological compounds are described.
.6
> +=
v)
z
w
n
.4
J
Q
0
k 0
.2
01
650
ZOO
Figure 1. Absorption Spectrum of Thiouracil and Propylthiouracil
Figure 2.
400
450 WAVE
500
550
000
LENGTH I N MILLIMICRONS
450 WAVE
550
500 LENGTH
IN
MILLIMICRONS
Absorption Spectrum of Uracil and Phenobarbital 4
and transfer to a volumetric flask. Dissolve the sample in dry chloroforni and dilute to volume. Transfer exactly 5 ml. to a dry 25-ml. volumetric flask, add 5 ml. of cobalt acetate reagent, and 5 ml. of isopropylamine reagent B, and dilute to volume with chloroform. Mix thoroughly and take readings a t 560 mp. Run a blank on reagents and subtract the value obtained from the sample reading. Prepare standard graphs using the dried standard compound dissolved in chloroform in suitable concentrations.
The data for the absorption curves were obtained using a Coleman Model 11 spectrophotomet.er and are represented in Figures 1 and 2. Figure 1 includes curves for thiouracil and propylthiouracil. Both compounds contain the -CONHCSgroup and show maximum absorption in the visible spectrum a t 530 mp and a t wme point in the ultraviolet spectrum. Figure 2 shows curves for uracil and phenobarbital, which contain the -COiYHCOgroup and exhibit maximum absorption a t 560 mp. The color forms almost immediately after addition of the reagents and is stable for several hours. For best results the medium used in this reaction should be a chloroform-methano1 mixture in the ratio of 3 to 2. Either methanol or chlo-
Table I. Determination of Uracil, Thiouracil, Propylthiouracil, and Phenobarbital
Uracil Thiouracil Propylthiouracil Phenobarbital
Added Mg. 3.75 3.i5 3.00
Found M 0.
Recovery
3.75 3 .i 6 2.99
100.0 100.3
70 95.6
5 00 5 00 1.00 2.00 , .a0 7.50
4.91
j.03 3 5.5 5.98 7.48 7.45
100.6
.i 00 , 5.00
4.98 4.58 2.48
99.6 99.6 99.2
2.50
,
t
c v)
z
W
n
d
EXPERIMENTAL
Compound
650
600
98.2 58.8
95.7 59.7 95.3
0 c
a 0
0
I 2
I 4
I 6
I 8
MILLIGRAMS I N 2 5 ML.
Figure 3. Calibration Curves for Phenobarbital and Propylthiouracil roform solvent may be used for dissolving the sample. The 3 to 2 chloroform-methanol ratio may be accomplished in either case by proper choice of diluent in preparing reagenta. If difficulty is experienced in dissolving a sample in either of these solvents, a small amount of isopropylamine added to the solvent often aids solution. It is important to carry out the reaction under anhydrous conditions, as the presence of moisture will cause a gradual fading of color. The sample, solvents, and reagents must be completely dry to ensure development of a maximum stable color. Figure 3 shows calibration curves for phenobarbital and propylthiouracil, made using a Coleman Model 11 spectrophotometer. The curves obey Beer’s law Tithin certain limits of concentration and fall off a t varying points of higher concentrstion, depending upon the compound tested. DISCUSSION
Numerous tests with various compounds indicate that the color reaction is rather specific for compounds possessing the -CON-
V O L U M E 2 1 , NO. 1 1 , N O V E M B E R 1 9 4 9 ECO- or -CONHCSgroup, which may be found in aliphatic compounds or aromatic heterocyclic compounds. Some examples of compounds having the -CONHCOgroup and forming a blue-violet color complex with cobalt are: phenobarbital, barbital, pentobarbital, theobromine, phthalimide, alloxan, biuret, and uracil. The red color complex formed with cobalt and the -COSECS- group is evidently a composite color, for the solution has more than one absorption maximum. Propylthiouracil, and thiopental are examples of compounds that contain this group and form the red color complex. Theophylline presents an interesting exception to the above :ouhvlline
1391 was found to give a blue-violet color complex similar to those compounds with a -CONHCOgroup. It is supposed that the functional group is formed with atoms 2, 6, and 7 to make the same complex as is formed with uracil and similar compounds. This reaction will find application in various fields of chemistry. particularly in biological and pharmaceutical chemistry. LITERATURE CITED
(1) Cohen, E. L., A m . J . Pharm., 118,40(1946). (2) Dille, J. M., and K o p p a n y i , T., J . Am. Pharm. Assoc., 23, 1079 (1934). (3) Green, M .W., Veitch, F. P., a n d K o p p a n y i , T., I b i d . , 32, 309 (1943). (4) Linegar, C. R., Dille, J. M., and Koppanyi, T., I b i d . , 24, 847 (1935). (5) M a t t s o n , L. N., a n d Holt, W. L., I b i d . , 38,55 (1949) (6) Merley, R. W., A m . J . CZin. Path., 18,906 (1948).
RECEIVED April 4 ,
1949.
Determination of Calcium on Soil Extracts and Plant Ash by Chloranilic Acid Compensating Errors Caused by Presence of Magnesium and Iron NATHAN GAMMON, J R . , A S D R. B. FORBES Florida Agricultural Experiment Station, Gainesville, Fla. Colorimetric determinations of calcium in soil extracts and plant ash by the chloranilic acid procedure are subject to error due to the presence of iron and magnesium. These errors may be compensating, as iron increases and magnesium decreases the intensity of the chloranilic acid color. Magnesium and iron cause additional errors by inhibiting precipitation of calcium chloranilate. A corrective procedure for use when all samples of a group have nearly the same magnesium and iron content is suggested.
T
H E authors have noted that the chloranilic acid colorimetric procedure for determination of calcium as proposed by Tyner ( 7 ) frequently gives results that are a t variance with standard oxalate procedures ( 1 ) by from 10 to 25% in analysis of soil extracts and plant ash. Investigation has shown that these errors were due to the presence of magnesium and/or iron and that the error may be positive or negative, large or small, dependIng on the relative concentration of these elements. Additions of magnesium reduce the intensity of the chloranilic acid color (Figure l), and prevent calcium precipitation (Figure 2). As reported by Tyner (Y),additions of ferric iron to chloranilic acid increase the intensity of the color (Figure 1) and also prevent precipitation of calcium (Figure 2). It may also be noted ln Figure 2 that the effects of iron and magnesium in depressing calcium precipitation are additive. Data for Figure 2 were obtained by additions of magnesium sulfate, ferric chloride, and magnesium sulfate plus ferric chloride to solutions containing 0.786 mg. of calcium. The calcium chloranilate was then precipitated by Tyner’s procedure ( 7 ) . This precipitate was dissolved and the calcium determined by reprecipitation as oxalate, using a volumetric micro procedure ( 1 ) . These points were also verified by washing the calcium chloranilate precipitates with dilute (25 p.p,m.) chloranilic acid, transferring the precipitate and filter paper to a beaker containing 50 ml. of
0.1 S hydrochloric acid, dissolving the precipitate and, after filtering, reading in the photometer. A straight line following Beer’s law was obtained over the range from 0 to 1.5 mg. of calcium in solutions where iron and magnesium were omitted. The inhibiting effects of iron and magnesium shown in Figure 2 were also noted for other concentrations of calcium. The effect of magnesium is contrary to the report of Tyner ( 7 ) . who stated that magnesium is coprecipitated or occluded in the calcium chloranilate precipitate. This was proved incorrect by spectrographic analysis of a calcium chloranilate precipitate produced in the presence of 1.25 mg. of magnesium. Rccovery of magnesium in the calcium chloranilate precipitate containing 0.629 mg. of calcium T ~ ofS the magnitude of 0.005 mg. of magnesium. Even this small amount is probably the result of incomplete washing of the filter paper and precipitate. Undoubtedly the lighter color of chloranilic acid caused by the presence of magnesium led Tyner to the erroneous conclusion that magnesium was coprecipitated. The possible errors which could be caused by small amounts of iron or magnesium and horv they may compensate for each other are clearly shown in Figure 3. Variations shown here are within the range of concentrations that might be expected in using the chloranilic acid procedure for calcium determination in plant tissues (2, 6). Greater variations are found in soil extracts. Plant
