the results of which are shown in Table VI. I n each experiment 0.50 ml. of 4.OM selenic acid was mixed with a given volume of 30% hydrogen peroxide before a given volume of 6% sulfurous acid was added. I n parallel experiments without hydrogen peroxide, the reaction was so fast that it could not be measured with a stopwatch. The inhibiting effect of the hydrogen peroxide is obvious. Similar experiments with mixtures of selenic and selenious acids of various concentrations showed that hydrogen peroxide always inhibits the reaction, often to such a n extent that the result is negative when the concentration of the selenic acid, the selenious acid, or both, is such that it
should be strongly positive. The data in Table VI also show that when the concentration of hydrogen peroxide is Iow enough, the test will give a positive result even when no selenious acid is present. For the purpose recommended by Gilbertson and King this test is therefore wholly unsuitable. LITERATURE CITED
(1) Benger, E. B., J . Am. Chem. SOC.39,
2171 (1917). (2) Berzelius, J. J., Acad. Handl. Slockholm 39, 13 (1818). (3) Gilbertson, L. I., King, G. B., J . I m . Chem. SOC.5 8 , 180 (1936). (4) Hillebrand, W. F., Lundell, G. E. F., Bright, H. A., Hoffman, J. I., “Applied Inorganic Analysis,” 2nd ed., p. 334, Wiley, New York, 1953.
(5) Mellor, J. W., “Comprehensive Trea-
tise on Inorganic and Theoretical Chem-
istry,” Vol. 10, p. 751, Longmans,
Green, London, 1930.
(6) Mitscherlich, E., Ann. physik. Chem. 9., 629 - - 11827). \ (7) Muller, E.,‘Z. physik. Chem. (Leipzig) 100, 346 (1922). ( 8 ) Rose, H., Ann. physik. Chem. 113, 472
(,1- A-6 -l , ). (9) Wenger, P., Duckert, R., eds., “Reactifs pour l’halyse Qualitative MinBrale Recommand& par la Commission Internationale des Reactions et Reactifs Analytiques Nouveaux de 1’UnionInternationale de Chimie,” 2nd Rapport, p. 52, Basle, 1945. (10) White, H. C., Ph.D. thesis The Ohio State University, Columbus, 6hio, 1950. RECEIVED for review September 23, 1959. Accepted February 29, 1960. Taken from the M.S. thesis submitted by Clayton L. Henderson to The Ohio State University,
1955.
Co I o rimetric Reactio ns of 3,5- Dinitro-0-toI ua mide and Related Compounds with Aliphatic Diamines GRANT N. SMITH AND MARLENE G. SWANK Agriculfural Chemical Research, The Dow Chemical Co., Midland, Mich. )The colorimetric reactions of 3 3 dinitro-o-toluamide and related dinitrobenzamides with aliphatic diamines are described. These reactions can b e used to estimate and distinguish the various dinitro compounds which give a postive test with this reaction. The factors influencing the formation of colored complexes by reaction of 3,5-dinitro-o-toluamide with various diamines and organic solvents are elucidated.
of the color. In initial investigations @), only the straight-chain primary aliphatic monoamines formed colored complexes with 3,5-dinitro-o-toluamide. The presence of a second functional group such as a hydroxyl or amino group appeared to stabilize the complex. A series of di-, tri-, and polyamines was, therefore, investigated to ascertain if stable complexes could be formed with 3,5-dinitro-o-toluamide. EXPERIMENTAL
T
of 3,5-dinitro-o-toluamide with methylamine in the presence of dimethylformamide has been used as a basis for a colorimetric procedure for the assay of 3,5-dinitro-otoluamide ( 2 , S ) , a drug (Zoalene, registered trade-mark of The Dow Chemical Co.) used to treat coccidiosis in chickens. This reaction has several advantages. The colored complex formed has a yery intense color which permits the detection of microgram quantities of the drug. The complex also exhibits an absorption peak at 550 mpL,a portion of the visible spectrum in which most of the naturally occurring pigments do not exhibit absorption bands. The test is also specific for 3,5-dinitro-o-toluamide in the presence of its possible metabolic degradation products. The one major disadvantage of the reaction is the evanescent properties HE REACTION
978
ANALYTICAL CHEMISTRY
To ascertain the influence of various diamines on the production of color complexes, a solution of 3,5-dinitro-otoluamide in dimethylformamide was mixed with an equal volume of amine solution. The absorbance of the colored solution was then determined in a Cary recording spectrophotometer Model 14 in the region from 350 to 650 mp. All solutions were maintained a t 20” C. and read as soon as possible after the color had been developed. The various factors influencing the intensity and stability of the colored complex were investigated by varying each factor independently. RESULTS A N D DISCUSSION
Influence of Various Diamines on Color Formation. The various amines shown in Table I gave a positive test in the color reaction. Of all the amines tested, only those compounds containing a primary
amino group produced colored complexes. Most of the higher molecular weight polyamino compounds were unsatisfactory for this test because they were solids or had a high visc osity . In general, the diamines were the most satisfactory reagents for the development of color. In this series, greater color intensity was obtained with diamines in which the amino groups were attached to the terminal carbons than with compounds in which one amino group was located along the chain. Most of the amines gave complexes
Table I. Comparison of Absorbance Readings for Colored Complex Formed with 3,5-Dinitro-o-toluamide, Dimethylformamide, and Various Amines
Absorbance at 560 Mfi (Corrected for Reagent Blank) 0,565 0.280 0.635 0.515 0.605 0.573 0,545
Amine 1,2-Ethanediamine 1,2-Propanediamine 1,3-Propanediamine 1,3-Butanediamine 1,4-Butanediamine 1,5-Pentanediamine 1,bHexanediamine 3,3 ’-Iminobispropylenediamin le 0.583 Triethylenetetramine 0.493 Methylamine 0.540
.. 350
Figure 1 .
400
450
550
500
€00
650
700 350 WAVE LENGTH IN MU
400
450
500
550
600
650
100
Spectral curves of colored complexes leff.
Formed with 3,5-dinitro-o-toluamide, dimethylformamide, and various amines Triethylenetetramine D. 1,2-Proponediamine E. 1,5-Pentanediomina E. 1,2-Ethanediamine C. 1,3-Propanediomine
A.
Right.
Formed with 1,3-propanediamine, dimethylformamide, and various dinitrobenzamides and dinitrotoluamides A. 2,4-Dinitrabenrarnide G. 3,5-Dinitro-p-toluamide E. 3,5-Dinitrobenramide H. 2,6-Dinitro-p-toluamide C. 2,5-Dinltrobenzamide 1. 4,6-Dinitro-m-toluamide D. 3,5-Dinitro-o-toluamide J. 3,5-Dinitro-o-toluoyI chloride E. 4,6-Dinitro-o-toluamide K. 3,5-Dinitro-o-toluic acid F. 4.5-Dinitro-o-toluamide
which exhibited two absorption peaks, generally in the 350- to 450- and the 500- to 600-mp regions. Most of the complexes gave similar absorption curves in the 500- to 600-mp region, but there was considerable variation in the absorption curves in the 350- to 450-mp region (Figure 1). Maximum color intensity was obtained with the 1,3-propanediamine complex in the 560-mp region, while the triethylenetetramine complex gave maximum color in the 400-mp region. The 3,3’-iminobispropylamine, 1,4-butanediamine, 1,3-butanediamine, and 1,6-hexanediamine gave spectral curves similar to that of lI3-propanediamine. Influence of Amine Concentration on Color Formation. The influence of amine concentration on color formation was studied by varying the concentration of dimethylformamide and amine while keeping the concentration of 3,5-dinitro-o-toluamide constant. For comparison, the color intensity of the various complexes was determined a t 560 mp (Table 11). The intensity of the color increased as the concentration of amine was increased, reaching a maximum with about 55 to 65% amine. Beyond this point there was a very slight change in color with an increase in diamine concentration. A colored complex was obtained by reaction of the diamines with 3,5-dinitro-o-toluamide without dimethylformamide. Apparently dimethylformamide is not essential for color formation, but acts as a solvent for the colored complex. Stability of Colored Complexes. When primary aliphatic monoamines
were used t o develop colored complexes n-ith 3,5-dinitro-o-toluamide, i t iyas difficult t o determine the absorbance of the solutions because of the evanescent properties of the color. This lack of stability was associated with the low boiling point of the amines, poor solubility of the amines in dimethylformamide, and the necessity of using aqueous solutions. Water tends to quench the formation of a colored complex. Because most polyamino compounds have considerably higher boiling points than monoamines, it seemed likely that the complexes formed with the polyamino compounds would be more stable. Results indicate that all the complexes formed with diamines are stable. In most cases only a slight decrease in color could be noted during an hour’s observation a t 2 O O C . At temperatures below freezing, it has been possible to maintain the colored complexes for as long as 8 weeks. Complexes formed with the diamines containing two terminal amino groups were more stable than those formed from diamines containing only a single terminal amino group. Because of the greater stability of the 3,5-dinitro-otoluamide-diamine complexes, diamines, especially 1,3-propanediamine, should be used to form the colored complexes. Influence of Solvents on Color Formation. I n investigating the metabolism of a n aryl dinitro compound in biological systems, i t is often necessary t o isolate the compound from the tissues by various solvent extraction procedures. If i t were possible t o carry out the color
reaction used t o identify the dinitro compound in various organic solvents, considerable time and effort mould be saved in tracing the compound through the extraction procedure. Table I1 shows a series of solvents which were investigated to ascertain if the colorimetric procedure for 3,5-dinitro-o-toluamide could be performed in other solvents besides dimethylformamide. The considerable variation in the response obtained depended on the amine uwd to develop the colored complex. Acetone, alcohol, and tetrahydrofuran gave poor results as far as color intensity was concerned. Dimethylformamide, acetonitrile, and pyridine gave the best results. Maximum color intensity was obtained with a combination of dimethylformamide and 1,3propanediamine. Influence of Contaminants on Color Formation. During investigations on the reaction of 3,5-dinitro-o-toluamide with various amines, i t became apparent t h a t contaminants would influence the formation of colored complexes. For example, small quantities of formic acid which may be present in dimethylformamide might seriously hinder color formation. By studying formation of the colored complex in the presence of formic acid, it would be possible t o speculate as to the susceptibility of the reaction to the presence of small quantities of contaminants. Table I1 illustrates the influence of formic acid. I n all cases, small quantities of formic acid seriously hinder formation of the colored complexes. Those diamines containing a VOL. 32, NO. 8, JULY 1960
979
Table II.
Effects of Varying Factors on Production of Color Complexes of 3,5-Dinitro-o-toluamide
Absorbance a t 560 Mp x 1000 Ethane 1,2-do
CONCN. OF AMINE, yo 5 10 15 20 25 30 35 40 45 50 55 60 65
io
75 SOLVENT Acetone Alcohol Acetonitrile Dimethylformamide Methyl ethyl ketone Pyridine Tetrahydrofuran CONCN.OF FORMIC ACID, % 0
0.5 1.0 2.0 3.0 4.0 5.0 7.5 10.0
128
284 400 460 512 549 573 586 597 608 610 615 626 625 630
Propane I,2-d
Propane 1,3-d
Butane
26 65 113 153 202 242 274 350 330 350 359 386 402 416 425
240 405 510 585 630 665 690 710 735 755 757 765 768
0 0
3,3’-Imino-
1,3-d
Butane 1,4d
Pentane 1,5-d
Hexane 1,6-d
770
143 245 345 390 440 472 495 513 531 545 554 560 562 560 565
163 267 396 479 544 582 598 620 640 660 680 680 680 680 680
100 225 335 425 495 530 560 590 605 625 625 622 625 628 630
100 180 250 305 370 390 458 505 498 558 581 592 632 630 643
233 285 375 450 500 562 580 605 610 620 635 640 645 640 645
142 30 311 526 174 172 39
455 188 533 659 456 392 62
100 32 448 628 220 245 32
292 122 247 575 270 335 93
167 72 505 615 300 306 102
10 34 305 520 188 203
618 445 340 275 222 187 175 105 580
550 280 219 180 100 80 25 6 5
604 539 503 464
537 445 373 297
770
285 195 572 625 397 495 278
106 348 275 90 28
533 365 620 755 580 600 315
605 573 525 458 410 356 312 230 158
345 165 135 95 80 65 45 15 5
750 650 580 520 465 425 380 280 200
540 414 370 265 220 180 146 80 47
685 500 400 375 340 315 275 175 100
625 400 297 255 225 202 90 25
580 320 264 204 190 130 100 54 40
635 560 535 465
365 285 210 80
760 720 665 610
535 435 360 312
650 630 610 280
625 600 558 540
565 555 510 445
1i o
bispropyl- Triethyleneenediamine tetramine 116 188 275 342 402 445 478 500 530 548 579 591 600
600 602
50
CONCN. OF WATER,yo 0 8
16 25 d
(I
=
diamine.
single terminal amino group were influenced more than the corresponding diamines containing two terminal amino groups. The absorbance reading obtained with the 1,2-propanediamine complex was reduced 52% by the addition of 0.5% formic acid, while the reading of the 1,3-propanediamine complex was reduced only 13YG. I n addition, the longer the carbon chain, the more the color reaction is suppressed by formic acid. Water will also influence formation of the color complex (Table 11). Here again diamines containing two terminal amino groups appeared to be influenced less by the presence of water than those containing only one terminal amino group. Comparison of Spectral Curves Obtained with Various Dinitrobenzamides and Dinitrotoluamides. Because complexes formed with 1,3propanediamine are stable, this reaction was investigated to determine the possibility of using i t for the identification and determination of 980
0
ANALYTICAL CHEMISTRY
dinitrobenzamide and dinitrotoluamide isomers. The spectral curves obtained with t h e various isomers are illustrated in Figure 1. In the dinitrobenzamide series, both the 2,4- and 3,5-dinitrobenzamides exhibit characteristic absorption peaks in the 500- to BOO-mp region. The 2,4-dinitrobenzamide exhibits peaks a t 355 (0.730 absorbance unit) and 550 mp (1.150 units) while the 3,5-dinitrobenzamide exhibits peaks a t 380 (0.910 unit) and 540 mp (1.090 units). It is, therefore, possible to identify these two isomers of dinitrobenzamide by their characteristic absorption peaks. The other dinitrobenzamide isomers did not give a positive test m-ith the 1,3-propanediamine reaction. I n the dinitrotoluamide series, 3,5dinitro-o-toluamide, 4,6-dinitro-o-toluamide, and 4,6-dinitro-m-toluamide formed colored complexes with 1,3propanediamine which exhibit absorption peaks at 550 to 600 m p . The 3,5dinitro-o-toluamide and 4,6-dinitro-o-
toluamide complexes have about the same color intensity at 560 mp (0.800 absorbance unit). The 3,5-dinitro-otoluamide complex exhibits a secondary peak a t 390 mp (0.660 absorbance unit) , while 4,6-dinitro-o-toluamide has a secondary peak a t 355 mp (0.580 unit). 4,6-Dinitro-m-toluamide has two absorption peaks, a t 625 (0.310 unit) and 410 mp (0.460 unit). Other isomers of dinitrotoluamide do not exhibit absorption peaks in the 550- to 600-mp range. Of the possible isomers of dinitrobenzamide and dinitro-o-toluamide, only five isomers give a positive test with the 1,3-propanediamine-dimethylformamide reaction. Because of the differences in absorption peaks exhibited by complexes formed from these five isomers, it is possible to distinguish them using this color reaction. In most biological investigations only 33dinitrobenzamide and 3,5-dinitro-o-toluamide will be encountered, as they are used as anticoccidial drugs. These two
compounds can be distinguished on the basis of their spectral curves. The results show that diamines will form more stable complexes n i t h 3,5dinitro-o-toluamide than did the monoamines. I n general, the complexes formed with those diamines containing two terminal amino groups are less susceptible to reaction conditions and are more intensely colored than those formed with other amines. Of the various diamines studied 1,3propanediamine gave the most intensely colored complex. Past use of this diamine has been limited because of its price and availability. However, recently it has become conimercially available in large quantities a t a low enough price t o permit its use in routine analyses. Caution should be taken in handling
these diamines. Most of the shortchain diamines are liquids which tend to fume and give off heat when mixed with small quantities of water. The diamines also react with carbon dioxide in the air to form a white precipitate which may present a problem in maintaining a solution free of insoluble particles. With proper precautions no difficulties have been encountered with the use of lJ3-propanediamine. The use of lJ3-propanediamine in place of the methylamine in the colorimetric procedures previously described for the determination of 3,S-dinitro-otoluamide has several advantages. The complex it forms is more stable and less susceptible to reaction conditions than was the methylamine complex. It is possible to obtain a complete spectral curve without the complicating
factor of color fading. I n addition, a somewhat more intensely colored solution is obtained with the diamine. Since the completion of this work, it has come to the attention of the authors, that XIarshall ( 1 ) has been using ethylenediamine in place of the methylamine previously recommended. LITERATURE CITED
(1) Marshall,
C. V., Department of Agriculture, Ottawa, Canada, private communication, January 1959. (2) Smith, G. N., ANAL. CHEM.32, 32 mfin) (3) Smith, G. N., J . l y r . Food Chem. 8, 224 (1960). \ ~ . _ _ , .
RECEIVED for review January 21, 1960. Accepted April 29, 1960.
Fluorometric Determination of Traces of Selenium J.
H. WATKINSON
Rukuhia Soil Research Station, Department o f Agriculfure, Hamilton, New Zealand
b A fluorometric method using 3,3’diaminobenzidine has been developed for determining as little as 0.02 pg. of selenium in plant and other materials.
STIL RECEKTLY biological interest in selenium has been concerned with toxicity, and determinations in animal tissues, herbage, and soils have been of comparatively macro amounts. Hoi\erer, with the finding, for example, that as little as 1 mg. of selenium per week, corresponding to about 0.1 p.p.m. of selenium in the feed, can control “white muscle” disease in lambs and markedly increase low growth rates in mature lambs (hoggets) under certain conditions ( 6 ) , research has demanded a more sensitive method of analysis. The most sensitive method in current use (apart from neutron activation) is that described by Cheng ( 2 ) , in which the absorbance of selenadiazole formed from 3,3’-diaminobenzidine in toluene is measured. Although this permits the determination of amounts as low as 0.2 pg. of selenium, it is still above the required range. Because selenadiazole formed from 3,3’-diaminobenzidine fluoresces strongly and would allow the measurement of selenium in amounts
