conversion of the benzoylglucuronic acid concentration determined b y the naphthoresorcinol reaction t o its corresponding ultraviolet absorption at 232 mp. The problem of interferences in the method is greatly simplified in studies in which hippurate is used rather than benzoate, as this eliminates biological conjugation. This makes the technique extremely useful for clearance studies of kidney function. The nieasurement of the absorption a t 287 mp makes it possible to follolv the effect of small doses of benzoate on the urinari- excretion of uric acid by the kidney. These results vi11 be reported elsewhere along with the liver and kidney function studies. The effects of other components in the urine n-hich might absorb ultraviolet light a t 232 mp h a r e been considered to be negligible, as dilutions of
1 to 200 through 1 to 5000 were used for the spectrophotometric analysis.
(6) Gaffney, G. W.,Schreur, K., Ferrante, N., Altman, K., J . BioE. Chem. 206, 695-8 (1954). ( 7 ) JIoore, S., Stein, W. H., Ibid., 192,
663-81 (1951).
ACKNOWLEDGMENT
The author is indebted to A. J. Quick, AIarquette University, Alilwaukee, Kis., for a sample of benzoylglucuronic acid. Discussions u-ith Hsien Wu, E. B. Carmichael, and J. TT’. Woods are acknon ledged ii-ith gratitude. LITERATURE CITED
(1) .irkin, H., Colton, R. R., “An Outline of Statistical Methods,” Barnes and Noble, S e w York, 1950.
(2) Bergmann, F., Dikstein, S., J . B d . Chenz. 211, 691-6 (1‘354). (3) Dickens, F., Pearson, J., Biochon. J . 48, 216-21(1951). (4) Elliott. H. C., Ph.D. thesis, Cniveieity of Alabama Medical Center. 1956. (5) Ewing, G., Parsons, T., ANAL.CHEN 20, 423-5 (1948).
(8) Praetorius, E., Poulsen, H., Scand. J . Clin. Lab. Invest. 5 , 2’73-80
(1953). (9) Pri.de, J., Williams, R. T., Biochem. J . 27, 1210-15 (1933). J . B i d . Chem. 69, 549-
(11) Teague, R. S., Dept. Pharmacology, Universitr of Alabama Medical Center, personal communication. (12) Cngnade, H. E., Lamb, R., J . Am. Chem. SOC.74, 3789-94 (1952). (13’1 Kall, J. S., ASAL. CHEJI. 25, 950-3 (1953). (14) K u , H., JT-u. D., Federatzon Proc. 11, 314 (1!)52). RECEIVED for review March 30, 1957. .iccepted July 1, l%i. Extracted from the thesis of H. C. Elliott, Jr., presented in partial fulfillment of the requirements for the Ph.D. degree in biochemistry to the 1-niversity of AAlabamahIedical Center.
Spot Tests Based on Nencki Synthesis of Rhodanine (Rhodanic Acid) FRITZ FEIGL and VICENTE GENTIL laboraforio da Producao Mineral, Ministerio da Agriculfura, Rio de laneiro, B r a d Translated b y RALPH E. OESPER, University of Cincinnati, Cincinnati, Ohio
b The color reaction of rhodanine (rhodanic acid) with 1,2-naphthoquinone-4-sulfonic acid i s used as the basis of indirect tests for compounds which can participate in the Nencki synthesis of rhodanine. These include rnonochloro(bromo)acetic acid, thiocyanates, thiourea and its N-monoalkylated or -arylated derivatives. Cyanamide and its salts also can be indirectly detected in this manner by virtue of their ready conversion into thiourea. All of these tests can be rapidly accomplished within the framework of spot test analysis. The limits of identification are within the rnicroanalytical bounds.
R
hodanine (I), as well as the numerous other compounds with a reactive methylene group, forms colored quinoidal compounds in alkaline solution on treatment with 1,2-naphthoquinone-4sulfonic acid (11) which is known as the Ehrlich-Herter reagent ( 2 ) . The practically instantaneous condensation 0
HN-CO
I
I
SC
CHz
‘d (1)
+~
I1 a O , S - ~ = o
c_> (11)
HS-CO
ONa
U
(111)
+ Sa2S03 + 2H20
(1)
yields the water-soluble blue-violet p-quinoidal compound (111) (7’) and permits the detection b y means of a spot test (3) of rhodanine with an identification limit of 0.6 y. During studies of the analytical usefulness of syntheses and methods of formation of various organic compounds (4) it was found that the color reaction cited above permits the sensitive and selective detection of those compounds which can participate in a synthesis of rhodanine. According to h’encki (e), rhodanine results if an aqueous solution of monochloroacetic acid is warmed with ammonium thiocyanate:
+ 2SHdCNS + HzO + 1 + NHiCl + COn + 2KH3 CHQ
CHzClCOOH HK-CO
I
SC
Although this reaction gives only about a 30% yield of rhodanine, even
a brief warming of the reaction mixture is sufficient t o cause color Reaction 1 to occur to a satisfactory extent after the addition of alkali and 1,2naphthoquinone-4-sulfonic acid. Either a slight quantity of monochloroacetic acid or ammonium thiocyanate can be employed in the starting mixture, provided a n excess of the other reacta n t is present. Rhodanine can be formed by Reaction 2 either in aqueous solution or in molten monochloroacetic acid (melting point 63” C.) Alkali thiocyanates can be substituted for the ammonium salt, and monobromoacetic acid for the chloro acid. Thiourea, which is isomeric and tautomeric with ammonium thiocyanate, shows a remarkable behavior. Volhard (8) observed that the socalled mustard oil-acetic acid is produced in aqueous solution by reaction with monochloroacetic acid:
CHzClCOOH HK-CO
I
OC
I
CH,
+ CS(NHz)z + iu”dC1
(3)
‘S/ This product, analogous to rhodanine, contains a reactivemethylene group and consequently is capable of reacting with 1,2 - naphthoquinone - 4 - sulVOL. 29, NO. 1 1 , NOVEMBER1957
1715
ionic acid. The condensation product is orange-red. Thiocyanatoacetic acid, which is tautomeric with mustard oil-acetic acid, gives a n analogous color reaction ( 3 ) . However, if thiourea (melting point 180” C.) is heated to 160’ to 180’ C. with monochloroacetic acid and the cooled melt is then taken up in water, the blue color characteristic of rhodanine appears on the addition of alkaline Ehrlich-Herter reagent solution. The difference in the behavior of the thiourea-monochloroacetic acid system in aqueous solution and in the melt is doubtless due to a shift, in the melt. of the tautomeric equilibrium CS(SHJ2 $?;H4CKS of sufficient degree toward the right so that the reaction with monochloroacetic acid folloJT s the Reaction 2 rather than Reaction 3 n i t h participation of moisture from the air. .\‘-Mono-alkylated or -arylated derivatives of thiourea appeared to react like the parent compound-that is, after fusion with monochloroacetic acid they also yield rhodanine. When melted with monochloroacetic acid, thiosemicarbazide reacts similarly to thiourea. This appears also to be the case for many derivatives of thiosemicarbazide. Because of their possession of an active methylene group, all these compounds give a colored (usually orange-red) product on treatment with alkaline Ehrlich-Herter reagent. According to Baumann ( I ) , cyanamide and its salts can be quantitatively converted to thiourea by evaporation Kith ammonium sulfide: SCSH2
+ (SHh),S+
+
+
+
CS(SH,), 2NH3 ( 4 )
(SH&S 2H20 + CS(SH2)2 Ca(OH)2 2SH3 ( 5 )
SCXCa
+
+
The detection of the thiourea formed by Reactions 4 and 5 through its participation in the synthesis of rhodanine accordingly makes possible an indirect detection of cyanamide and its salts. Dicyandiamide reacts in the same manner as cyanamide. DETECTION OF MONOCHLORO(BROM0)ACETIC ACID
Procedure. T h e test is conducted in a micro test tube. A drop of t h e test solution is treated with one drop of 1% ammonium thiocyanate and evaporated a t 120” C. If basic solutions are t o be tested, they should be carefully taken to dryness after adding dilute hydrochloric acid, before t h e addition of t h e ammonium thiocyanate. After cooling, one drop of t h e reagent solution and one drop of 21V sodium hydroxide solution are introduced. T h e blue or violet color varies with t h e content of monochloro(bromo)acetic acid. T h e Ehr17 16
ANALYTICAL CHEMISTRY
lich-Herter reagent solution is a 0.05% aqueous solution of t h e sodium salt of 1,2-naphthoquinone-4-sulfonic acid (freshly prepared). T h e limit of identification is 5 -/ of monochloro(bromo)acetic acid. Monoiodoacetic acid may be expected to react in t h e same manner; however, no test material is available t o t h e authors. DETECTION
OF
ALKALI THIOCYANATES
Procedure. A drop of the neutral or faintly alkaline test solution is treated in a micro test tube with 1 drop of a 0.5% solution of monochloroacetic acid. T h e rest of the procedure is as described above. Depending on the quantity of thiocyanate present, the color is a more or less intenqive red-violet. The limit of identification is 10 y of ammonium thiocyanate. The procedure given here is not as sensitive as the ne11 known test based on the formation of ferrithiocyanate, but it has the advantage that fluoride or phosphate does not int erf ere. DETECTION OF THIOUREA A N D ITS DERIVATIVES
Procedure. A grain of t h e substance t o be tested or t h e evaporation residue of a solution is brought together with one drop of 10% alcoholic solution of monochloroacetic acid, and evaporated. T h e micro test tube containing t h e mixture is then inimersed in a glycerol bath whose teniperature is gradually brought to 180” C. After 1 to 2 minutes the mixture is cooled and the reacted mass carried through t h e procedure given for monochloro(bromo)acetic acid, but using 0.5% Ehrlich-Herter reagent. A more or less intense violet color appears, t h e shade depending on the amount of thiourea or its derivatives present.
drop of yellow ammonium sulfide solution is added and taken to dryness carefully. Thiourea results. The remainder of the procedure is t h e same as given for thiourea. T h e formation of a violet color signals a positiye response. T h e liniit of itlentification is 6 y of cyanamide. The procedure can be applied to fertilizers to reveal the presence of cyanamide or its calcium salt. .I definit,e thiourea test is given by urea after the latter is evaporated to dryness with ammonium sulfide solution. Therefore, when fertilizers are being examined with respect to a content of calcium cyanamide, bhe presence of urea must be considered. If necessary, the latter can be removed by extracting the sample with ethyl alcohol. The procedure heretofore recommended for the spot test detection of calcium cyanamide and urea in fertilizers ( 5 ) involved evaporation of the sample u-ith hydrochloric acid and heating the residue (guanidine and urea) to 250’ C. ilmmonia is evolved and can be detected by means of Sessler’s reagent. A combination of this procedure with the one described here might be of value in the examinnt.ion of fertilizers. Dicyandiamide hehaves analogously. LITERATURE CITED
(1) Baumann, E., Ber. 8, 26 (187s). (2) Ehrlich, P., Herter, C. X., 2. physzol. Chem. 41, 379 (1904). (3) Feigl, F., “Spot Tests in Organic Analysis,” 5th ed., p. 303, Elsevier,
Kew York, 1956.
(4)Ibid., Chapter 1. ( 5 ) Ibid., p. 459. (6) Nencki, M., J . prakt. C h e w [2] 16, 2 (1876). (7) Sachs, F., Craveri, XI., Bo.. 38,
3685 (1905). (8)T’olhard, J., J . prakt. Chem. [2] 9 , 9 (1874).
The procedure detects 2.5 y of thiourea, 3 y of allylthiourea, 2.5 y of acetylthiourea, 5 y of malylthiourea, 10 y of ethylenethiourea, 10 y of 1,3 diethylthiourea, and 50 y of a-naphthylthiourea. For this case, and also for monochloro (bromo)acetic acid compounds with reactive amino or methylene groups must not be present, as such materials react directly n-ith the Ehrlich-Herter reagent and alkali to give colored products. This blank test should be carried out prior to the actual test. DETECTION OF DICYANAMIDE A N D ITS SALTS
Procedure. A grain of the test material is placed in a micro test tube, or a n appropriate volume of t h e test solution is evaporated therein. A
RECEIVED for review Febriary 27, 1957. Accepted July 12, 1957.
Separation of Bismuth from Uranium Using Thioacetamide Precipitation Correction
-
I n the article on “Separation of Bismuth from Uranium Using Thioacetamide Precipitation” [Stoner. G. A, Finston, H. L., ANAL. CHEJI. 29, 570 (1957)] , reference 4 should read. Williams, C., Miles, F. T., Kucleonics 12, No. 7 , 11-13 (1954).
