ANALYTICAL CHEMISTRY
1494
APPLIC4TIOY OF T H E METHOD
Table 11. Analysis of Commercial Products Spread of Spot Test Met hod. Zirconium, Reagent
Fe-Zr-.il alloy Cupierron Mandelic acid p-Bromomandelic acid m-Azo-beta-naphtholmandelie acid Zirconium sulfate liquor p-Bromomandelic acid m-Azo-beta-nanhtholniandelic acid
%
%
(Four Analyses)
46.44 47.59 47.06 16.47
2 5
0.40 0.36
0.04
0 05 0.04
0.01
Effect of Acidity. Spots containing 0.05 mi. of the reagent solution and 0.05 ml. of the zirconium solution (10 micrograms of zir. conium in 1 to 6 A- hydrochloric acid) were prepared and allowed t o stand 15 minutes. The excess solvent was then removed and the p a p m were dried in an oven. Each spot 4 a s then leached with methanol. The spots which were 1 S and 2 1\' in hydrochloric acid 11 ere slightl) more intense. Solutions 2 Ai in hydrochloric acid were selected as the optimum acidity because more foreign ions would be soluble a t this concentration. rlcid of this concentration did not harm the confined spot test papers. Higher concentrations were likely t o decompose the excess reagent during the drying and could also affect the paper. Effect of Cosolutes. Spots Containing 0.05 ml. of the reagent solution and 0.05 ml. (2 S in hydrochloric acid) containing 10 micrograms of zirconium and 100 micrograms of iron, aluminum, tin, copper, barium, calcium, bismuth, antimony, cerium, thorium, vanadium, titanium, chromium, cadmium, and magnesium, respectively, were prepared. After drying in a forced draft air oven a t 60' C. for 5 minutes and leaching with methanol it was found that the spots were of approximately the same color intensity as that of a 10-microgram spot prepared with pure zirconium solution. The only exception was vanadium which interfered through destruction of the reagent. Solutions containing above 0.01% of vanadium(V) ion n-ere found t o interfere with the determination. Effect of Sulfuric Acid. Spots were prepared containing 0.05 ml. of the reagent solution and 0.05 ml. ( 2 S in hydrochloric acid) containing 10 micrograms of zirconium and varying concentrations of sulfuric acid. A sulfuric acid content up to 0.4% by weight does not interfere with the spot test.
An iron-qmonium-aluminum alloy, a commercial zirconium sulfate liquor, and a chromium low alloy steel mere run by the foregoing spot test procedure. Iron-Zirconium-Aluminum Alloy. Approximately 0.20 gram of the finely ground sample was placed in a 150-ml. beaker and treated with 15 ml. of concentrated hydrochloric acid. The beaker was covered with a watch glass and the mixture was boiled until the sample dissolved (approximately 1 hour). The solution n a s diluted with 50 ml. of distilled water, filtered into a 100ml. volumetric flask, and brought up to the mark with distilled water. T h e percentage of zirconium in the alloy could be closely estimated by spotting different dilutions of the test solution and h of by comparinj the intensities of the resulting spots ~ i t those the standard scale. Zirconium Sulfate Liquor. The acidity of the solution \\as adjusted until it was approximately 2 in hydrochloric acid. The solution a a s then diluted with 2 S hydrochloric acid until the spotting procedure shop ed the concentration of the solution t o be in the 0- to 10-microgram region. Chromium Low Alloy Steel. One gram of the steel was dissolved by prolonged boiling with concentrated hydrochloric acid in a 50-ml. beaker. The solution was taken t o dryness on a steam bath. The residue JTas then dissolved in 10 ml. ot water and filtered. The determination was carried out by the spot test procedure as previously described. The results obtained by the above procedures as compared with those yielded by other methods are shown in Table 11. LITERATURE CITED
Brode, W., and Adams, R., J . Am. Chem. Soc., 48, 2202-6 (1926). Feigl, F.,"Manual of Spot Tests," p. 251-9, S e w York, d c a demic Press, 1943. Fredga, A , , and Andersson, E., Ai-!&. Kemi,Mineral. Geol., 14B,S o . 38 (1941). Gavioli, G., and Traldi, E., M e t . ital., 42,179-81 (1950). Hahn, R. B., AXIL. CHEY.,21, 1579 (1949). HeIler, G., Ber., 46, 3976 (1913). Kumins, C. A,, AVIL. CHEM.,19,376 (1947). Oesper, R. E., and Klingenberg, J. J., Ibid., 21, 1509 (1949). Porter, C., and Ihrig, H., J . A m . Chem. SOC.,45, 1990 (1923). . 9, 79 (1937). Yagoda, H., IND.ENG.CHEM.,- ~ N A L ED., RECEIVED for review February 5 , 1952. Accepted June 7, 1952. Abstracted in part from a dissertation submitted b y Raymond A . Dunleavy, to the Graduate School of t h e University of Cincinnati, in partial fulfillment oi the requirements for the degree of h1.8.in chemistry, June 1951.
New Color Reaction for Steroids with Perchloric Acid HENRY TAUBER Venereal Disease Experimental Laboratory, U . S . Public Health Service, School of Public Health, University of North Carolina, Chapel Hill, 11'. C.
T has been reported that tryptophan (and tryptophan in
I untreated proteins) is converted by perchloric acid, a t room temperature, to a yellowish green fluorescent compound (6).
tion spectra of certain steroids after shaking solutions of these steroids in chloroform for 5 minutes a t room temperature with perchloric acid.
A trace of dichromate increases the sensitivity of the test (6). Some samples of perchloiic acid fail to give the test without dichromate. This reaction has been applied t o the quantitative estimation of tryptophan ( 2 ) . Tryptophan and the pigment produced from it are insoluble in chloroform and acetone. Steroids should be removed from biological materials prior to the application of the test. Carbohydrates form colored condensation products when heated ~ i t htryptophan for 10 minutes a t 100" C. in 30% perchloric acid ( 1 ) . In the tryptophan test, and in the reaction described, boiling is not an essential factor. Schaltegger ( 4 ) heated certain steroids and aldehydes in the presence of perchloric acid and obtained colored condensation products. Lange and associates (3) obtained different absorp-
COLOR REACTIOh FOR STEROIDS
Chloroform (0.5 ml.) containing 0.5 nig. of a steroid was placed in a test tube and in a second tube was placed 0.5 ml. of chloroform alone, to serve as a control. To each tube, 1 ml. of perchloric acid (c.P., 70 t o 72%) was added. T h e tubes were stoppered with aluminum-covered cork8 and their contents were mixed. They 15 ere placed in a water bath a t 56' C. for 10 minutes and were shaken occasionally. Then both layers were observed for color and for fluorescence. One milliliter of distilled water wap added and, after gentle mixing, the appearance of color, if any, was noted. After thorough mixing the colors were noted again. The tubes were returned to the water bath and were shaken occasionally. After 2.5 hours they were removed once more and examined for color and fluorescence. The colors were stable for
V O L U M E 2 4 , N O . 9, S E P T E M B E R 1 9 5 2 Table I. HI. Wile Br. Brown BK. Brick red Ca R . Carinin red Cr. Crimson cr R , ~~i~~~~~ red
1495
Color Keactions of Steroids with Perchloric Acid D BI. D Or.
Deep Blue Deep Orange
Gr, Green G r Y . Greenish yellow
Or. Orange Or r e . Orange vermillion P. Pink Pu. Purple ' R R . Rose red
L B1.
Sc.
fl. Fluorescence
Light blue
Ultraviolet
U.V.
Violet
1'.
T. Yellow Xone
0.
Scarlet
Color after Color after Addition o f 1 Color after 10 hfin. a t A l l . of V a t e r , Color after 2.5 Hours a t 5G0 C. and XIixing Mixing 5Go C. ChloroChioroChloroChloro.icid form Acid form Acid form Acid form layer layer layer layer layer layer layer layer
Compound
0 Gr Y
Y
0 Gr 0 Y
0 0 0 Y
0 Y P Or
0 L B1 Y B1
0
Yfl D BI 0
0 D BI 0
P
Pu
B1
0 D B1 0 0 0
0 D B1 0 0 Cr
Y 0 Yfl Yfl 0
Br 0 B R Y 0 Gr Y 0 P 0 P P Y 0
Br 0 Or Y Y 0 Gr P 0
10 Cr Y 0 0
Y
Y
R R Yfl
T fl Yfl
onate Dehydroisoandrosterone acetate Progesterone Pregnenolone 11-Desoxycorticosterone 17-Hydroxycorticosterone 21acetate Cortisone (alcohol) Estrone Estradiol Estriol Estriol benzoate Vitamin Ds, crystalline Diethylstilbestrol Indol Skatol Catechol Resorcinol Pyrogallol Ergosterol Cholic acid
0 Y 0 Y
T Br fl
T T T fl
0 Yfl 0 Or 0
I'
P Y Y Y Y
Yfl
Y
1-
T
Br Or 0
T
0 0
O Br Y
1-
Y
Y
Gr Gr 0
1-
1-
0 0 0 Br
0 0 0 Br
T
a few hours, Some of the colors faded when the tubes were cooled to about 3" C. but their brilliancy reappeared at room temperature. The two layers were dissolved in j ml. of acetone. Most, of these solutions showed int,ense fluorescence under ultrayiolet light. In Tatile I are recorded the colors given by a series of steroids
.\liter Addition of 5 RII. of Acetone, and Mixing Under I n day- U . V . light light
0 Ca R
RR
0 SC R R
L BI L BI 0 0 Y
Br D BI Br
P D BI n
Y
V Br
R R
Y
Y R R
Ca R
1-fl
Y
0
0 Or ve
0
0
0
Bl 0
0 D Or P 0 P 0 0
Cr 1-
u
Pi!
Pfl 0
7-
Y 0 0 0 0 D Br
Y
I'
0 0 0 0 Br
0 0 0 0
T
I'
1-
0
P fl
Or fl Yfl
Y fl B1 fl Yfl Yfl D Or fl Yfl 0 Y fl Yfl 0 Yfl 0 0
Y fi
0 0 0 0 0 0
and a few other compoundt'he uerchloric acid test. The test' s h o w color differences even hetrveen closelj. related compounds. Cortisone acetate gave no color. I t i-. hoped that, with the eliniiiiation of interfering sutwtances arid the application ot proper optical controls, the new mior reaction u-ill be useful for the quantitative e s t i m a t i o n of st,eroids in biologic materials and in reaction mixtures. %CKNOWLEDG51EFT
The steroids employed in this investigation n-ere kindly iurnished by Chemical Specialties Co., Merck and Co., Inc., Schering Corp., and the Cpjohn Co. LITEKITLR E CITED
C'ohen. S ,
\ , J . B d . Chem., 156, 091 (1914). (2) Gordon, M., and Mitchell. H. K., Ihid.. 180, 1005 (1949). (3) Lange, IT.,Folzenlogen, R. G., and Kolp, D. G., ,J. A m . C h e m
(1)
Sol., 71, 1733 (1949).
Schaltegger, H., Helz.. Chim. dctcc, 29, 285 (1946). ( 5 ) Tauber, Henry, J . Ani. Clzem. SOC.,70, 2615 (1948). ( 6 ) Tauber, H., J . Biol. Chem., 177, 337 (1949). (4)
RLCEIVED
for review xarFi1 6, 1952.
Accepted June 7 , 1932
Alkalimetric Method for the Estimation of Cadmium and Zinc MAHADEO M. TILLU' Department of Chemistry, Ramnarain Ruia College, Bombay, India ( 3 )have given a method for the estimaRAYtionandof Das-Gupta mercuric oxide using sodium thiosulfate as the reagent. Double salts of cadmium and zinc: K2S203 CdSz03 HzO and K2S203ZnSzOs H,O, have been reported by Fock and Kluss ( 1 ) and by Raseinheim and Davidson ( 2 ) . I t u a s therefore thought interesting to t r y to extend the use of sodium thiosulfate t o the estimation of cadmium and zinc, t o replace potassium iodide which \$as previously used (4,5 ) . It has been found that cadmium hydroxide and basic zinc carbonate completely dissolve in sodium thiosulfate, in the cold, u i t h the liberation of an equivalent amount of alkali, and the formation of double salts. The reactions involved may be represented as follows:
+ +
+ + +
Cd(0H)t 2Na&O3 % i a ~[Cd(S203).] 2 S a O H 3Zn(OH)z.2ZnC03 10 NaXS203@ 5?;a2[Zn(Sz0,)1] ZKanCOa 6NaOH
gradually as possible. This brings about a progressive react ion b e t m e n sodium thiosulfate and the unreacted cadmium hj-dro\ide and basic zinc carbonate. EXPERIMENTAL
Estimation of Cadmium. Cadmium hydroxide was pi"ipitt~ted from an aliquot of calcium chloride stock solution by adding a slight excess of sodium hydroxide. To free it from chlorides and escf'ss sodium hydroxide, the precipitete was xashcd wit 11 hot water in a rintercd-glass funnel. applying suctioii. Table 1.
1 2 3
Estimation of Cadmium L-sing Sodium Thiosulfate
0 IOfi'I 0 1601 0 2138
0 1072 0 1608 0 2144
.4 slight excess of sodium thiosulfate does not interfere Kith the titration of the liberated alkali. As the suspension of cadmium hydroxide and basic zinc carbonate does not dissolve at first, titration of the liberated alkali, Rith 0.1 N hydrochloric acid, should be carried out as 1 Present address, ' Sahas," Plot KO. 21. East Sion, Bombay, India.
1 2 3
- 0 18 -0 37 -0 28
-0
?I
_ _
~__ Estimation of Z i n c ( ' s i n g Sodium Thiosulfate ~~
Table 11.
-0 3 - 0 fi -0 6
0.1149 0 2298
0,3447
~
0.1142 0.2286, 0.3446
-0 7 -1.2 -0.1
-0.60 -0 50 -0.03
-0.38
.
.
