centrations were made from the corresponding acetates or nitrates. A conventional H-cell was used with a saturated calomel electrode as reference. Solutions were purged with nitrogen before the polarogram was recorded.
Table 1. Effect of Silver Ion Concentration on Sulfur Current Ag +,Moles/Liter Sulfur Wave," x 104 pa. 0 1.90 1 1.50 2 1.20 4 0.70
(1
6 Sulfur concentration-0.20
DISCUSSION
0.30 mmole/liter.
The effect of silver ion concentration on the sulfur wave was examined in
Table II. Effect of Metal Ion-Sulfide Interaction on Polarographic W a v e Heights
Metal Sulfur only Cd +* Cd +2 c u +2 Hg +2
Pb +*
Eiiz
S.C.E., Volts
US.
Concn., Mmole/Liter
-0.58
-0.65 -0.65 -0.04 >O
-0.35 -1.25
0.20 0.39 0.39" 0.50"
0 . 24a 0.22a
0.22 Zn + 2 -1.25 0.22" Sulfur concentration--0.20 mmole liter. Zn +2
the precipitation of metal sulfides with sulfide ions produced by reduction of free sulfur. EXPERIMENTAL
Polarograms were recorded with a Sargent Model 20 polarograph. Methanol, 0.lM in either sodium or ammonium acetate and acetic acid, was used as solvent. A stock solution of sulfur in benzene was diluted 20-fold with the methanol solvent t o give solutions 0.20 millimolar in sulfur. Solutions of metal ions of equivalent con-
S o Wave, pa.
M +2 Wave, pa.
1.90 1:90 0.40 0
0.90 1
: !to
2 . 40 0 . 40 2 . 80
1.50 1.70 1.10
detail. Current from silver ion reduction is flowing at zero volts os. S.C.E.; for reduction of sulfur, E l l z = -0.58 volt us. S.C.E. The decrease in sulfur current with iiicrease in silver ion concentration is linear (Table I). A small increase in current at the sulfur potential is still evident when the silver ion concentration is three times the sulfur concentration, which is 1.5 times the amount necessary to react with the sulfide produced. This probably results from differences in the diffusion coefficients (1). When chloride is added to remove the silver, the sulfur wave attains its normal height.
Other metal ions that form insoluble sulfides also d e e t the sulfur wave (Table 11). Zinc and cadmium were the only metal ions studied which were reduced a t a potential more negative than that of sulfur. In these cases, the sulfur wave appears with the expected height, although somea.hat changed in shape; it shows a shoulder and a decomposition potential slightly less negative than usual. The increase in current a t the cadmium potential is very small, while the zinc wave is decreased in height by only 357,. Removal of the elemental sulfur from solution by adding triphenylphosphine or by shaking with mercury restores the cadmium and zinc waves to their usual heights. These effects are attributed to the decrease in the contribution the metal ions make to the diffusion current. The incoming metal ion is precipitated as the sulfide by interaction with a sulfide ion from reduction of sulfur. Sulfur reduction is believed to occur as usual, with the total wave height being lowered as a result of the decreased current from the reduction of the metal ions. LITERATURE CITED
(1) Kolthoff, I. M., Lingane, J. J., "Polarography, 2nd ed., pp. 105-8, Inter-
science, New York, 1952. ( 2 ) Lingane, J. J., Niedrach, L. W., J . Am. Chem. SOC.71, 196 (1949). (3) . . Kemula. W., Grabowski. Z. R.. Roczniki Chem. 26; 266 (1952).' Ethyl Corp. Detroit, Mich.
R. E. HUMPHREY' Research Laboratories
Present address, Sam Houston State Teachers College, Huntsville, Tex.
Spectrophotometric Determination of Androsterones by a Modified Pyridine Procedure SIR: In a new spectrophotometric method for the determination of androsterones in urine, the reagents, pyridine and aqueous KOH, are substituted for ethyl alcohol and alcoholic KOH, respectively. J17endland and Lohmann (3)were the first to substitute pyridine for ethyl alcohol as a solvent in the Zimmermann reaction. They dissolved the steroid in pyridine and allowed this solution to react with solid m-dinitrobenzene and aqueous 0.1N KaOH solution. They eliminated the red color of the blank by adding small amounts of solid boric acid and measured the absorbance a t 585 mp against pyridine or distilled water. 168
ANALYTICAL CHEMISTRY
To avoid the inexact addition of solid reagents, a method is presented in n-hich m-dinitrobenzene is dissolved in pyridine. The reaction is started by adding 2 . 5 5 aqueous KOH; the end product of the reaction is dissolved in a mixture of ethyl acetate and pyridine. EXPERIMENTAL
Reagents. m-Dinitrobenzene solution in pyridine, 10%; 2 . 5 N aqueous KOH solution; mixture of ethyl acetate with pyridine 1 t o 1 (v./v.). Performance. Dissolve t h e steroid in 0.1 ml. of a solution of m-dinitrobenzene in pyridine. Add 0.05 ml. of 2.5-V K O H solution. Place in a water b a t h of 45' C. for 30 minutes.
Dilute with 5 ml. of the mixture of ethyl acetate and pyridine. Measure the absorbance a t 572.5 mp. (Avoid contact with pyridine by aspirating through a rubber tube placed over the pipet.) RESULTS A N D DISCUSSION
T h e n the blue complex, which is formed by this reaction, is dissolved in ethyl acetate, it turns red. When this blue complex is dissolved in pyridine, i t remains blue. I n the mixture of these t x o solvents, both forms of this complex are in balance. -4s this reaction follows Beer's law and no side reaction takes place (as shown by the almost colorless blank), all absorbance curves
Table I. Demonstration That the New Pyridine Reaction Follows Beer’s Law After 5 ml. of the mixture of pyridine and ethyl acetate (1 t o 1) were added to the
colored complex, the colored solution was diluted again with the same 1 t o 1 mixture of pyridine and ethyl acetate rlbsorbanco at 572.5 A l p MeasCalcu- Difference, 6Dilution ured lated 10 0 0,840 1:2 0,421 0:420 +0:2 1:4 0.214 0.210 fl.9 1:8 0.104 0.105 -0 9 1:16 0.049 0.053 +6 i
0.6
Y
0 z
a 0.4 v) 0
m
4
0.2
Table 11. Absorbance Values of Dehydroepiandrosterone and Androsterone
DHEA, Pg. 400
Figure 1 .
500
600
WAVELENGTH IN m y
Absorption spectra of colored complex obtained from urine extract
- Callow reaction
____
New pyridine reaction, ethyl acetate-pyridine in 1 to 2 ratio
20.13 29.13 58.25 58,25 Androsterone, rg
Absorbance at 572.5 M p Calculated Measured for 1 pg. 0 416 0,0143 0.419 0.0144 0.840 0.0144 0.838 0.0144
.
26.03 26 03 52.05 52 03
0.352 0.355
0.680 0.700
0.0135 0.0136 0.0131 0,0134
the Callow reaction (1) arc shown in Figures 1 and 2 . I n the Callow reaction, the reaction end product is disso.ved in 5 ml. of ethyl alcohol, whereas here the reaction end product is diesolved in 5 ml. of an ethyl acetate-pyridine mixture. Absorbance values are given in Tables I and 11. Both tables demonstrate that the react on follows Beer’s lam. The method has the following advantages over the Callow reaction ( 1 ) : better specificity and sensitivity (Figures 1 and 2); elimination of the problem of maintaining the stability of alcoholic KOH (aqueous KOH used here); and greater solubility of m-dinitrobenzene in pyridine than in ethanol (R). LITERATURE CITED WAVELENGTH IN m y
Figure 2. Absorption spectra of colored complex from 43 pg. of dehydroepiandrosterone ___
Callow reaction New pyridine reaction with different ethyl acetate-pyridine ratios
_.____--________._ 1:2 2:l - - - ._5:1 1 :1
_____
cross a t one point, tlic isosbestic point (4).[The absorption curve of the Callow reaction ( 1 ) passcs close to this same point, too.] The isosb(>sticpoint (point of constant extinction) “is an easily recognized criterion of an equilibrium between two absorbing substances with
__ __
overlapping absorption bands.” Its presence shows t h a t “two and o d y ti70 colored species participate in the equilibrium” (4). The procedure was used for the determination of androsterone and dehydroepiandrosterone. Comparisons with
(1) Callow, S . H., Callow, R. K., Emmens, C. IT7., Baochem. J . 32, 1312 (1938). (2), Munson, P. L., Kenny, A. D., in ’ Recent Progress in Hormone Research,” G. Pincus, ed., Vol. IX, p. 142, Academic Press, New York, 1954. (3) Wendland, W., Lohniann, K., Rzochem. 2 328, 420 (1957). (4) Kest, W., in “Technique of Organic Chemistry,” A. Weissberger, ed., Irol. IX, p. 68, Interscience, Sew Tork, 1956. DIETERJ. P O X T I U S ~
?;euro-Endocrine Research Unit \Tillowbrook State School Staten Island, N. Y. Present address, Fairmount Chemical Co., 117 Blanchard St., ?;em-ark, S . J. VOL. 34, NO. 1, JANUARY 1962
169
