RESULTS. $11 the halides tested with the exception of chlorobenzene produced single absorption peaks with maxima in the region of 360 to 375 m,u. There was, however, considerable variation in E BS calculated per mole of alkyl halide used; the results are shown in Table IV. As a check the quaternary salts from pyridine and methyl bromide and methyl iodide mere isolated and used to prepare standard solutions for treatment in the routine way. The result for iYrnethylpyridinium iodide (32,000) agrees with that using methyl iodide itself but the result for iV-methylpyridiniuni bromide (28,000) is substantially lower than that obtained with the parent methyl bromide. I n general the results for the bromides mere higher than for the other halides and i t was noticed that the solutions of the bromides in pyridinealcohol were often yellow before the addition of alkali. I n the one case tested, the quaternary salt itself was not colored; it may be that there has been Borne oxidation to molecular bromine before the usual reaction and that this interferes by producing spuriously high absorption during the final stage. This point has not been further investigated. DISCUSSION
Some light is thrown on the mechanism of the modified Fujiwara reaction by the results of these investigations. The primary stage of the reaction is condensation of khe methyl chloride with pyridine to form N-methylpyridinium chloride. Under the influence of alkali in anaerobic conditions this is converted to the substance or substances absorbing maximally a t 365 mp and this conversion involves the participation of further molecules of pyridine used as solvent. Evidence presented suggests that the a-positions of both the quaternary salt and the solvent base are of importance in the reaction since block-
Table IV.
Use of Other Organic Halides in Standard Procedure
Chlorides TYPO Monohalides Methyl Ethyl n-Propyl
n-Butyl tert-Butyl Cetyl Benzyl Phenyl Dihalides CHzXa CHICHXZ CH2X.CHzX Polyhalides CHX,
cx4
CXSCOOH
E
mp
31,400 365 8,370 365 11,500 370 8,860 365 5,970 365 34,600 365 52,800 375 No peak above 320 mp
Iodides
Bromides ma%.,
Luax.,
rn8x.t
e
mw
e
w
47,600 27,900 112,000
365 365 365
30,200 33,400
365 366
...
... ... ... ... ...
... ... ... ... ... ...
... ... ... *..
...
... ...
-.. ... ...
890 5,510 8,110
370 365 365
33,400
370
16,000
375
20,000
370
...
...
...
4,370 1,074 3,080
3G5 365 365
35,000
365
6,390
370
ing of these positions inhibits the form* tion of the usual product. The reaction is sensitive to the presence of small amounts of compounds containing CYmethyl groups as shown by the inhibition produced by trivial contamination of pyridine with a-picoline. The results for different organic halides may be complicated by the fact that they were all investigated under the standard reaction conditions and that there may well be differences in ease of reaction which would be ignored by this procedure. The result for any individual halide may possibly be improved by investigation along the lines carried out for methyl chloride in this paper. With this proviso, the reaction appears to lead to similar products with a wide variety of groups attached to the nitrogen atom of the initial pyridinium salt and suggests that the group is not involved in any ring formation. The group may well alter the reactivity of the a-position of the quaternary salt thus affecting the further reaction with Solvent pyridine. Under the conditions
... ...
...
... ...
...
...
... ...
...
used, the modified Fujiwara reaction is certainly not specific for methyl chloride and serious interference from other alkyl halides is t o be expected if they are present in comparable amounts. ACKNOWLEDGMENT
The authors are indebted to J. & E. Hall, Ltd., Dartford, England, for a gift of methyl chloride. Gas chromatography was carried out in collaboration with Petrochemicals, Ltd., Irlam, England and with T. H. Quibell, Chemistry Department, Manchester University. We are grateful to I. M. ITa Simpson and J. Gill for able technical assistance. LITERATURE CITED
(1) Allison, V. C., Meighan, hf. Ind. Eng. Chem. 11, 943 1919).
H.,J .
b
(2) Chalmers, J. N. M., illam, A. E., Kench, J. E., Lancet 239, 806 (1940). (3) Fujiwara, K., Sztzber. Abhandl. naturforsch. Ges. Rostock 6 , 33 (1914). (4) Meyer, F. R.,Ronge, G., Angew. Chem. 5 2 , 637 (1939). R~~~~~~~ for review April 7, 1960. Accepted August 8, 1960.
ination o
%so LEWIS J. TWROOP’ Research Division, Syntex, S. A., Mexico,
A method for the determination of ganic selenium compounds in steroids has been developed. Selenium is separated from the organic compound using Raney nickel and is then
1 presentaddress, lIea,j johngon and Go., Evanavilie, Ind.
D. F., Mexico oxidized to selenous acid with concentrated nitric acid. The selenous acid is o-phenylene-
diamine to form 18283-benzoseienodiazo’ which has an absorbance maxim~mQ t 3 3 0 ~ P L The . method i 5 fast, and applicable to 10 to 150 p p m . of selenium.
DETERMINATION of selenium as a contaminant in steroids requires a method applicable to the microgram range. Several methods for the determination of larger amounts of selenium have been reported ( I , 6, 8) and in 1956 Cheng ( 2 ) developed a procedure for the determination of small amounts using 3,3’-diaminobenzidine HE
VOL. 32, NO. 13, DECEMBER 1960
@
8887
Table 1.
Av. Deviation, P.P.M.
P.P. M. CorAbsorbance Selenium rected 0.310 0.305 0.295 0.122 0.117 0.118
0 O
m
0
-
m
0
N
m
0
m
m
T
r
0 m
n
m
0 i
r
0 O
n
Figure 1. Absorption spectra of 1,2,3benzoselenodiazole in 9670 ethyl al-
cohol
hydrochloride. Sawicki ( 7 ) described a test for selenium with a limit of detection of 0.05 cg. but did not report a quantitive study. Mozingo et al. (6) described the hydrogenolysis of sulfur compounds by Raney nickel cata!yst. A similar reaction was used by Wiseman and Gould (9) for the elimination of selenium from various organic compounds. I n this work, the method used was essentially that of Mozingo for the removal of the selenium from the organic molecule. AFPARATUS AND REAGENTS
Gary Model 11 recording spectrophotonieter with 1-em. silica cells. Beckman Model H-2 meter. Ground joint glassware. o-Phenylenediamine, Distillation Products Industries, practical grade. The dihydrochloride was prepared with concentrated hydsochloric acid and crystallized from 9.5% ethyl alcohol. Eelenium dioxide, c.P., purified by sublimrztion. Chioroform, reagent grade.
42.4 41.2 40.0 17.0 16.0 16.2
47.1 45.8 44.5 18.9 17.8 18.0
I
/---I
I
I
.
I
1808
e
ANALYTICAL CWEhllSTRY
1%5
00
300
320
340
WAVE
LENGTH
350
380
y
Figure 3. Absorption spectra of 1,2,3benzoselenodiazole in 98% H&i04
with chloroform. The solution is read at 330 mu against a chloroform blank. A blank determination is run simultaneously on the Raney nickel. The absorbance of the blank is subtracted from that of the sample and the quantity of selenium is calculated using 791 as the E::m, value of the benzoselenodiazol. The factor for conversion of benxoselenodiazol to selenium is 0.432. The quantity of selenium found is multiplied by 1.1 to correct for the incompleteness of the reaction between the o-phenylenediamine and the selenous acid. Micrograms of selenium = abeorbance X 0.432 X 1.1 791 X 4 DISCUSSION
When the selenium was in solution as selenous acid, 3,3'-diaminobenzidine hydrochloride was tried as a reagent ( 2 ) . However, the copper present in the Raney nickel interfered and attempts to complex the copper with (ethylenedinitri1o)tetraacetic acid were unsuccessful. RESULTS
PROCEDURE
Preparation of 1,2,3-Benzoselenodiazol. To 1.11 grams of o-phenylenediamine dissolved in 50 ml. of distilled water was added a solution of 1.08 grams of selenium dioxide dissolved in 20 ml. of distilled water in a 125-ml. Erlenmeyer Zask. The slightly soluble benzoselenodiazol was filtered OE and mashed with cold water; Yield, 1.0 gram, melting point 69 to 73" C. Sublimation of this material yielded 0.9 gram of product, melting point 74" to 76" C.; X,,. 330 mp, log E 4.18. Absorption spectra are shown in Figure 4.
I
k0.4
1 1-
80
I
zk0.8
Determination of Selenium. A sample containing from 20 to 100 pg. of selenium is placed in a 100-ml. roundbottomed flask with a 24/40 joint. Fifty milliliters of ethyl alcohol are added to dissolve the sample and about 300 mg. of Raney nickel (fresh) prepared by the method of Drake (8) are added. The flask is fitted with a condenser and the solution gently refluxed for 30 minutes. The flask is cooled and the ethyl alcohol decanted from the Raney nickel which is then washed by decantation with 30 ml. of ethyl alcohol. The flask is fitted with an air condenser and 4 ml. of nitric acid (D = 1.42) are added dropwise with caution (hood). The flask is warmed to complete the solution of the Raney nickel. After cooling, 10 ml. of distilled water are added and the solution is boiled to expel oxides of nitrogen. The solution is cooled, 20 ml. of distilled water are added and 10 mg. of o-phenylenediamine dihydrochloride. After standing for 15 minutes, the solution is adjusted to pH 2.5 =t0.1 with amnionium hydroxide, transferred to a 125-ml. extraction funnel, and extracted twice with 10-ml. portions of chloroform. The chloroform extract is dried with sodium sulfate, transferred to a 25-ml. volumetric flask and diluted to volume
100
1
Defermination of Selenium in Prednisone (Sample weight, 1 gram)
I
/
/'
