sorption spectrum is shown in Figure 3. The recovery of selenium from seleno1-dehydrotestosterone acetate [prepared by method A of Flory and Restivo (4)1 was checked by eight duplicate analyses. Csing 0.500 mg. of the steroid, 98 pg. of selenium were present, 88.1 i 2.1 pg. of selenium were found, with an average recovery of 89.8%. Table I gives the results of three determinations of selenium in two production lots of prednisone. This method has been used in this laboratory for the determination of
selenium in prednisone and 6~y-chloroprednisone for a period of 8 months, ACKNOWLEDGMENT
The author thanks Maria de 10s Angeles Cervantes G6mez for her help inrunningtheanalyses. LITERATURE CITED
(‘1 Brady, kv. E., Lyons, R. E*, J . Am* Ch6WL. SOC. 48,2642 (1926). (2) Cheng, K. I,., Chemist Analyst 45, 67 (1956).
(3) Drake, K.L., Org. Sy~zthesis21, 15. (4) Flory, K. G., Reetivo, A. R., J . Qrg. Chem. 22,406 (1957). ( 5 ) Kan, Mmami, Ann. Repts. Takeda Research Lab. 11, 54 (1952). (6) Mozingo, Ralph, Q’olf, D. E., Harvis, S. A., Falkere, Karl, J. Am. Chem. Soc. 6 5 , 1013 (1943). (7) Sawicki, Eugene, ANAL. CHEY. 29, 1376 (1957). (8) Shakhov, A. El., Zauodskaya Lab. 11, 893 (1945). (9) Wiseman, G. E., Gouid, E. S., J . Am. Chem. SOC.76, 1706 (1954).
RECEIVEDfor review June 1, 1959. Accepted September 8, 1960.
ectrophotornetric Determination of Stercdic Aci A. J. DEUTSCHMAN, Jr., and IRVIN S. KLAUS1 Deparfmenf o f Agricultural Biochemisfry, Universify o f Arizona, Tucson, Ariz.
b
The cyclopropene configuration present in sterculic and malvalic acids has been found to b e toxic to nonruminants. The reaction conditions for currying out the Halphen test for this structure have been siudied and a procedure is proposed that results in reproducible color development. The reaction has been used as the basis for a spectrophotometric determination of the cyclopropene structure. The absorbonce cur veof the solution is presented. Purified sterculic acid was used for the preparation of a standard adsorbance curve at 505 mp. Standard error estimate is A0.2 X gram of sfercuk acid in 10 ml. of sohtion with an accuracy of *lo% at 95% confidence a t the mid-point of the range for which the procedure is suggested.
T THE TURN OF THE CENTURY the
Halphen test was used to determine whether a material was contaminated with cottonseed oil. It was believed that this test was specific for cottonseed oil and indicated the concentration of the oil by the intensity of the color that developed during the test. A number of investigators have modified the test initially reported by Halphen. Kuhn and Bengen (6) indicated sulfur and carbon disulfide were necessary for the color development. Other materials such as pyridine and amyl alcohol simplified the experimental procedure. Rupp (8) used pressure to achieve a high enough temperature for Present address, Chemistry Department, Northwestern LTniversity, Evanston, 111.
rapid color development. Garnier (S) used amyl alcohol. Gastaldi (4) proposed a procedure utilizing pyridine. It would appear that the various procedures and solvents suggested are devices to raise the temperature of the reagents involved in the color reaction (CSZ, oil) to a sufficient level for rapid color development. Recent work by Nunn ( 7 ) , Faure ( I ) , and Faure and Smith (a) on the nature of the Halphen test and the structure of sterculic acid, CHa(CH2)7-(CH&-COOH, permits a better understanding of the limitations and applications of the test. The Halphen reaction appears to be specific for the cyclopropene structure and may be used as the basis of an analytical procedure for determination of this structure in oils. The physiological activity of materials containing the cyclopropene structure is marked even at low concentrations in chickens (6, 9, 10). Large amounts of cottonseed meal are being fed to livestock as a protein source. Apparently, ruminants can utilize this protein source without any obvious problem. Cottonseed meal cannot be fed to nonruminants. Gossypol is known to bring about darkening of hen eggs. Products similar to sterculic acid are also present. Sterculic acid causes abnormalities in chicken eggs and death of the embryo (9, 10). If advances are to be made toward a better understanding of the mechanism by which the cyclopropene configuration functions physiologically, an analytical procedure must be developed. The use of cottonseed meal as a poult r y feed has been limited because, of growth inhibition, toxicity, and the discoloration of eggs produced by laying
hens on a diet rich in such a feed. The pink discoloration of the whites has been definitely traced to the presence of the eyclopropene structure, the structure which gives the Halphen test. Shenstone and Vickery (11, 18) and Masson et al. ( 6 ) fed malvalic and sterculic acids to hens, and the whites of the eggs became pink when stored. Hydrogenation of the acids to destroy the cyclopropene structure resulted in an end product that did not give a Halphen test or cause pink discoloration of the egg whites. Schneider et at. (9, IO) studied the effect of Sterculia foetida oil on yolk weights and water uptake of the hen egg, as well as on hatchability. The deviation of these eggs from normal eggs was extreme, and the embryo died after 5 days. Kone of the Halphen procedures described in the literature permit the quantitative determination of the cyclopropene concentration in an oil. Therefore, the effect of different variables on the Halphen reaction was studied and a quantitrttive procedure developed. REAGENTS AND APPARATUS
Maliinckrodt reagent grade sulfur flowers, carbon disulfide, and pyridine were used in all determinations. Carbon disulfide was saturated with sulfur at room temperature; small amounts of sulfur suspended in the carbon disulfide did not interfere. -4 0.0193-gram sample of sterculic acid purified by the procedure of Nunn ( 7 ) consumed 6.29 ml. of 0.0103N barium hydroxide. This corresponds to an equivalent weight of 298 as compared with 295 for sterculic acid with a structural formula of CI9Hs4O2, or a purity of 9970, assuming no acid impurities. A Beckman Model DU spectrophotometer was used, but any spectrophoVOL. 32, NO. 13. DECEMBER 1960
1809
analytical balance. The volume of oil was adjusted t o 1 ml. with corn oil after weighing each sample. The Halphen test was carried out as outlined above to obtain the data shown in the calibration curve in Figure 2.
0 3
02
w
z m
= 02
DISCUSSION
w
d: 01
01
0 450
470
490
W A V E LENGTH
Figure 1. reaction
530
510
550
mp
Absorbance
of
Halphen
toineter operating in the 300- to 700-mp range would be satisfactory. Tenmillimeter silica cells were used for the samples. All absorbance measurements were carried out at 505 m,u, the wave length of maximum absorbance of the Halphen color in the visible range (Figure 1). PROCEDURE
Halphen Reaction. Approximately 1 ml. of oil was accurately weighed into a tared 50-ml. Erlenmeyer flask on a direct-reading analytical balance. Five milliliters of carbon disulfidesulfur reagent and 5 ml. of pyridine were pipctted into the flask. T h e mixture was placed in a water bath a t 48' C. for 1 hour, then placed in an oil bath a t 108' C. for an additional 45 minutes. The residue was quantitatively transferred to a 10-ml. volumetric flask and diluted to volume with pyridine. The absorbances of the solutions were measured a t 505 mp after the mixture had been allowed to stand a t room temperature for 2 hours. A blank sample using 1 ml. of corn oil was run a i t h all determinations. Sterculic Acid Standard Curve. I n a 150-ml. ground glass-stoppered Erlenmeyer flask was placed 0.1823 gram of sterculic acid and t o i t was added 115.27 grams of corn oil (Mazola). Aliquots of this solution were weighed into tared 150-ml. Erlenmeyer flasks on a direct-reading
1 8 10
4
ANALYTICAL CHEMISTRY
T'ariations of reported Halphen procedures were studied because of the conflicting information in the literature. Isopentyl alcohol m s first studied in a procedure that approximated that outlined above. Samples giving essentially identical absorbance readings had over a 100% difference in sample neights. Furthermore, sulfur crystallized from the isopentyl alcohol as the solution cooled. This precipitate had to be removed and caused further manipulation problems that were avoided by the use of pyridine. Studies of time and temperature variables indicated that the carbon disulfide boiled off too rapidly if the water bath temperature exceeded 48" C. and poor color development resulted. At the oil bath temperature of 108' C., which was slightly below the boiling point of pyridine, the color development was most rapid and the loss of pyridine by vaporization was negligible. After the final heating, cooling, and dilution to volume, 2 hours were required before the absorbance stabilized. An effective measurable concentration of sterculic acid was between 1 and 7 X loF4 gram of sterculic acid per gram of oil. At lower concentrations slight deviations produced large errors. The intensity of the color was so great a t the higher concentrations that small variations in absorbance could not be measured; thus, dilution of an oil with corn oil to the approximate range indicated above was necessary. The procedure outlined mas applied by three analysts to reference samples of known sterculic acid concentration. The results were within 10% of the known concentration. Replicate analyses of corn oil samples with known added concentrations of sterculic acid were carried out as described above. The standard error estimate for 17 determinations is -i: 0.2
7
02L-
o
l
I
I
I
I
I
e
3
4
5
6
I
1
GRAMS OF STERCULIC ACID IN IO ml SOLUTION X IO-.
Figure 2. Concentration of sterculic acid as u function of absorbance in the Halphen reaction
X gram of sterculic acid in 10 ml. of solution and is based upon the deviation of the individual analyses from the absorbance curve derived from the determinations. An accuracy of i10% with 95% confidence can be expected a t the mid-point of the concentration range indicated. LITERATURE CITED
(1) Faure, P. K., Nature 178, 372 1956).
(2) Faure, P. K., Smith, J. C., J Chern. SOC.1956, 1818. (3) Garnier, L., J . pharm. chim. ,5) 29, 273 (1909). (4) Gastaldi, E., Ciorn. farm. china. 61, 289 (1912). (5) Kuhn, B., Bengen, F., 2. A-ahr. Genussm. 12, 145 (1907). (6) Masson, J. C., Vavich, &I.G., Heywang, B. 'CV., Kemmerer, A. R., Science 126, 751 (1957). (7) Kunn, J. R., J . Chenz. SOC.1952, 313. (8) Rupp, E., Z . Nahr. Genussm. 13, 74 (1907). (9) Schneider, D. L., Doberena, A. R., Kurnick, A. A,, Vavich, M. G., Kemmerer, A. R., Federation Proc. 19, 221 (1960). (10) Schneider, D. L., Vavich, RI. Kurnick, -4. A., Doberena, A. Kemmerer, A. R., Poultry Sci., in pi (11) Shenstone, F. S., Vickery, J. Nature 177, 94 (1956). (12) Shenstone, F. S., Vickery, J. Poultry Sci. 38, 1055 (1959). RECEIVED for review February 26, 1960. Accepted July 25, 1960.
