V O L U M E 25, NO, 9, S E P T E M B E R 1 9 5 3 contains a comparison of colorimetric uranium values obtained by burning mixtures of uranium and graphite in a small furnace. These samples are divided into tn.0 sets, I n the first set, the furnace was set a t maximum and no subsequent adjustment ninde, while in the second set, the temperature was manually controlled a t approximately 850’ C. -1 comparison of results by the 8-quinolinol colorimetric method and the alpha-counting technique mas made. Table V contains these values. Effect of Impurities in 8-Quinolinol. Some samples of 8quinolinol gave colored solutions in chloroform, but part of the original color remained in the aqueous extract. Better grades of the rengeiit werr colorleqs in chloroform solution. Effect of Illumination. The chloroform-quinolinol solution is greatly affected by illumination from fluorescent lighting, and poor grades of 8-quinolinol solution must be discarded after 4 days. On the other hand, uranyl quinolinolate solutions extracted into chloroform from colorless chloroform-8-quinolinol stable if mnintaincd a t 25’ C. for 16 solutions weie found to hours. LITER.4TURE CITED
(1)
Blitz, W.,and Muller, IT., Z . anorg.
16.
allgem. Cheni., 163, 296
(1 927).
( 2 ) Currah. J. E.. and Beamish. F. E.. AXAL.CHEM..19. 609 (1947). ( 3 ) Gerhold, M., ’and Hecht, F., Microchemie veT. Mikrochim. Acta, 36, 1100-5 (1951). (4) Greenspan, J., Schuler, 31. J., Goldenberg, H., Taub, D., and
1373
(5) (6) (7)
(8) (9) (10) (11) (12) (13) (14) (15)
Carlson, A. S., L7.S. Department of Commerce, Office of Technical Services, Washington, D. C., R e p t . D-12 (April 1 , 1946). Henicksman, A. L., I b i d . , R e p t . LA-1394 (March 15, 1952). Hillebrand, W. F., and Lundell, G. E. F., “Applied Inorganic Analysis,” iiew York, John Wiley & Sons, 1929. Hubbard, B., E. S. Department of Commerce, OfficeofTechnical Services, Washington, D. C., R e p t . CC-2134 (Sept. 15,19441,; Kats, J. J., and Rabinowitz, E., “The Chemistry of Uranium, Part 1 , 1st ed., Kew York. RlcGraw-Hill Book Co., 1961. Lafferty, R. H., McCormick, T . J . , and Myers, H., U.S.DeparG ment of Commerce, Office of Technical Services, Washington, D. C., R e p t . XAC-54-1348 (April 1 , 1946). Rloeller, Therald, IHD.ENG.CHEM..ASAL. ED., 15, 346 (1943). hIuntz, J. A, V. S. Department of Commerce, Office of Technical Services, Washington, D. C., Rept. MUC- JIW-599. Ostroumow, E. -4., 2. anal. Chem., 106, 244-8 (1936). Rodden, C. J., “Bnalytical Chemistry of the Manhattan Project,” Chap. 1, New York, AIcGraw-Hill Book Co., 1950. Sandell, E. B., “Colorimetric Determination of Traces of Metals,” 2nd ed., Kew York, Interscience Publishers, 1950. Smales A. A, U. S. Department of Commerce, Office of Technical Services, Washington, D. C., Rept. BR-r12 (April 17,
1944). (16) Smales, A. A., and Wilson, H. X . , I b i d . , Rept. BR-150 (Feb. 22, 1943). (17) Tissier, Marguerite, and Benard, Henri, C‘ompt. Tend. SOC. biol., 99, 1144 (1928). (18) . , Ware. E., V. S. Department of Commerce, Office of Technical Services, TTTashington, D. C’., Rept. MDDC-1432 (August 1945, declassified Nov. 7, 1947). (19) White, L. J., Coke and Gas, 14, 285-8 (1942). RECEIVED for review April 16, 1953. Accepted June 22, 1953. Based on studies conducted for the Atomic Energy Commission under C o n t r a c t A T - l l 1-Gen-8. Presented at -4nalytical Chemistry Information Meeting, Oak Ridge, Tenn., M a y 19 to 21, 1953.
Determination of Diacetyl DUANE T. ENGLIS, E,MILY J. FISC“, AND SHIRLEY L. B.4SH University of Illinois, Urbana, I l l . Dimethylglyoxime shows an intense absorption peak near 226 mp in the ultraviolet region of the spectrum. The absorption by the compound follows Beer’s law and serves as a basis for a direct determination of diacetyl in starter distillates or distillates from other food products after its conversion to dimethylglyoxime, but without the supplementary formation of a metal complex. The results obtained with this spectrophotometric procedure compare favorably with those obtained with the gravimetric method in which the nickel compound is formed. The spectrophotometric procedure is of high sensitivity. Concentrations of 0 to 10 p.p.m. may be easily estimated. The method should be advantageous for laboratories equipped with instruments for work in the ultraviolet range.
D
ISCETYL has long been recognized as a constituent occurring in small amounts in vinegar, fermented cane juice, cultured milk products, and a number of other food materials. Because the amount normally present is small, relatively large samples are usually necessary to furnish enough of the compound to make possible a quantitative determination. The widely used method of Barnicoat (2) involves a steam distillation of the diacetyl, its conversion to dimethylglyoxime, precipitation with a nickel salt, and final gravimetric estimation as the nickel compound. Colorimetric methods have also been employed. As an alternative procedure, Barnicoat has dissolved the nickel precipitate in chloroform and estimated the material by a standard series comparison with similarly prepared solutions of known concentration. Stotz and Raborg (8) suggested a sensitive method based upon an assumed tetravalent nickel dimethylglyoxime compound. Okac and Polster (5) confirmed the sensitivity of the method, but differed as to the nature of the colored compound. Mohler and 1
Present address, University of California Loa .4ngeles. Calif
dlmasy (4)detected diacetyl by the specific absorption of the ferric complex. Prill and Hammer (6) employed the rose-red ferrous complex for the quantitative microdetermination of diacetyl. They reported that it was possible to detect as little as 0.001 mg. of diacetyl per 5 ml. of solution and to measure conveniently 0.01 to 0.5 mg. per 10 ml. (1 to 50 p.p.m.) of solution. More recentlv, Speck ( 7 ) has developed a method for diacetyl based on the formation of a purple dye with chromotropic acid in the presence of concentrated sulfuric acid. This method may be used for quantities from 0.03 to 0.10 mg. per ml. (30 to 100 p.p.m.), which is not quite as sensitive as that of Prill and Hammer. A considerable number of substances interfere or similarly respond to the reagents. Diacetyl has only a moderate absorption in the ultraviolet region of the spectrum ( 3 ) . However, when converted to the dimethylglyoxime it has intense absorption. This property appeared to offer promise as a means of determination of diacetyl in various food products without the necessity of conversion to a metallic compound.
ANALYTICAL CHEMISTRY
1374 Table 1. Absorbance of Aqueous Dimethylglyoxime Solutions at 226 mp Mg. per 100 M1. Dimethylglyoxlme
1.130 0.565 0.451 0.226
Diacetyl
0.838 0.419 0.335 0.168
Absorbance,
1-Cm.Cell 1.348 0.655 0.540 0.265
EXPERIMENTAL
A Beckman Model DUspectrophotometer and a Cary Model 11 recording instrument were employed for the study. In order to establish the absorption curve of pure dimethylglyoxime, the reagent-grade material was f i s t dried a t 100' to 105" C. for 1 hour. A carefully weighed portion was then dissolved in water, and the solution was examined with the Cary spectrophotometer. In this aqueous solution an absorption peak (Figure 1) was observed a t 226 mp and the calculated molecular extinction coefficient was 13,400. A similar experiment with 95% ethanol as the solvent yielded an absorption curve of similar nature, but the peak was shifted slightly (about 2 mp) toward the longer wave-length side and uyas of higher intensity. The molecular extinction coefficient for the compound in the ethanol solution was of the order of 18,300. Because of the greater intensity of absorption in the ethanol solvent, some experiments were carried out in the early stages of the investigation in which the oximation reactions and the final spectrophotometric evaluations were made in ethanol solutions. Satisfactory results were obtained, but no marked advantage was apparent ovpr the use of aqueous solutions. -4sa consequence, all data arc for aqueous solutions. The experimental results on coilcentration and absorbance relationships for the aqueous solutions (Table I ) show good conformance to Beer's law. The wave length for the absorption maximum of dimethylglyoxime is subject to change with pH. The peak observed a t 226 mp a t pH 5.4 tor the aqueous solution shifted to a position near 260 mp when thesolution u a s r m d e basic with potassium hydroxide to a pH of 10.6 However, the solution buffered with sodium acetate a t pH 7 . 0 0 shoncd uo displacement of the peak from its position a t pH 5.4. For the preparation of the glyoxime compound, Vizern and Guillot (9) specify the use of 1 ml. of 10% hydroxylamine hydrochloride and 1.7 ml. of 0.1 M sodium hydroxide for each milligram of diacetyl present. Preliminary tests with the spectrophotometry qhowed that the reagents themselves absorb strongly. For both sodium acetate and hydroxylamine hydrochloride, an abaorption maximum is indicated below 210 mp, which is beyond the working range of the instrument. With the high concentration of these reagents which are employed, the side band of the oxiniation reagent mixture entirely obliterated the characteristic peak of the dimethylglyoxime. However, when the reagents were mixed with pure dimethylglyoxime and compared to a blank solution containing the same quantities of reagents, the characteristic dimethylglyoxime curve was observed. A similar observation was made wrhen diacetyl was converted to the glyoxime and the solution compared to a blank containing the same amounts of reagents. It was evident from this experiment that the quantity of reagent consumed in the formation of the reaction product, dimethylglyoxime, was insignificant in comparison to the excess of reagent left in solution. The addition of potassium hydroxide to the hydroxylamine hydrochloride caused a shift in the absorption toward the longer wavelength region. Hence, control of pH in the determination is necessary. In actual practice, this acetate buffer has accomplished this control very satisfactorily, and no difficulty has been experienced with the pH factor.
Other preliminary experiments employing the conditions of conversion specified by Vizern and Guillot served to indicate a slow rate of reaction and apparent incomplete conversion on some occasions. The modified Barnicoat method (8) calls for equal amounts by weight of sodium acetate and hydroxylamine hydrochloride as the converting reagents. The final step involves a concentration to a small volume, so that a heat treatment is a p plied which probably contributes to the completeness of the reaction. Prill and Hammer (6) use hydroxylamine acetate and prescribe heating a t 85" C. for 1 hour to form the dioxime. In the present work, experiments on conversion were carried out in which the quantities of reagents, pH, and periods of reaction were varied. PROCEDURE
Weigh out a sample of such size as to contain 0.1 t o 1.0 mg. of diacetyl. Remove the diacetyl from the food product by distillation in the usual way. Convert the diacetyl with 20 ml. of 10% hydroxylamine hydrochloride and 10 ml. of 40y0 sodium acetate. Heat the reaction mixture for 1 hour a t 85' C. according to the directions of Prill and Hammer, or allow to stand overnight a t room temperature. Dilute the solution to 100 ml. and determine the absorbance a t 226 mp, using for a comparison solution one containing identical quantities of reagents in a final volume
I
0
I
I
200
250
I
WAVE LENGTH, M p
I 300
I
Figure 1. Absorption Curve for Dimethylglyoxime 1.38 mg. per 100 ml. in water
equal to that used for the sample. Refer to the prepared working curve based on spectrophotometric evaluation of standard solutions of pure dimethylglyoxime (as shown in Table I ) for indication of the diacetyl content. COMPARISON OF SPECTROPHOTOMETRIC AND GRAVIMETRIC METHODS
In order t o test the proposed method, samples of starter distillates from cultured milk were secured from a commercial source. These had been analyzed by the producer, but no information wa furnished as to the method used. Upon their arrival, the samples were stored in a refrigerator, and considerable time elapsed after the receipt of the samples before they were subjected to examination. Although slight changes may have taken place during storage, the conditions for preservation were such as to assure reasonable stability. Some of the samples were exhausted before
V O L U M E 2 5 , NO. 9, S E P T E M B E R 1 9 5 3 the final procedure was developed, but data for a final comparison of methods are given for one sample. The sample was analyzed first by the Barnicoat gravimetric method in which the precipitated nickel dimethylglyoxime was filtered out on a tared sintered-glass crucible, washed with ether, dried, and finally weighed. The spectrophotometric analysis was carried out as previously described. The results are given in Table 11. Slightly higher values are to be noted for the spectrophotometric method. However, in view of the small amounts of material being determined the results are in reasonably good agreement. Several advantages may be cited for the spectrophotometric method. It requires less time t o make a determination, it is more sensitive, and it is free of errors resulting from solubility losses which are inherent in the gravimetric method.
Table 11. Ikterniinatioii of Diacetyl in Starter Distillate GravimetricQ Nickel Dirriethylglyoximc, Grains 0.1263 0.1249 0.1246 0.1261 0.124i 0 1247
Diacetyl, hlp: In portion P G n i . of analyzed original saniple i4 6 37.3 i4 4 37 2 .37 1 74 3 37.3 74 6 74.0 74 3
37 0
37,o
bi)e~troF)liotornetricb 1Lg. ~ - - _ Diacetyl, _ _ Per 100 ml. of Per ml. of Absorbance. l-Ciii portion anaoriginal samCell a t 226 n i ~ lyzed Serien 4? 1 . 2 6 39.8 0.798 1.25 1.24 1.31 1.24
Series B d 1.25
1.%8 1 26
1.27
0.792 0.785 0.830
0,785
39.6 39.2 41.5 39.2
0.792 0.806 0.796 0.804
39.6 40.3 39.8 40.2
Gquivalent oi 2 1111.ui .-ldrlt‘r sutrjrclcd to analyais. 5 Equivalent of 1 nil. oi ~tnrtmdiluted to 50 i d . ; thpn 1 1111.rnkrn. converted to dimethylglyoririie,and ruade up 10 100 ml. e Analyzed by the outlined procedure. d Only half the quantities of reagents used in Series A were einployed. a
For a high conceiitratioii of diacetyl, such as W&S characteristic of t,he sample whose analysis is reported, a considerable dilution is necessary before the spectrophotometric method may be employed. Any error in the estimation is then multiplied by the dilution factor, even though the relative effect remains the same on the final result. For high concentrations the gravimetric method may be advantageous. The quantities of reagents are not critical since in Series B, which employed only half the amounts of hydroxylamine hydrochloride and sodium acetate used in Series A, the results were essentially the same. The commercial distillates, as sold to the butter manufact,urera, have usually been subjected to oxidation so that the acetoin (3-hydroxy-2-butanone) originally present has been oxidized to diacetyl. In certain food products, one may be concerned with distillate mixtures in which the acetoin is present in quantities many times greater than the diacetyl. The question may then be raised as to the possible interference of the oximation products of these other substances. As an illustration of a monoxime, acetoxime shows high absorption ( ~ ism5000), ~ but this is a t 190 m r in the f a r ultraviolet region (1). One might predict that acetoin would have similar absorption, although the adjacent hydroxyl group might be expected to affect it to some degree. To test the possible interference of acetoin, a sample of t,his mat.erial was secured and an aqueous solution prepared. A port,ion of the solution was added t o a sample of starter distillate of known
1375 diacetyl content so that the ratio of acetoin to diacetyl w a 5 alJOUt 25 to 1. The mixture was then subjected to the oximatioii procedure and subsequent spectrophotometric analysis. An apparent diacetyl value equivalent to about double the amount known to be present in the distillate was observed. Hon ever, an examination of the oximated acetoin alone showed an alisorption curve identical with that of dimethylglyoxime. The typical red precipitate was given with a nickel salt, and it was apparent that the acetoin contained about 4% diacetyl. Ai3uming that the balance of the sample was acetoin, there wai no evidence of its presence as a result of the oximation reaction. Attempts were made to determine diacetyl in butter and butte1 substitutes without a preliminary distillation ?;one of these a t tempts were successful. The determination of acetoin after the usual oxidation with ferric chloride and sulivqueiit distillation constitutes a simple extension of the proceduie. During the progresb of the work, an attempt !\a> made to use diacetyl procured from a chemical supply source a3 a primary standard for the preparation of the dimethylglyoxime and establishment of a working curve. An analysis for diacetyl by the gravimetric method indicated a purity of only 96%. Pure dimethylglyoxime is a much more satisfactory standard. Some interesting differences in the rate of formation and other factors incidental to the precipitation of the nickel compound were noted, as the original materials were varied. Even though each test represented about the same concentration in terms of diacetyl and all precipitations were made under as nearly identical conditions as possible, the results were slightly different. For example, the precipitate from pure dimethylglyoxime formed most readily and had a rose-red or slightly bluish color. The precipitate from the “pure” diacetyl formed more readily than did that from the starter distillate. Furthermore, it was a true red, while that from the starter distillate was a brownish red color and more lumpy in character. It is possible that some slight impurity in the starter distillate retarded the formation of the precipitate and may, in some cases, have caused it t o be incomplete. The filtrate from the distillate reaction product was of a reddish color, while with the other materials the greenish color of the excess nickel ion predominated. The most reliable standard curve for spectrophotometric determinations i4 one prepared from pure dimethylglyoxime. Since concentrations of the order of 1 mg. per 100 ml. (10 p.p.m.) and less can easily be determined with a I-cm. absorption cell, the sensitivity of the method can be proportionately increased with the use of cells of greater length. In many cases, a total volume of reaction mixture smaller than 100 ml. could be used to attain a similar result. The method compares very favorably with that of Prill and Hammer (6) in sensitivity. I t is believed the procedure will be advantageous wherever suitable ultraviolet equipment is availahle ACKNOWLEDGMENT
Starter distillates from cultured milk were furnished by courtesy of C. H. Hansen Co., Milwaukee, Wis. LITERATURE CITED
(1) Ann. Bepts. on Progr. Chem. (Chem. Soc. London), 42, 112 (1946). (2) Barnicoat, C. R., Analyst, 60, 653 (1935). (3) International Critical Tables, Vol. 5,p. 377,New York, iMcGrawHill Book Co., 1929. (4) Mohler, Herm., and .4lmasy, Felix, 2. anal. Chem.. 96,399 (1934). (5) Okac, A., and Polster, &I., Collection Czechoslov. Chem. Communs., 13,561 (1948). (6) Prill, E.A.,and Hammer, B. IT., I o w a State Coll. J . Sci., 12,385 (1938). (7) Speck, J. C., ANAL.CHEX.,20,647 (1948). (8) Stotz, Elmer, and Raborg, Jane, J. Biol. Chem., 150, 25 (1943). (9) Vizern and Guillot, Ann. fals. et fraudes, 25,459 (1932) RECEIVED for review June 23, 1952. Accepted June 22, 1953.
