Adaptation of the Iodoform Reaction to Microgram Quantities for Determination of Organic Structures SIR: The iodoform reaction has been used extensively for the qualitative determination of methyl ketones and compounds readily oxidized to methyl ketones, and attempts have been made to use it for the quantitative estimation of appropriately reacting compounds. Extensive studies have been reported, too, on the conditions necessary for the quantitative estimation of acetone by the same reaction. Kramer (4) developed a gravimetric procedure for this purpose, while Messinger (6) utilized a volumetric technique to titrate the excess iodine used in the formation of the iodoform. Dal Nogare, Norris, and Mitchell (2) used the iodoform reaction for the quantitative estimation of acetaldehyde and acetone measuring the optical density of the iodoform at 3470 A. Considerable study has been expended upon the various aspects of the iodoform reaction including specificity and optimum reaction conditions. RRsults are not quantitative unless the experimental conditions are properly adjusted to minimize the occurrence of side reactions. Morgan, Bardwell, and Cullis (6) have investigated the limiting experimental conditions needed to obtain quantitative results in the formation of iodoform from acetone. Their investigations, as well as those of Cullis and Hashmi (I), indicated that the main factors which must be carefully controlled are the order and rate of addition and the relative concentrations of the reagents. In the present study, we were interested in devising a technique whereby the number of active methyl groups, in a compound of unknown structure, could be determined on microgram amounts of sample. This requires a semiquantita-
Table 1.
Weight of Ketone, pg.
Acetophenone ZOctanone Acetylacetone Mesityl oxide
1 174
EXPERIMENTAL
Reagents and Apparatus. Each of the ketones was freshly distilled before use. All of the solvents used were either “spectro grade” or were freshly distilled before use. The iodine solution was prepared by dissolving 20 g. of potassium iodide and 10.12 g. of iodine (Merck and Co., Inc., resublimed) in 100 ml. of water. A microburet was used for the delivery of the iodine solution. The gas chromatographic instrument employed for these investigations was the Perkin-Elmer Model 154 Vapor Fractometer with helium as the carrier gas. The iodoform was chromatographed using a 1-meter long by 4 m m . i.d. glass column packed with 30- to 60-mesh chromosorb (Johns-Manville) coated with 22% silicone 550 oil (Dow Corning) containing 10% stearic acid.
Quantitative Estimation of the Iodoform Obtained from Microquantities of Known Samples of Methyl Ketones
Ketone Acetone
donone
tive estimation of the iodoform produced. &presentative compounds were selected from the various types of methyl ketones to establish whether high yields of iodoform could be obtained from the different types of compounds which undergo this reaction. The volatility of iodoform at temperatures below that of explosive decomposition (210’ C.) permitted its estimation by gas chromatography, which proved reliable for the quantitative determination of microgram amounts of iodoform. The experimental conditions for the iodoform reaction used in the present work were essentially the same as described by Morgan and coworkers. The use of microgram amounts of acetone, Zoctanone, acetophenone, acetylacetone, mesityl oxide, and a-ionone indicated that the experimental conditions could be so controlled as to give high yields of iodoform.
13.3 13.3 38 38 38.4 38.4 42.8 42.8 39.2 39.2 24.6 56.4 64.4
ANALYTICAL CHEMISTRY
Weight of Iodoform, pg. Obtained
Theory 90 90 124.3 124.3 117.8 117.8 168.2 168.2 157.2 157.2 98.6 115.6 132
82.4 82 94 95.2 84.8 85.0 145 151.4 105.2 105.2 66 116 123.2
Recovery,
%
92 91 76 77 72 72
86 90 67 67 67 100 93
The temperature of the column was maintained at 170’ C. The helium flow rate was 50 cc. per minute. Procedure. To a solution of the methyl ketone in dioxane in a small test tube was added a 50-fold stoichiometric excess of 2.5N sodium hydroxide and a two-fold stoichiometric excess of the iodine solution (in small increments). The reaction mixture was shaken vigorously during the addition of the iodine solution by rot& ing the test tube between the palms of the hands of the operator. After the addition of the appropriate amount of iodine, the test tube was stoppered and the mixture was shaken for an additional 2 minutes and allowed to stand in the dark for 20 minutes. To the aqueous reaction mixture waa added one-half or one-third volume of carbon tetrachloride. After solution of the iodoform was complete, the aqueous layer was removed with a pipet and an aliquot of the organic layer was removed with a Hamilton 50-pl. syringe (No. 705) and injected into the Vapor Fraotometer. Carbon tetrachloride was chosen as the solvent, since the initial decomposition of iodoform is a t a minimum in this solvent (7). The iodoform emerged from the column showing a constant retention time of 6.7 minutes. The area under the iodoform peak was measured with a planimeter and the quantity of the iodoform was determined from a working curve. The curve was prepared using known amounts of iodoform, by partitioning between the blank reaction mixture and carbon tetrachloride. Partitioning waa necessary to compensate for the solun the aqueous bility of the iodoform i reaction mixture. The curve was linear over the range 60 to 280 pg. of iodoform with a standard deviation of 5.6 pg. for 140-pg. samples of :odoform. RESULTS AND DISCUSSION
The results obtained with the methyl ketones which were tested are listed in Table I. In all cases, adequate yields were obtained for the purpose of structural characterization. a-Ionone was not very soluble in the reaction mixture; however, by prolonging the time of reaction to about 30 minutes, excellent results were also obtained with this compound. We have utilized this procedure to advantage in the characterization of unknown terpenoid materials isolated from tobacco. Only 1 mole of iodoform is produced per mole of acetylacetone. ,%Diketones containing the grouping -COCH&Oappear to be iodinated first at the methylene group rather than on either of the methyl groups. The chain is subsequently cleaved forming only one
iodoform-producing species (3). Low yields of iodoform may also be produced from ketones without the a-methyl grouping. Diethyl ketone gave less than 17% yield when determined by this method. Such low yields could not be used to establish the presence of a reacting function in an unknown molecule. When possible, a preliminary study should be carried out with either the known ketone or one of similar structure t o establish conditions for maximum yields.
ACKNOWLEDGMENT
The authors thank John F. Williams for helpful suggestions and advice and Robert Lax and Dwight Galloway for technical assistance. LITERATURE CITED
(1) Cullis, C. F., Hashmi, H. M., J . Chem. Soc. 2512 (1956). (2) Dal Nogare, Stephen, Norris, T. O., Mitchell, J.. ANAL. CHEM. 23, 1473 (1951). ' ' (3) Fuson, R. C., Bull, B. A., Chem. Rev. 15,275 (1934).
(4) Kramer, G.,Ber. 13, lo00 (1880). (5) Messinger, J., Ibid., 21,3366(1888). (6) Morgan, K. J., Bardwell, J., Cullis, C. F., J . Chem. SOC.3190 (1950). (7) Verman, R. M., Bose, S., J . Indian Chem. SOC.37, 540 (1960).
Presented a t the Meeting-in-Miniature of the North Carolina Section, ACS, Raleigh, N. C., May 6,1961. ANDREWG. KALLIANOS JAMES D. MOLD Research Department Liggett and Myers Tobacco Co. Durham, N. C.
Liquid Scintillation Counting of Insoluble Samples with Applications to Carbohydrate and Sterol Derivatives SIR: The success reported by Funt and Hetherington (2) and others, whose work is reviewed by Funt (I), in counting radioactive samples adsorbed on filter paper, wet with a scintillator solution, suggested that heat-sealing the samples between two disks of plastic sheet would give an improved scintillator system to count solid, insoluble derivatives such as cbolesterol digitonide and D-glucose phenylosotriazole. The method evolved combines some of the advantages of both liquid scintillation and dry counting systems in that there is no selfabsorption present and in that the sample, which can be recovered, is sealed in a dry disk, easily handled and stored. Another advantage of this technique, which may prove important when many samples are assayed, is that it prevents contamination of the phototube or of other samples.
so that the heated die would cut and seal the two layers of Saran Wrap simultaneously to form a disk. The counter used was a refrigerated single channel liquid scintillation counter, Model 745 A, manufactured by BairdAtomic, Inc. Procedure. The cholesterol digitonide samples were prepared, in the
Volume of solution in microliters = 20 pl. 4 pl. per nig. of sample
+
EXPERIMENTAL
Materials. Cbolesterol-4-C14 digitonide was prepared according to the method of Windaus (6). FructoseU-cl4 was converted to phenyl-Dglucosazone (4) and the osazone was converted to D-glucose phenylosotriasole (3). The scintillator solution consisted of the solvent, 4isopropylbiphenyl, and other solutes as given by Funt and Hetherington (2). Millipore (Millipore Filter Corp., Bedford, Mass.) filters were used in counting the digitonide disks, and Whatman No. 50 filter papers 3/* inch in diameter were used in counting the osotriazole derivative of fructose. The radioactive sample and filter were contained in two layers of Saran Wrap (vinylidene chloridevinyl chloride copolymer, The Dow Chemical Co., Midland, Mich.). The Saran Wrap was heat-sealed with the device shown in Figure 1. The upper part of the sealing device consists of the handle and heating element of a small soldering iron. The lower part is the It was machined from cold die. rolled steel and the edges were sharpened
form of disks 1.5 cm. in diameter, by filtering the'suspension of the newly precipitated material onto fritted glass disks and washing the precipitate with 90% ethanol and 1: 1 ethyl ether-acetone. The flocculent digitonide mats to form a papery disk that can be removed intact from the fritted glass disk for weighing. Each digitonide disk was then placed on a Millipore filter disk and enough scintillator solution was applied to saturate the filter and disk. Each wet sample was placed between two layers of Saran Wrap, heat-sealed, and cut out with the die. The samples could be counted immediately a t -8" C.; a cold and dark adapt time was found unnecessary. The D-glucose phenylosotriazole samples were prepared in the following way. The fluffy crystals were transferred with tweezers onto a filter paper disk and weighed. The samples were then wet with a volume of scintillator solution determined by the following formula:
The samples were then packaged in the same manner as the digitonide disks. No adapt time was found necessary. Both compounds were counted in a sufficient range of weights to test for the presence of self-absorption or loss of efficiency with increasing sample weight. In most cases enough counts were taken to reduce the statistical error to less than 1%. RESULTS AND DISCUSSION
Figure 1. Tool for sealing samples in Saran disks
Plots of sample weights against total net counts per minute indicate no dependence of efficiency upon surface density (self-absorption) over a wide range (from approximately 1 to 35 mg.) of surface densities. Unless an excess of the scintillator solution was used, a variation in the ratio of sample weight to scintillator solution volume affected the counting rate. An empirical formula was developed in counting the osotriazole to provide just the right VOL. 34, NO. 9, AUGUST 1962
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