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
1\75
amount of solution to eliminate any loss in efficiency due t,o this cause. The activity loss after a four-month period of storage in B freezer a t -8' C. was determined to be 13%. The osotriazole was counted a t an efficiencv of more than 45% when first mad; up; the absolute activity was determined according to the method of Wilzbach and Sykes (6'). In preliminary experiments, cholesterol-HS adsorbed on filter paper was counted with 20% efficiency. While this is not a real test of the efficiency of an insoluble tritium-labeled sample, it did indicate that the sealed disk was convenient for tritium counting. D-Glucose phenylosotriazole and cholesterol digitonide were chosen as insoluhle, colorless, easily prepared derivatives of physiologically important
compounds. An attempt was made to count the intermediate phenyl+ g~ucosazone, but, as expected, the intense yellow color precluded scintillation counting.
(4) Hassid, W. Z., McCready, R. M., IND.ENQ.CHEM.,ANAL. ED. 14, 683 (1942). (5) Wilzbach, K. E., sykes, W. y., Science 120, 494 (1954). (6) Windaus, A., Ber. Deut. Chem. Ges. 42,238 (1909).
ACKNOWLEDGMENT
The author is indebted to F. C. Hickey, O.P., for his determination of the absolute activity of the D-glucose phenylosotriazole and to W. M. Stokes for his preparation of the cholesterol and glucose derivatives. LITERATURE CITED
(1) Funt, B. L., Can. J . Chem. 39, 711 (1961). (2)' Funt, B. L., Hetherington, A., Science 131, 1608 (1960).
(3) Hann, R. M., Hudson, C. S., J . Am. Chem. SOC.66, 735 (1944).
EDWARD T. GRIMES' Medical Research Laboratory Providence College Providence 8, R. I. Present address, 24 West Hunt St., Central Falls, R. I. TAKENfrom Senior Research carried out aa part of the N. I. H. Honors Science Program, Providence College. Work supported in part by grants (20-454) from the Division of General Medical Sciences, (C-2851) from the National Cancer Institute, U. S. Public Health Service, and the American Cancer Society, Rhode Island Division.
Spectrophotometric Determination of Zirconium in T horium-Zirconiu m Alloys SIR: In hydrochloric acid solution, zirconium is precipitated by p ( p dimethylamin o p h en ylaz 0 )benzenearsonic acid (pararsonic acid), DPB (1, 3, L), When the precipitate is treated with either a basic (2, 4) or an acid fluoride solution (3), the free DPB, which is yellow in basic solution and red in acidic solution, is released. The red color is somewhat more sensitive, Figure 1. Procedures based upon these reactions have been described for the determination of zirconium in steel (2) and uranium (3). Although thorium is also precipitated to a small extent under conditions leading to the formation of the zirconium complex (1, 4 ) , we have found that the coprecipitation is constant and reproducible for a wide range of thorium concentrations when specific precipitation conditions are used. Thus, low zirconium-thorium alloys can be analyzed simply and nccurately.
mixture, now 1.80N in hydrochloric acid, on a steam bath for 60 minutes. Filter the hot solution using vacuum through a fritted-disk filter of fine porosity. Wash the precipitate four times with 10-ml. portions of 0.1N hydrochloric acid. Dissolve the precipitate with two 25-m1. portions of a mixture composed of 50 ml. of concentrated hydrochloric acid and 2 ml. of 1% hydrofluoric acid. Wash the filter with several small portions of water. Dilute the filtrate and washings to 250 ml. with water. Measure the absorbance in 1-cm. cells a t 560 mp against water as a reference. Standard Curve. Precipitate 50 to 1000 pg. of zirconium in 45 ml. of 1.33N hydrochloric acid containing 100 mg. of thorium with 5.0 ml. of the DPB reagent. Digest and dissolve the precipitate, dilute the solution, and measure
PROCEDURE
Samples. Dissolve 0.1- to 1-gram samples of alloy in 10 ml. of 7Oy0 perchloric acid, 10 ml. of concentrated nitric acid, and 2 ml. of 1% hydrofluoric acid. Fume strongly t o near dryness, dissolve the salts in 10 ml. of concentrated hydrochloric acid, and dilute the solution to 100 ml. To an aliquot of 40 ml. or less containing 100 to 1000 pg. of zirconium and 25 to 250 mg. of thorium, add hydrochloric acid and water to bring the volume to about 45 ml. and the quantity of hydrochloric acid to 60 meq. Add 5.0 ml. of 0.5% DPB in 6N hydrochloric acid. Digest the resulting 1176
0
ANALYTICAL CHEMISTRY
W A V E L E N G T H , Yp
Figure 1 . Absorption spectra of acidic and basic forms of DPB. Precipitation of 100 pg. Zr as in procedure
Table 1.
Analyses of Zirconium-Thorium Mixtures
Zirconium, % Present 0.050 0.100 0.200 0.500 0.800 1.50 2.00
Found 0.050 0.100
0.208 0.502 0.812 1.51 1.98
the absorbance as described above for samples. The standard curve is linear. RESULTS AND DISCUSSION
Analysis of synthetic zirconiumthorium mixtures gave the values shown in Table I. Tungsten, tantalum, niobium, scandium, vanadium, plutonium, fluoride, phosphate, large amounts of sulfate, and strong oxidizing agents are potential interferences. Contrary to the experience of Grimaldi and White (I), it has been found possible to prevent fluoride interference by fuming with perchloric acid in the sample preparation. With the 5.0-ml. volume of DPB reagent specified, the thorium concentration can range from 25 to 250 mg. in the final 50-ml. volume. With 10.0 ml. of reagent, only 25 to 150 mg. of thorium can be tolerated without significant effect on the absorbance. Digestion periods shorter than 45 minutes increase the thorium con-
