Pass the solution through the bed of Dowex 50 a t 0.8 ml. per minute. Wash the bed with several column-volumes of water. Elute yttriuzn and the rare earths with 100 ml. of 5Y0 citric acid, pH 3.i0, a t 0.8 ml. per minute, and discard the rare earth fraction. Elute actinium with 35 ml. of 5% citric acid, pH 6.2, a t 0.8 ml. per minute. Add 2 ml. of indicator to the actinium fraction and adjust the acidity t o p H 2 with concentrated nitric acid. Transfer the solution to a separatory funnel and extract fcr 2 minutes with 50 ml. of l.5M EHPA. Discard the aqueous phase and wash the solvent twice with 15-ml. liortions of 0.lM nitric acid. Strip the actinium from the solvent by extracting for 2 minutes with two 20-ml. portions of 0 831 hydrobromic acid. S o t e the time and discard the solvent. Combine the two strip solutions in a separatory funnel arid extract for 2 minutes with 50 nil. of 3oyO Aliquat-336. Draw off the aqueous phase into a beaker. Wash the solvent with 5 ml. of 0.8-11 hydrobromic acid and add the wash 5olution to the beaker. Discard the solvent. Evaporate the solution to dryness and bake on a high-temperature hot plate to char any organic residue. Add 10 ml. of concentrated nitric acid and evaporate to 1 ml. Transfer the solution t o a 2-inch itainless steel planchet mith an eye dropper and evaporate t o dryne- under a heat lamp.
Rinse the beaker with a little concentrated nitric acid and evaporate this solution on the planchet. Flame the planchet over a burner and alpha count. Follow the in-growth of alpha activity over a period of days, with elapsed time calculated from the time previously noted. Using an actinium yield of 8470 and the in-growth rate given in Table I, calculate the actinium activity. DISCUSSION
To determine the actinium recovery, sixteen samples of known actinium content were analyzed by the procedure. The results are given in Table 11. To evaluate the procedure, samples of varying actinium concentrations were analyzed. The results are given in Table 111. Effluents from five different mills were also analyzed. All five mills used an acid leach process and the samples were all unneutralized mill effluents. The data are given in Table IV. Decontamination factors for uranium and uranium daughter activities were determined. These factors are given in Table V. If it is known that the solution to be analyzed contains less than 5 p.p.m. of cerium-group rare earths, the ion exchange step can be eliminated. Instlead, the yttrium hydroxide is dis-
solved in acid, the acidity adjusted to pH 2, and the procedure continued from the point where the actinium fraction was collected from the ion exchange column. The method is also applicable to the analysis of ores and ore residues. The ore sample should be dissolved in a nitric acid-hydrofluoric acid mixture, fumed with perchloric acid, and the solution diluted to 200 ml. with water. This solution can then be treated in an identical manner as a mill solution. Several ore samples were analyzed in this manner. The results for two ore samples, one a standard pitchblende sample known to be in equilibrium, and the second a carnotite ore from Colorado, of unknown history, are given in Table VI. LITERATURE CITED
(1) Hagemann, F., Symposium on “The
Chemistry of the New Elements,” Division of Physical and Inorganic N. Y., Chemistrv. A.C.S.., Syracuse, ” June 30, 1948. ( 2 ) Peppard, D. F., Mason, G. W., Maier, J. L., Driscoll, W. J., J . Inorg. Nucl. Chem. 4, 334 (1957). (3) Petrow, H. G., Sohn, B., Allen, R. J., ANAL.CHEX33, 1301 (1961). RECEIVED for review October 18, 1962. Accepted March 4, 1963. Work supported by U. S. Atomic Energy Commission, Division of Biology and
Medicine, under Contract AT(30-1)-2470.
Estimation of Strontium-84 in Biological Material by Neutron Activation Analysis HAMILTON SMITH Department of Forensic Medicine, The University of Glasgow, Scotland, and Western Regional Hospital Board, Regional Physics Department, Glasgow, Scotland
b Neutron activation analysis combined with chemical separation and scintillation spectrometry is a quick, accurate method for the estimation of added strontium-84 in biological materials. After nitric acid digestion of the activated sarriple a precipitation separation i s combined with a gravimetric yield dei,ermination. The activity is detected by counting over a limited y-energy raiige.
N
studies in calcium and strontium metabolism were made by administering radioactive isotopes of either element and then detecting its presence in biological samples by direct counting methods. This practice was not allowed in some types of people, for example young children, ORMALLS,
so it was necebsary to use a rare stable isotope of reasonable cost which could be used in place of the unstable one and which could be detected easily in small amounts against the normal body background. This is most important because the intake of even a milligram of strontium may upset the normal balance and so make the studies invalid. Strontium enriched 50% in respect to strontium-84 was used. The problems in the quantitative determination of strontium-84 in biological material were: the development of an efficient separation procedure; the yield recovery; and the activity determination using a r-energy scintillation spectrometer. The following outline covered these points and acted as a basis for the investigation. After neutron activation samples were
placed in a beaker with some inactive carrier and dissolved in IGN nitric acid, calcium was removed by precipitation with potassium ferrocyanide and then barium was removed by precipitation as the chromate. The strontium n-as finally precipitated as the carbonate and the recovery was determined gravimetrically. The activity of the strontium-85 was measured using a scintillation spectrometer and compared with a standard. EXPERIMENTAL
Preparation and Irradiation of Samples. Calcium oxalate precipitates were prepared from body fluids or digested materials and, after careful purification by solution in nitric acid and reprecipitation of the oxalate by ammonium oxalate in neutral solution VOL. 35, NO. 6, MAY 1963
749
Table 1.
Effect of Ferrocyanide on Calcium and Strontium under Varying Conditions
Series
Isotope present
1
Calcium-47
2
Calcium47
3
Calcium47
4
Calcium47
5 6
Strontium-85 Strontium-85 Strontium-85 Strontium-85
7
8
or one slightly alkaline with ammonia, the weight was determined. The oxalates of calcium and strontium were completely precipitated under these conditions (8). About 40 mg. of the samples were weighed into aluminum tubes which were then sealed. A sample (about 0.3 gram) of high purity strontium carbonate was used as the standard as the natural abundance of strontium-84 has been established a t 0.56% (2). The samples and standard were packed into a standard aluminum can and irradiated in a reactor a t a thermal neutron flux of 10l2 neutrons per square em. per second for 1 week. The unit was returned and processed as described below. The standard was diluted as necessary. X o accurate knowledge of neutron flux or activation cross section was required as the method is an example of the standard comparator method of activation analysis in which measurements are gauged against a standard sample, irradiated a t the same time and in the same unit pack as the samples, and in which after chemical recovery, yjelds are standardized. Strontmm Isotopes. Three isotopes were available for study by activation ) They analysis using the ( n , ~reaction. were as follows: Srs4(n,-y)Sr86 Srs8(n,-y) fir8* (n,?)Sr89
Strontium-87m rapidly disappeared from the activity of the irradiated natural element due to its short half life of 2.8 hours. Strontium-89 (t1/*-51 days) and strontium-85 (t1/*-65 days) gave comparable activities when the natural element was irradiated though the natural abundancies were 82.56% and 0.56%, respectively. There was the possibility of interference when estimating strontium-89 in yttrium matrix produced by the reaction Ye9 (n, p ) Sr*9 and in a zirconium matrix by the reaction Zr9* (n, cy) Sr*9. There was also the possibility of interference from second order reactions or from fission products. In the production of strontium435 there was no apparent interference. The cross section for
750
ANALYTICAL CHEMISTRY
Precipitating conditions Ammonium ferrocyanide and ammonium salts from earlier steps Same as series 1 excess ammonium chloride Potassium ferrocyanide and ammonium salts from earlier steps Same as series 3 excess ammonium chloride As series 1 As series 2 As series 3 As series 4
+
+
Recovery in supernatant, 7 0 0
0 0 C-1
52-69 61-75 82-91 80-92
thermal neutron capture by strontium84 was 1.4 barns. Reagents and Apparatus. Where possible, “AnalaR” reagents were used. Measurements of activity were carried out using a Marshall 100-channel pulse height analyzer and 21/2-inch crystal. Preliminary Separation of Samples. The active calcium oxalate was placed in a beaker with 20 mg. of strontium carrier and dissolved in the minimum volume of 16N nitric acid. Any oxalate t h a t remained and which would interfere in the following precipitation was destroyed by boiling the acid solution with 2 ml. of sodium bromate solution (saturated a t room temperature) for a few minutes. If all the oxalate was not destroyed a precipitate formed when ammonia was added in the next step. The solution was then made alkaline with 15N ammonia solution, boiled for a few minutes and transferred to a 50-ml. centrifuge tube. Acetic acid (0.1.V) was added until the solution was neutral to bromo-thymol blue. The tube was placed on a boiling water bath for a few minutes and calcium was precipitated by adding 10 ml. of 0.331 potassium ferrocyanide. Heating was continued for a further 10 minutes, then the precipitate was removed by centrifuging and the supernatant was filtered into clean tubes for the next step. The precipitate m-as calcium potassium ammonium ferrocyanide and was of variable composition (1j. There was some loss of strontium a t this step, but this was considered reasonable to obtain a good separation with a single simple technique. Greater losses were found if the concentration of ammonium salts increased-e.g., when the precipitation of calcium was attempted using ammonium ferrocyanide. Table I shows the effect of varying the precipitating conditions. Filtration after centrifuging is recommended a? this removes any small particles of precipitate which may have remained on the surface of the liquid or on the sides of the tube. Wetting agents may be used to make centrifug-
ing complete, but they are not so reliable. The ferrocyanide precipitate was often found in small amounts in solutions which had not been filtered even though reasonable quantities of wetting agent (Teepol) were present. Separation of Barium. The filtrate from the previous step was made alkaline with 1 ml. of 1 5 s ammonia and strontium precipitated by adding 1 ml. of oxalic acid solution (saturated a t room temperature). The precipitate was centrifuged from solution, washed with water, and dissolved in 16Ar nitric acid. The solution was transferred to a beaker and the remaining oxalate was destroyed by heating with sodium bromate on a boiling water bath as described above. Ammonia (15iV) was added and the heating was continued for a few minutes, then the solution was neutralized with 0.1N acetic acid, and 20 mg. of barium carrier was mixed with the solution and precipitated with 0.5 ml. of 0.5M potassium chromate solution. The precipitate was spun down and the supernatant was filtered into a clean tube for the final separation. Under these conditions barium chromate was insoluble and strontium chromate was soluble (4). There was some carrying down of the strontium on the barium precipitate so there was a loss a t this step varying from 5 to 15y0. Final Precipitation of Strontium. The filtrate was made alkaline with 1 ml. of 2 N ammonia, and strontium was precipitated as the carbonate b y adding 4 ml. of 1M sodium carbonate solution. The tube was heated for 10 minutes on a boiling water bath and then centrifuged. The precipitate was washed twice with water and once with acetone. Strontium carbonate was insoluble (3) in the reaction medium so there was no loss except in the transfer stage, and this depended on the care taken. RESULTS A N D DISCUSSION
Recovery and Activity Estimation.
It was possible t o calculate the recovery gravimetrically from the 20 mg. of strontium carrier. dfter washing with acetone the precipitate was slurried into a weighed planchet with acetone and dried under infrared lamps, the temperature being 90” C. The activity of the samples, with respect to strontium-85, was estimated by counting pulses across the peak activity around 0.51 m.e.v. The strontium-84 content was calculated by comparing the recovery weight and count rates with the standard sample. There was no significant interference from strontium-89 as the initial total activity due to this isotope was much lower than the strontium45 isotope, and the only y-ray from it (0.91 m.e.v.) occurred to the extent of about 0.01%. No other activities were observed in strontium separated from biological material when the usual y-energy spectrum was examined.
Sensitivity. The application of activation analysis t o strontium-84 micro estimations allowed amounts of the order of gr:tm to be detected with ease. As the half life was relatively long, the samples could be processed in large batches and their activity detected later when all the samples were completed. Tests on solutions of known strontium-84 content gave experimental results which were in the range ztlyo of the (calculated value. Biological samples varying in known strontium-84 content from gram to 10-6 gram were malyzed by the above method and gave experimental results which were in the range *2Q/, of the calculated value.
CONCLUSION
This type of analysis was made possible by the use of activation analysis and made simple by the ability to add relatively large amounts of carrier strontium, thus removing the need for micro separation techniques. The mixed ferrocyanide was useful in the separation of calcium from barium and strontium.
ACKNOWLEDGMENT
The author thanks John Glaister, J.M.A. Lenihan, and Edgar Rentoul
for support and laboratory facilities during the investigation. LITERATURE CITED
(1) Feigl, F., “Spot Tests in Inorganic Analysis,” 5th Ed. p. 220, Elsevier,
London and New York, 1958.
(2) Nier, A. O., Phys. Rev. 54,275 (1938). (3) Sunderman, D. X., Townley, C. W.,
U. S. At. Energy Comm. Rept. NAS-NS 6, 1960. (4)Vogel, A. I., “Qualitative Inorganic :Analysis,” 4th Ed., p. 302, Longmans, .Green, and Co., London and New York, ,1954. 3010 p.
RECEIVED for review September 17, 1962. Accepted January 29, 1963. The work was supported by a grant from the Medical Research Council.
Identificatiion and Origin Determinations of Cannabis by Gas aind Paper Chromatography T. W. M. DAVIS and C. G. FARMILO Organic Chemistry and Narcotic Section, Food and Drug Directorate, Department of National Health and Welfare, Ottawa, Ontario
MIROSLAW OSADCHUK The Regional laboratory, Food and Drug Directorate, Toronto 5, Ontario, Canada
b The cannabinols present in the leaves and flowering tops ccin be characterized by relative retention times and Rf values. Pyrahexyl, a commercially available tetrahydrocannabinol homologue, may b e used as a reference. The Beam reaction diffcrentiatescannabidiol from the other ccinnabinols in gas chromatographic fralctions and on paper chromatograms. This test may now be considered ,characteristic of cannabis. A plot of peak areas of cannabidiol vs. cannabinol plus tetrahydrocannabinol permits differentiation between cannabis of different geog ra p hica I origins.
T
FOLLOWING !cannabinols are contained in Cannabis sativa L.: HE
i=-0
CH/
‘CH3
Tetrahydrocannabinol (THC)
Cannabinol (CBN)
extract of cannabis. Crystalline THC derivatives have been described (3, 16). Some doubt still exists regarding the position of the double bond in the cyclohexene ring of CBD, CBDA, and THC, although it is not conjugated ( 1 , 19) with the benzene ring. It would be useful for court purposes to present evidence particularly of the presence of T H C in cannabis samples. THC is assumed to be a mixture of isomers whose structures differ, both in the position of the alicyclic double bond and in optical and steric properties ( I S , 19, 20). THC has paper and gas chromatographic values similar to those of the low-melting synthetic standard ( 5 ) . In reviews of the analytical chemistry of cannabis, it was shown (2,6) that further characterization of cannabinols was required. Results of recent work on the isolation, purification, and identification of cannabis extracts from plants of different origins by means of paper and gas chromatography are reported here. Their use in the determination of origin is discussed.
Cannabidiol (CBD)
Sample Preparation. Fresh cannabis samples of Canadian origin were stored a t - 30” C. for 15 months. The samples were air-dried just before analysis. The other samples were received as dry material. At the time of analysis, the ages of the samples in months were: Canadian Seizure and
Reviews of the chemistry and pharmacology of CBD, CBN, and T H C (14, 20), and of CBDA (17), show certain properties of the cannabinols which are important to the drug analyst faced with the problem of identification of large numbers of marihuana samples of different origins. CBD, CBDA, and CBN are solids with definite melting points, which suggests that they are homogeneous, pure, crystalline materials. THC, the physiologically active component (14, 21), however, was obtained until recently as a resin or viscous oil. Two synthetic T H C isomers, m.p. 62’ to 63’ C. and 125’ C., have been reported (11), along with a crystalline THC from plants, (m.p. 120’ to 125’ C.). An earlier worker ( 9 ) had isolated a similar crystalline substance from an
c-0 C H j ‘CH3
EXPERIMENTAL
Cannabidiol acid (CBDA)
VOL. 35, NO. 6, MAY 1963
751
