Estimation of Molybdenum in Biological Material by Neutron Activation Analysis H. D. Livingston and Hamilton Smith Department of Forensic Medicine, Glasgow University, Glasgow, W.2, Scotland
MOLYBDENUM OCCURS in biological materials in amounts which require a highly sensitive analytical technique such as activation analysis for reliable estimations. 1olM0 (TII2-14.6 minutes) and g g M(7'~112-67 hours) are produced on thermal neutron irradiation of naturally occurring molybdenum. g9Mois the more useful isotope for activation analysis as it gives greater sensitivity and more time for processing a batch of samples. The natural abundance of 98Mois 23.8 and the thermal neutron capture cross-section is 0.12 barn. The equivalent figures for looMoare 9.6 and 0.02 barn. 99Mo may be measured by P- or y detection. The latter was used in this work because it permitted detection of 99mTc (T1/2-6.00 hours)-the daughter isotope of 9 9 M ~ . g9mTc is a monoenergetic y-emitter and is the most abundant y-ray in the spectrum of g9Mo(photopeak maximum at 0.14 MeV). The advantage of using 99mTclay in its relationship with its parent isotope 99Mo. At the final stage of the radiochemical separation, a separation step for technetium was made twice under the same conditions. During the first separation all the daughter 99mTcactivity was separated plus other nuclides extractable under the chosen conditions. After a delay to allow re-equilibration of the 99Mo/Tc9" couple, the second extraction separated the ggmTc radiochemically pure. EXPERJMENTAL AND RESULTS
Samples and Standards. Samples analyzed by this technique included human teeth, dry human tissues, dry vegetable tissues, and dried, powdered kale. The latter (obtainable from H. J. M. Bowen, University of Reading) is available as a homogeneous biological standard. To avoid sample contamination the only pre-irradiation treatment used was vacuum drying of wet human and vegetable tissues. For irradiation, samples were weighed and wrapped in aluminum foil. Two forms of standard were used and both found suitable. The first was a solid standard which consisted of about 1 mg of high purity molybdenum trioxide, weighed and heat sealed in a silica ampoule. The second was a solution of either ammonium molybdate (ANALAR) or molybdenum trioxide. About 0.1 ml of either solution (containing about 1 mg of molybdenum) was weighed and sealed in a silica ampoule as for the solid standards. Samples and standards were packed with silica wool in an aluminum irradiation can. Irradiations were made for 3 or 7 days in a reactor with a thermal neutron flux of 1012n/cm2/sec. After irradiation the molybdenum standards were dissolved and diluted to 1000 ml. One milliliter of the resultant solution was taken and processed with the samples as the molybdenum standard. Reagents. High purity reagents were used where possible. Cupferron solutions are not stable and were prepared freshly when required. Chemical Separation. After irradiation each sample was placed in a 125-1111 conical beaker containing 3 ml of 18M sulfuric acid and 5 mg of molybdenum carrier (as ammonium molybdate solution). The sample was heated until white fumes of sulfur trioxide appeared and then solid sodium 538
ANALYTICAL CHEMISTRY
nitrate (20 to 30 mg) was added to oxidize charred organic material. When the solution was clear it was allowed to cool and was diluted to 30 ml with water. If any calcium sulfate precipitated it was centrifuged off and discarded. The solution was transferred to a 100-ml separating funnel and 2 ml of ammonium cupferrate solution ( 5 % w/v) were added. The white precipitate of molybdenum cupferrate which formed was extracted into 15 ml of chloroform. Cupferron is a reagent which is known to give quantitative separations of molybdenum ( I ) . After the chloroform layer was washed with dilute acid in a fresh funnel, it was transferred to a 125-ml tall form conical beaker. The chloroform was evaporated by heating on a hot plate and the black organic residue which remained was re-digested as for initial sample digestion. This digestion step was necessary to destroy the cupferron extracted with the molybdenum. Although molybdenum could be directly back-extracted from chloroform into sodium hydroxide solution, the presence of cupferron was found to interfere with the molybdenum recovery estimation, On obtaining a clear solution, the sulfuric acid was evaporated to dryness leaving a blue residue. This residue was dissolved in 10 ml of 1M sodium hydroxide and 100 pg. of rhenium were added, as a carrier for technetium. The solution was transferred to a separating funnel, two drops of tetraphenylarsonium chloride solution (2% w/v) were added, and the resulting solution was extracted with 15 ml of chloroform. The organic layer was discarded, the aqueous layer washed with a further portion of chloroform and transferred to a 50-ml centrifuge tube containing 10 mg of rhenium carrier. In the extraction step described above technetium and rhenium are completely removed from molybdenum which remains in the aqueous phase (2). The sodium hydroxide solution was allowed to stand for 18 hours to permit 99mTcgrowth. This was the minimum time required for 99Mo/99mT~ equilibration. After this waiting period, 2 ml of tetraphenylarsonium chloride solution (2% w/v) were added. It was shown that this volume of reagent was sufficient to precipitate all of the rhenium and technetium from solution, leaving all of the molybdenum in the supernate. The precipitate was centrifuged off and the supernate plus washings were saved for molybdenum recovery estimation. The precipitate was washed with water and iso-propanol. It was finally transferred to a weighed aluminum planchette with iso-propanol and dried under an infrared lamp. The weight of the tetraphenylarsonium perrhenate precipitate gave the chemical recovery of the separated technetium. Tracer experiments with the long lived 9 9 Tisotope ~ and 99mT~ derived from 99Mo, showed that, under the experimental conditions, rhenium acted as a valid carrier for technetium. This was demonstrated by the agreement between chemical recoveries of rhenium and radiochemical recoveries of technetium. Some workers (3) recommend the use of hydrogen peroxide in
(1) N. H. Furman, W. B. Mason, and J. S. Pekola, ANAL.CHEM., 21, 1325 (1949). (2) S.Tribalat and J. Beydon, Anal. Chim. Acta, 8,22 (1953). (3) Radiochemistry of Technetium, National Academy of Sciences Publication, U.S.A.E.C., NAS/NS 3021, p. 15.
technetium separations using tetraphenylarsonium chloride to ensure that the element remains in the VII-valent extractable state, viz., TcOh-. It was not found necessary because separations both in the presence and absence of peroxide gave the same yields of :racer. Molybdenum Recovery. The aqueous fraction remaining after separation of rhtnium and technetium was analyzed by colorimetry to g h e the molybdenum recovery. The combined supernate 2nd washings following perrhenate precipitation were dilated to 100 ml with water. A 10-ml aliquot of the diluted solution was placed in a 100-ml separating funnel containing a bout 10 ml of chloroform, The pH of the solution was adjusted to about 2 with 2M hydrochloric acid and 1 ml of potassium ethyl xanthate (4)solution (10% w/v) was added. The purple precipitate of molybdenum ethyl xanthate was extrs cted into chloroform and the extract plus washings made u p to 20 ml with chloroform. The absorbance of the resulting solution was measured at 520 mp with a colorimeter using 5-mm quartz cells and a Kodak No. 4 filter. The color was found to be stable and Beer’s law was obeyed. Recoveries of molybdenum were usually greater than 80%. Recoveries of rhenium were normally in the range 95-100 %. Activity Measurements. The activity of the tetraphenylarsonium perrhenate precipitates was measured using a sodium iodide (Tl) cryst31 and a pulse height analyzer. The y-spectra obtained from samples which contained detectable amounts of molybdenum such as liver, kale, or parsley showed ~ at 0.14 MeV. The radiochemical only a 9 9 m Tphotopeak purity of the separated activity was confirmed by measuring the decay of this photopeak using an automatic decay follower. The decay obtained showed the characteristic 6.00 hour halflife of egmTc. Calculation of Molybdenum Content. The molybdenum content of a sample was calculated by comparison of the 9 9 m Tphotopeak ~ activity of sample and standard after various corrections had been made. The measured activity was corrected for background radiation, technetium recovery, molybdenum recovery, end technetium decay. It was convenient to make the technetium decay correction to the time of gg**Tcseparation from the 99Moparent but if all the separations are made at the same time, decay corrections relative to each other are sufficiert. A 99Mo decay correction is not necessary if separation of technetium is made from all samples within a short period but would be required if the separations were distributed over a period greater than 1 to 2 hours. Precision and Accuracy. AnalyTes were made on standard amounts of molybdenum Varying amounts, of a Specpure molybdenum trioxide solution, were weighed, sealed, and irradiated in silica ampoules. The contents of each ampoule
were analyzed for molybdenum after irradiation using a normal molybdenum standard. The agreement found between the actual and measured amounts of molybdenum gave an average error between the actual and measured value Of 3.5 %. A series of replicate analyses was made on a powdered kale biological standard. The molybdenum content of the kale was estimated as being 2.59 f 0.23 p.p.m. The molybdenum content of the kale has been previously estimated by other workers as being 2.33 =t0.47 ppm (5). These results seem to be in reasonable agreement despite the rather wide spread. Sensitivity of Analysis. The experimental limit for molybdenum detection at a thermal neutron flux of lO”h/cmz/sec was found to be 0.01 pg. For samples containing smaller amounts of molybdenum, analysis became possible when iradiation was made at a thermal neutron flux of 1014n/cm2/ sec. For this irradiation samples were enclosed in a silica ampoule 2.5 cm in length and 3 mm in internal diameter. The standard was about 100 pg of Specpure molybdenum trioxide and was enclosed in the ampule in a sealed silica capillary. Silica wool was used to separate samples in the ampoule. The limit of molybdenum detection using this high flux irradiation was found to be increased to gram. DISCUSSION
By using the favorable parent/daughter half-lives of the nuclides in the 99Mo/agmTc couple, it was found possible to devise a separation producing ggmTcin a radiochemically pure form. The second separation of newly equilibrated 99*Tc under the same conditions as the first separation ensured that no radio-contaminant could be inadvertently separated and counted. By use of this analytical scheme it was possible to make highly sensitive molybdenum analyses of biological material. The method benefited from all the usual advantages of activation analyses-viz., avoidance of blanks or microseparations and the opportunity of analyzing a large number of samples during one analysis. ACKNOWLEDGMENT
The authors thank Gilbert Forbes, George Nixon, and J. M. A. Lenihan for the help and encouragement given throughout the course of this work. RECEIVED for review December 19, 1966. Accepted January 30, 1967. H. D. Livingston received financial support from the Scottish Hospitals Endowment Research Trust.
(4) F. Pavelka and A. Laghi, Mikrochem. Mikrochim. Acta, 31,
138(1943).
( 5 ) H. J. M. Bowen, Analyst, 92, 124(1967).
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