118 Analyst, February, 1967, Vol. 92, $$. 118-123 The Determination of Antimony, Cadmium, Cerium, Iridium and Silver in Biological Material by Radioactivation BY H. J. M. BOWEN (Chemistry Department, T h e University, Reading, Bevks.) A method for determining antimony, cadmium, cerium, iridium and silver simultaneously in a sample of biological material is described. The method involves radioactivation to long-lived radionuclides by using thermal neutrons from a reactor, and has a high sensitivity. The technique has been used to measure these five elements in a standard biological material. THE elements studied in the present work are all rare constituents of plants and animals that defy the ingenuity of the analyst. While methods of analysis are available for traces of these elements at the microgram level, they are insufficiently sensitive for determinations at the milli-microgram or micro-microgram level.Methods that are capable of determining these elements in natural biological materials are set out in Table I, with an indication of their minimum sensitivity. TABLE I SEXSITIVITY OF DETECTION OF SILVER, CADMIUM, CERIUM, IRIDIUM AND ANTIMONY, GRAMS Element r Method Ag Cd Ce I r Sb ’ Activation1* . . .. 5 x 10-9 10-9 10-9 10-11 10-10 Colorimetry2 . . . . 10-7 10-8 2 x 10-7 2 x 10-6 3 x 10-8 Mass spectrometry3 . . 2 x 10-6 3 x 10-8 4 x 10-8 9 x 10-7 Polarography4 95 .. - 1.5 x 10-G - Spectroscopy2 9 6 . . .. 10-7 5 x 10-9t 5 x 10-7 5 x 10-6 4 x 10-6 X-ray fluorescence7 . . 2 x 9 x 10-8 10-7 5 x 10-8 1.2 x 10-7 - 10-7 * Assuming a flux of 1012 neutrons cm-2 second-l.t After chemical concentration. It is clear that activation analysis is the most sensitive method available for determining these elements. It also has the advantage of avoiding contamination by reagents. Activation analysis has already been used to measure all five of the elements discussed here in biological materials, as follows- Antimony-In soft mammalian tissues8 y 9 ; in bloodlo; and in urine.ll Cadmium-In bone12; in soft tissue^^,^; and in blood.1° Cerium-In soft t i s ~ u e s . ~ , ~ ~ Iridiuwz-In soft Silver-In soft tissues.8,9 However, a full radiochemical procedure has been reported only for antimony, the other determinations being based wholly or partly on y-ray spectrometry. In this work an attempt has been made to devise a separation procedure after activation that will yield all five elements in a radiochemically pure state, and to apply the procedure to a standard biological material.NUCLEAR RESULTS *All five elements are activated to long-lived radionuclides when exposed to thermal neutrons. ,4ntimony--On activation this gives a good yield of antimony-124 (half-life 60 days), together with much antimony-122 (half-life 2-8 days) wliicli soon decays away. The 0.60-MeV y-ray of antimony-124 is convenient for counting. Tellurium and iodine are too Some characteristics of these radionuclides are as follows.BOWEN 119 rare in most biological material to interfere, although iodine in thyroids could generate antimony-124 by an (n,a) reaction. Cadmium-On activation this gives a moderate yield of cadmium-1 15m (half-life 43 days) together with cadmium-115 (half-life 2.3 days).The decay is complex and involves indium- 115m (half-life 4-5 hours). In practice, the y-rays from cadmium-115m are of low intensity and P-counting is best for its determination. Iridium and tin are too rare to interfere by com- peting nuclear reactions. Standard solutions must be very dilute to avoid self-shielding. Cerium-This gives a moderate yield of cerium-141 (half-life 32.5 days), together with cerium-143 (half-life 33 hours) which decays via praseodymium-143 (half-life 13.7 days). The 0.145-MeV y-ray of cerium-141 can be used for counting (praseodymium-143 is a pure p-emitter). Uranium could interfere as cerium-141, cerium-143 and cerium-144 (half-life 284 days) are all fission products produced with a yield of 4 to 6 per cent.(nuclides of silver, cadmium and antimony are also fission products with yields of GO-04 per cent.). Iridium-This gives an excellent yield of both iridium-192 (half-life 74 days) and iridium-194 (half-life 19 hours). The former has an 0.32-MeV y-ray, which is useful for counting. Gold and platinum are too rare to interfere by competing nuclear reactions. The iridium standard solutions must be dilute to avoid self-shielding. Silver-This gives an excellent yield of silver-1 10m (half-life 253 days) together with some short-lived activities. Indium and cadmium are too rare to interfere by competing nuclear reactions. BEFORE ACTIVATION- Samples of standard kale14 were ashed at 450" C in silica crucibles in a furnace lined with silica.The ash was thoroughly mixed with a plastic spatula, and weighed aliquot parts of about 0.7 g were placed in small silica tubes capped with aluminium foil. The silica tubes were cleaned by boiling with nitric acid, and then washed with water until no sodium could be detected in the rinsings with a flame photometer sensitive to about lO-7g of sodium. The ashing procedure could have resulted in changes in the antimony and cadmium contents of the material as these elements have volatile derivatives that could distil into, or out of, the samples at the temperature concerned. Adsorption losses might also have occurred, although the ash did not adhere to the crucible walls. Ashing before activation is an undesirable step.In this work it was necessary because of the high operating tem- perature of the reactor. Standards were prepared by dissolving weighed amounts of Johnson Matthey Specpure silver nitrate, cadmium oxide, cerium( IV) oxide, ammonium iridmm chloride [ (NH,) JrC16] and antimony in water or concentrated or dilute hydrochloric or nitric acids, distilled from a silica still, to make solutions as follows- 0.4 mg of silver per ml; 50 pg of cadmium per ml; 0.5 mg of cerium per ml; 1 pg of iridium One or two drops of each solution were then weighed into a silica tube by using a polythene transfer pipette. Alternatively, the drops were weighed on to squares of aluminium foil. The water was evaporated off at about 60" C and the tubes were capped with aluminium foil. Four samples, and two standards per element, were activated for 28 days in a flux of about 1-5 x 1012 neutrons cm-2 second-l in the Harwell reactor BEPO.REAGENTS- The 0.66-MeV y-ray is convenient for counting. METHOD per ml; and 50 pg of antimony per ml. All reagents were of recognised analytical grade. Ammonia solution, 18 N. Ammonium acetate, 30 per cent. w l v . Ammonium oxalate, 4 per cevzt. w/v. Ammonium reinzeckate, 4 per cent. W/LL (fyeshly prepared). Ammoniwn sulphide, yellow. Iron(III) nitrate, 10 per cent. w/v. Formic acid, 100 pev cent. Hydrochloric acid, 12 and 2 N. Hydrogen peroxide, 30 $ev ceiqt. w / i ~ . Iodic acid, 50 pev cmt. X J / I ! .120 BOWEN : DETERMINATION OF ANTIMONY, CADMIUM, CERIUM, [Ana,@St, VOl. 92 Yitric acid, 16 and 6 N. Sodium bronzate, saturated solution.Sodium hydroxide, 40 per cent. w l v . Sodium hypochlorite, 10 per cent. w / v . Sodium sulphite, 10 per cent. w/v. Sulphuric acid, 36 and 2 N. Teepol, 1 per cent. wIv. Thiourea, 5 per cent. v / v . Zinc acetate, 10 per cent. w / v . Zirconium nitrate, 10 per cent. w/v in dilute nitric acid. Isopropyl ether. Silver nitrate carrier solution-Prepare a 3.15 per cent. w/v solution of silver nitrate in water. 1 ml of solution = 20 mg of silver. Cadmium acetate carrier solution-Prepare a 4-74 per cent. w/v solution of cadmium acetate, Cd(C,H,O,) ,.2H,O, in water. 1 ml of solution = 20 mg of cadmium. Cerium.(IV) sulphate carrier solution-Prepare a 2.885 per cent. w/v solution of cerium(1V) sulphate, Ce(S0,) ,.4H,O in dilute nitric acid. 1 ml of solution = 10 mg of cerium(1V).ammonium chloroiridate, (NH,) ,1rC16, in water. A mmonium chloroiridate carrier solution-Prepare a 2.289 per cent. w/v solution of 1 ml of solution = 10 mg of iridium. ,4 ntimony trichloride carrier solution-Prepare a 3.545 per cent. w/v solution of antimony trichloride in dilute hydrochloric acid. 1 ml of solution = 20 mg of antimony. RADIOCHEMICAL SEPARATION SCHEME SAMPLES- Step 1-Transfer each activated sample into a 50-ml centrifuge tube and dissolve it in a few millilitres of 12 N hydrochloric acid. Add 1 ml each of antimony, cadmium, cerium, iridium and silver carrier solutions in that order, and dilute to about 3 N. Spin to collect the silver chloride precipitate and wash it twice with water. For treatment of the precipitate see step 5.Step 2-Add 1 ml of Teepol solution to the supernatant liquid, and pass hydrogen sulphide through it. Dilute the solution with water and continue to pass hydrogen sulphide until both the antimony and cadmium sulphides are fully precipitated. Spin to collect sulphides and wash the precipitate twice with 2 N hydrochloric acid. For treatment of the precipitate, see step 9. Step 3-Transfer the solution to a 150-ml beaker, add 5ml of ammonium acetate and 5 ml of formic acid, and boil for 10 minutes to precipitate iridium. Transfer the suspension to a clean centrifuge tube, spin it and wash the collected precipitate once with 12 N hydro- chloric acid and three times with water. Transfer the precipitate to a weighed counting tray, dry and weigh. Step 4-Add ammonia solution to the supernatant liquid until it is alkaline, spin to collect the cerium(II1) hydroxide and wash it twice with water.Discard the supernatant liquid. For treatment of the precipitate, see step 12. Step 5-Dissolve the silver chloride from step 1 in hot ammonia solution, add 1 drop of iron( 111) nitrate solution and spin to collect iron(II1) hydroxide. Reject the precipitate. Step 6-Pass hydrogen sulphide through the solution for 1 minute, spin to collect silver sulphide and wash it twice with 2 N sulphuric acid. Step 7-Dissolve the silver sulphide in hot 16 N nitric acid, dilute with water and add excess of sodium hydroxide. Discard the supernatant liquid. Step 8-Dissolve the silver oxide in hot 36 N sulphuric acid, dilute with water and add 0-5 ml of iodic acid solution.Spin to collect silver iodate and wash it three times with water. Transfer the precipitate to a weighed counting tray, dry and weigh. Reject the supernatant liquid. Reject the supernatant liquid. Spin and wash the collected silver oxide with water.February, 19671 IRIDIUM AND SILVER I N BIOLOGICAL MATERIAL 121 Step 9-Extract the sulphide precipitate from step 2 twice with hot ammonium sulphide, spinning to collect cadmium sulphide each time. For treatment of the supernatant liquid, see step 16. Step 10-Dissolve the cadmium sulphide in hot 12 N hydrochloric acid and boil out hydrogen sulphide. Dilute the solution to N, add 2 ml of thiourea solution, 1 drop of zinc acetate and 2.5 ml of ammonium reineckate. Spin, collect the cadmium reineckate and wash it with water.Step 11-Dissolve the precipitate in hot 16 N nitric acid, dilute the solution to N and pass hydrogen sulphide. Spin to collect cadmium sulphide and wash it twice with water. Transfer the precipitate to a weighed counting tray, dry and weigh. Step 12-Dissolve the precipitate from step 4 in 2 N hydrochloric acid and add ammonia solution until the solution is neutral. Reject the supernatant liquid. Step 13-Dissolve the precipitate in 6 N nitric acid, add 1 drop of hydrogen peroxide [to reduce any cerium(IV)], 1 drop of zirconium nitrate and 1 ml of iodic acid solution. Spin to separate zirconium iodate and discard it. Step 14-Add 1 ml of sodium bromate to the solution, boil and spin to collect cerium(1V) iodate. Reject the supernatant liquid.Step 15-Dissolve the precipitate in 12 N hydrochloric acid, and add 1 ml of sodium sulphite, 2 ml of ammonium oxalate and ammonia solution until the pH is about 6. Spin to collect cerium(II1) oxalate and wash it twice with water. Transfer the precipitate to a weighed counting tray, dry and weigh. Step 16-Add 12 N hydrochloric acid slowly and carefully to the thioantimonate solution from step 9 until it is acidic, then pass hydrogen sulphide through the solution and spin to collect the antimony sulphide. Cool, add an equal volume of water and 1 ml of sodium hypochlorite solution. Transfer the solution to a 50-ml separating funnel and extract three times with di-isopropyl ether. Reject the aqueous phase. Step 18-Add 2 N hydrochloric acid to the di-isopropyl ether and cautiously evaporate off the ether.Pass hydrogen sulphide through the solution, spin to collect antimony sulphide and wash it twice with water. Transfer the precipitate to a weighed counting tray, dry and weigh. Reject the supernatant liquid. Discard the supernatant liquid. Spin to collect cerium(II1) hydroxide. Discard the supernatant liquid. Discard the supernatant liquid. Step 17-Dissolve the antimony sulphide in hot 12 N hydrochloric acid. STAKDARDS- carrier, and hydrogen sulphide was used to precipitate antimony sulphide. carrier, and hydrogen sulphide was used to precipitate cadmium sulphide. carrier, and ammonium oxalate at pH 6 was used to precipitate cerium oxalate. carrier, neutralised with ammonia and boiled with formic acid to precipitate iridium.carrier. was finally precipitated as silver iodate. The antimony standards were dissolved in hydrochloric acid containing 1 ml of antimony The cadmium standards were dissolved in hydrochloric acid containing 1 ml of cadmium The cerium standards were dissolved in hydrochloric acid containing 1 ml of cerium The iridium standards were dissolved in hydrochloric acid containing 1 ml of iridium The silver standards were dissolved in water and nitric acid containing 1 ml of silver Silver chloride was precipitated and dissolved in ammonia solution, and the silver TESTING THE RADIOCHEMICAL PROCEDURES- The radiochemical procedures were tested by carrying out each operation with anti- mony-124, cadmium-1 15m, cerium-1 14, iridium-194 or silver-ll0m so as to estimate the chemi- cal yields and the losses involved in each step.In addition, some decontamination factors were measured by using iron-59, phosphorus-32, sulphur-35 and zinc-65 as tracers, as these are the main activities in activated biological material that has been allowed to decay for a week or so. The results of these tests can be summarised as follows. A ntimony-Chemical yield, 86 per cent. ; phosphorus-32 carried through procedure, 1.4 x Iron and zinc are removed by the precipitation of sulphides in acidic solution. per cent. The main loss of antimony occurs in the solvent extraction, step 17.122 BOWEN : DETERMINATION OF ANTIMONY, CADMIUM, CERIUM, [Analyst, Vol. 92 Cadmium-Chemical yield, 93 per cent. ; phosphorus-32 carried through procedure, 2 x Iron and zinc are not precipitated as reineckates or as sulphides in acid solution.Cerium-Chemical yield, 86 per cent. ; phosphorus-32 carried through procedure, 0-0625 per cent. The main loss of cerium occurred by adsorption on the zirconium iodate precipitate. Iridium-Chemical yield, 57 per cent. ; phosphorus-32 carried through procedure, 0.03 per cent. The most likely radioactive contaminants are other platinum metals that might be precipitated by boiling formic acid. The main loss of iridium occurred at this stage. Silver-Chemical yield, 87 per cent. ; phosphorus-32 carried through procedure, 8.3 x per cent.; zinc-65 carried through procedure, 2.7 x The main loss of silver occurred in the precipitation of silver oxide. per cent. Iron, zinc and other lanthanides are not precipitated as iodates.per cent.; sulphur-35 carried through procedure, <2.7 x per cent. RESULTS The method was used to determine the elements in four samples of standard kale powder, with the following results, expressed in terms of p.p.m. of dry kale tissue- Antimony . . . . . . 0.0930, 0.0972, 0.0976, 0.1101 mean 0.0995 Cadmium . . .. . . 0.347, 0.386, 0.392, 0.410 mean 0.384 Cerium . . . . . . 0.379, 0.430, 0.507, 0.510 mean 0.457 Iridium . . . . . . 0.0098, 0.0101, 0.0171, 0.0162 mean 0.0133 mean 0-0295. Silver . . .. . . 0.0264, 0.0235, 0-0386, There are few comparable results in the literature; results from unpublished work by F. Girardi, H. D. Livingston, H. P. Yule, J. F. C. Tyler and K. Samsahl are available for this material- Antimony .. 0-059 and 0.065 p.p.m. by activation analysis. Cadmium . . Iridium . . . . <0.021 p.p.m. and <0.0006 p.p.ni. by y-ray spectrometry after activation. Lanthanum . . Silver . . . . 0.50 p.p.m. and (0-45 p.p.m. by y-ray spectrometry after activation. 1-0 p.p.m. by polarography and 0-63 p.p.m. by activation analysis. 0.08 p.p.m. by activation analysis ; the terrestrial cerium-to-lanthanum ratio is about 3. The reasons for the discrepancies must await further analysis, preferably by other techniques. The y-ray spectrometric results are not reliable, so that the main discrepancies occur for antimony and cadmium, both of which may have been affected by the ashing procedure. DISCUSSION OF THE METHOD The method described appears to be quite satisfactory for antimony and silver,l5 especially in view of the excellent decontamination from radioactive phosphorus.For cadmium, it is advisable to repeat steps 10 and 11 in the radiochemical separation at least once, to remove traces of phosphorus-32, if the cadmium is to be determined by p-count ing . The method is not altogether satisfactory for cerium in view of the relatively poor decontamination from phosphorus-32 and the untested decontamination from other radio- active lanthanides, such as neodymium-147, europium-152, europium-154, terbium-160, dysprosium-159, erbium-169, thulium-170 and ytterbium-169. These impurities can be neglected if the sample is shielded from the counter by 3 mm of aluminium to eliminate 13-rays from phosphorus-32 and by using a scintillation spectrometer focused on the 0.14-MeV y-ray of cerium-141.Among the long-lived lanthanide radionuclides, ytterbium-169 has a 0.13-MeV y-ray and neodymium-147 a 0-16-MeV y-ray which might interfere. The method is not ideal for iridium, but more work must be carried out on the radio- chemistry of this element before a really satisfactory procedure can be evolved.16 In this. work it was expected from a study of the literature that iridium would be precipitated by silver (as AgJrCl,) and by hydrogen sulphide, but only 2-3 per cent. and 4-8 per cent., respectively, of the iridium present was lost from solution during these precipitations. Tracer studies showed that most of the small amount of iridium precipitated as sulphide was soluble in yellow ammonium sulphide solution; less than 1 per cent.of this iridium was extracted from 6 N hydrochloric acid by isopropyl ether; and less than 1 per cent. of iridium in solution co-precipitated with cerium(II1) hydroxide (step 4).February, 19671 IRIDIUM AND SILVER IN BIOLOGICAL MATERIAL 123 REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. Bowen, H. J. M., and Gibbons, D., “Radioactivation Analysis,” Oxford University Press, 1963. Meinke, W. W., Science, 1955, 121, 177. Wolstenholme, W. A., Nature, 1964, 203, 1284. Cholak, J., and Hubbard, D. M., Ind. Engng Chem. Analyt. Edn, 1944, 16, 333. Goodwin, L. G., and Page, J . E., Biochem. J . , 1943, 37, 198. Mullin, J. B., and Riley, J. P., J . Mar. Res., 1956, 15, 103. Gofmann, J. W., de Lalla, 0. F., Johnson, G., Kovich, E. L., Lowe, O., Piluso, D. L., Tandy, Samsahl, K., Rrune, D., and Wester, P. 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