506 BOWEN AND CAWSE: DETERMINATION OF SODIUM, POTASSIUM AND [VOl. 86 Determination of Sodium, Potassium and Phosphorus in Biological Material by Radioactivation BY H. J. M. BOWEN AND P. A. CAWSE ( U . K . Atomic Energy Authority, Wantage Research Laboratory, Wantage, Berks.) Neutron-activation analysis has been applied to the determination of sodium, potassium and phosphorus in biological material. When a flux of 1012 neutrons per sq. cm per second for activation and an anti-coincidence counting unit were used, the ultimate limits of sensitivity for the three elements were approximately 10-lo, and 10-lo g, respectively. Radio- chemical separation procedures were used, and it was possible to analyse eight samples for all three elements in an 8-hour working day. WHEN biological material is exposed to thermal neutrons, a large percentage of the induced radioactivity is produced by four nuclides, viz., sodium-24, phosphorus-32, chlorine-38 and potassium-42 ; the half-lives and characteristic radiations of these nuclides are shown in Table I. The only other nuclide frequently contributing a major proportion of the induced TABLE I HALF-LIVES AND RADIATIONS OF "a, "P, 38c1 AND 42K Maximum Nuclide Half-life beta energy, Gamma energy, MeV MeV . . 15 hours 1.39 1-37 and 2.76 Sodium-24 . . .. Chlorine-38 . . .. . . 35 minutes 4.81 1.60 and 2.15 Potassium-42 .. . . 12.4 hours 3-60 1.53 Phosphorus-32 . . .. 14 days 1.71 - activity is manganese46 (half-life 2.6 hours), the gamma spectrum of which can often be observed after plant material has been exposed to neutrons.The gamma spectra of activatedAugust, 19611 PHOSPHORUS IN BIOLOGICAL MATERIAL BY RADIOACTIVATION 507 tomato seeds at three different times after activation are shown in Fig. 1, in which the decay of the peak caused by manganese-56 at 0.85 MeV can clearly be seen; the peaks caused by sodium-24 and potassium-42 are also prominent. Several methods are available for determining the relative contributions of these nuclides to the total activity. Keynes and Lewis1 and Reiffel and Stone2 have utilised simple forms of beta spectroscopy. Chlorine-38 was allowed to decay away, and the hard P-rays from the potassium-42 were determined by counting through a filter sufficiently thick to eliminate the softer /?-rays from the other nuclides. Phosphorus-32 was then determined as the residual beta-activity after decay for 1 week, and sodium-24 was found by difference.Gamma ~pectroscopy3~~~~ is another possible method of analysis, but, unfortunately, it cannot be used 1 I I 1 1 1 0 I .o 2.0 3. Gamma energy, MeV Fig. 1. Gamma spectra of activated tomato seeds: curve A, 3.5 hours after activation; curve B, 7.5 hours after activation; curve C, 25 hours after activation to measure phosphorus-32 (a pure beta-emitter), and the main gamma energies of sodium-24 and potassium-42 are so close together that they are not well resolved by most gamma spectrometers. A third technique, used by Salmon,B involves radiochemical separation of the three nuclides after the addition of carriers. We have used this principle in the work described here; it requires more time and effort than do the other techniques, but is necessary to attain maximum accuracy and sensitivity.It is therefore suitable for analysing small samples of biological material, but offers no obvious advantages when large samples are available. A rapid radiochemical separation of chlorine-38 has been described elsewhere? and will not be discussed here. IRRADIATION- Liquid standards were sealed into 6-cm lengths of polythene tubing (0.5 mm bore), and samples of seeds were sealed in polythene film and then packed in polythene bags in 3-inch x 1-inch aluminium cans. (Before it was filled, the polythene tubing was washed with 6 N hydrochloric acid and then with water distilled from quartz apparatus; it was subse- quently handled with clean Perspex forceps).Each can was irradiated for 15 hours in a flux of about 1012 thermal neutrons per sq. cm per second in the Hanvell reactor BEPO. This period of irradiation activates about half the theoretical maximum number of sodium and potassium atoms, but only 3 per cent. of the corresponding number of phosphorus atoms. In order to attain higher sensitivity for phosphorus, activation should be for about 14 days, and all samples should then be sealed in silica rather than in polythene, which degrades after several days inside the reactor. EXPERIMENTAL508 [Vol. 86 STANDARDS- The standards used consisted of 0:005-ml aliquots of a solution containing 0.5 pg of sodium and 1Opg each of potassium and phosphorus. The standards were prepared by dissolving the calculated amounts of Specpure sodium hydrogen carbonate and potassium carbonate and analytical-reagent grade ammonium dihydrogen orthophosphate in water distilled from quartz apparatus ; they were stored in polythene bottles.Self-shielding during activation was negligible. Some standards were also prepared by absorbing aliquots of the solution in small squares of Whatman No. 541 filter-paper. This technique was satisfactory for amounts of potassium and phosphorus down to lO-'g, but was useless for sodium because of the amount of this element in the paper. Attempts to wash this residual sodium out of the paper were un- successful. It was estimated that the filter-paper contained about 0.35pg of sodium per sq. cm, which is within the range of values measured by Born and Stark.* Possibly, the sodium-24 found was produced by an (n,a) reaction from aluminium present in the paper.PRELIMINARY TREATMENT AFTER ACTIVATION- After irradiation, the exterior of each polythene tube was washed with 6 N hydrochloric acid containing sodium as carrier and then with distilled water to remove active contaminants ; if this precaution was neglected, the results for sodium were often erratic. The tube was then cut open at each end, and its contents were washed into a 50-ml centrifuge tube with water from a hypodermic syringe, about 2 ml of water being ample for this purpose. Each centrifuge tube contained 1 ml of a carrier solution containing sodium, potassium and phos- phorus. Biological material was wet ashed in 1 ml of nitric acid on a sand-bath at 180" to 200" C, 1 ml of sulphuric acid was then added, and heating was continued for 1 hour, by which time fumes of sulphur trioxide were visible.(Any active halogens were expelled during the wet ashing, which was therefore carried out in a fume cupboard.) The tubes were then cooled to room temperature, and chemical separation was begun. BOWEN AND CAWSE: DETERMINATION OF SODIUM, POTASSIUM AND METHOD REAGENTS- All reagents were of recognised analytical grade. Sulphuric acid, 36 N. Nitric acid, 16 N. Hydrochloric acid, 12 N. Perchloric acid, 70 per cent. Ammonia solution, 15 N. Magnesium chloride solution, 50 $er cent. w/v-Prepared from magnesium chloride Barium chloride solution, 30 per cent. w/v-Prepared from barium chloride dihydrate.Ammonium carbonate solution, 10 per cent. w/v. Sulphuric acid, 5 per cent. w/v, in diethyl ether. n-Butyl alcohol-Saturated with hydrogen chloride gas. n-Bzctyl alcohol - ethyl acetate mixture (1 + 1 v/v). Wash solution-A solution 6 N in hydrochloric acid and 1 N in sodium chloride. Combined carrier solution-Prepared by dissolving 37.1304 g of ammonium dihydrogen orthophosphate, 44.5652 g of potassium sulphate and 61.7689 g of anhydrous sodium sulphate in distilled water and diluting to 1 litre. 1 ml = 20 mg each of sodium and potassium and 10 mg of phosphorus. hexah y dr at e . Potassium carrier solution-Prepared by dissolving 38.1381 g of potassium chloride in distilled water and diluting to 1 litre. 1 ml 3 20 mg of potassium. Sodium carrier solution-Prepared by dissolving 225-4164 g of sodium chloride in distilled water and diluting to 1 litre.1 ml = 10 mg of sodium. Combined standard solzction-Prepared by dissolving 7.4261 g of ammonium dihydrogen orthophosphate, 3.5345 g of Specpure potassium carbonate and 0.3653 g of Specpure sodiumAugust, 196 11 PHOSPHORUS IK BIOLOGICAL MATERIAL BY RADIOACTIVATIOK 509 hydrogen carbonate in water distilled from quartz apparatus and diluting to 1 litre in polythene apparatus. 0-005 ml 3 10 pg each of phosphorus and potassium and 0.5 pg of sodium. SEPARATION OF PHOSPHORUS- The separation of phosphorus was based on the solubility of dry orthophosphoric acid in diethyl etherg; salts of alkali metals are insoluble in ether. The phosphate was purified and then finally precipitated as ammonium magnesium phosphate.A 15-ml portion of diethyl ether, previously dried over calcium chloride, was added to the contents of each centrifuge tube, and the tube was spun in a centrifuge to coagulate the metal sulphates. The supernatant ether was transferred to a clean tube, and the precipitate was washed twice with 4 ml of ether containing 5 per cent. of sulphuric acid. The precipitate was retained for determining the alkali metals, and the supernatant ether and washings were evaporated by means of a current of air at room temperature. The pH of the residual acid was brought to 9 by adding ammonia solution, and, when cool, 1 ml of magnesium chloride solution was added; the solution was then diluted to 10ml and swirled. After 30 minutes in a water bath at 20" C, the precipitate was separated by centrifugation and washed three times with water.Finally, it was transferred, as a slurry with acetone, to a weighed aluminium counting tray, dried, weighed and counted; the chemical yield averaged 90 per cent. SEPARATION OF POTASSIUM- This separation of potassium was based on the insolubility of its perchlorate, so that the sulphates present had first to be removed. The precipitate from the extraction with ether was dissolved in water, 2-5 ml of barium chloride solution were added, and the volume was made up to 10ml. After the tube had been set aside for 10 minutes in a bath of boiling water, the precipitated barium sulphate was separated by centrifugation and rejected, and the supernatant liquid was poured into another tube containing 5ml of perchloric acid.After being cooled for 15 minutes in a bath of ice, the precipitated potassium perchlorate was separated by centrifugation, and the supernatant liquid was reserved for determining sodium. The potassium perchlorate was freed from traces of sodium by recrystallisation, 0.1 ml of sodium carrier solution and 1 ml of perchloric acid were added to it, and the solution was diluted to 10ml with water. The precipitate was dissolved by heating at 90" C for 10 minutes and was then re-precipitated by cooling at 0" C for 20 minutes. It was then separated by centrifugation and washed three times with the n-butyl alcohol - ethyl acetate mixture. Finally, it was transferred, as a slurry with ether, to a weighed aluminium counting tray, dried, weighed and counted; the mean chemical yield was 40 per cent.SEPARATION OF SODIUM- This separation was based on the insolubility of sodium chloride in n-butyl alcohol saturated with hydrogen chloride; it was rendered somewhat tedious by the necessity for removing traces of active potassium and inactive barium left from the previous steps. A 1-ml portion of potassium carrier solution was added to the supernatant liquid from the first precipitation of potassium perchlorate, and the solution was evaporated to dryness in a 100-ml beaker on a hot-plate at 150" C. When cool, the sodium perchlorate was dissolved by boiling with 15ml of hot n-butyl alcohol, the solution was cooled, and the residue of potassium perchlorate was separated by centrifugation. The supernatant liquid was poured into a fresh tube containing 5 ml of n-butyl alcohol saturated with hydrogen chloride and was kept at 100" C for 10 minutes.The precipitated sodium chloride was separated by centrifugation, and the supernatant liquid was rejected. The sodium chloride was dissolved in 2 ml of water, 10 ml of ammonium carbonate solution were added, and the solution was boiled for a further 10 minutes. Some barium carbonate was precipitated at this stage, and this was separated by centrifugation. The remaining solution was transferred to a 100-ml beaker, acidified with 0.5 ml of hydrochloric acid, covered with a watch-glass and evaporated to dryness on a hot-plate. Ammonium chloride was removed by heating strongly with a bunsen burner for 5 minutes, and the residue of sodium chloride was cooled, transferred, as a slurry with acetone, to a weighed aluminium counting tray, dried, weighed and counted.510 DETERMINATION O F RADIOACTIVITY- ROWEN AND CAWSE: DETERMINATION OF SODIUM, POTASSIUM AND [VOl.86 The beta-activities of the precipitates were counted with either a 2B2 end-window Geiger counter (efficiency, about 40 per cent.; background, 30 counts per minute) or an anti-coincidence counter (efficiency, about 10 per cent. ; background, 1.5 counts per minute). The low-background counter was suitable only for low count rates and was not often used. Radiochemical purity was checked by counting at intervals for several half-lives (and by gamma spectrometry for phosphorus). The entire procedure, including counting, could be carried out on eight samples by a single individual in 8 hours.DISCUSSION OF THE METHOD COMPARISON WITH OTHER TECHNIQUES- The method was compared with a flame-photometric technique for sodium and potassium and with the molybdophosphate colorimetric technique for phosphorus ; these techniques have been described elsewhere.1° Three replicate determinations were made by each technique, and, as shown in Table 11, good agreement was found. TABLE I1 COMPARISON BETWEEN RESULTS BY ACTIVATION AND OTHER TECHNIQUES Amount of element found by- Amount of , A \ Element element present, activation, conventional technique, tLg P.g tLg 0.0 0.1 0.2 0.4 0.6 0 . . { : 8 Sodium . . Potassium . . 10 Phosphorus . . 4 0.0006 0.112 0.193 0.399 0.620 0-0035 2-12 4.32 8.0 1 9.80 0.01 2-14 4.20 8.2 1 10.7 <0*012 0.088 0.202 0.428 0.587 <0-013 1-92 4.23 8-13 9.97 <Om15 2.06 4.00 7.87 10.1 SENSITIVITY AND ACCURACY- When 0-l-pg portions of sodium, potassium and phosphorus were activated for 15 hours, the respective count rates were approximately 3500, 250 and 200 counts per minute on the 2B2 counter or 1000, 80 and 50 counts per minute on the anti-coincidence counter.These figures are practical and allow for the low chemical yields in precipitating the alkali metals and for decay during the separation. The minimum detectable amounts of the three elements (sufficient to double the background of the anti-coincidence counter) were therefore 0-086 mpg of sodium, 1-2mpg of potassium and 16mpug of phosphorus. I t is possible to attain such sensitivity for sodium and potassium by flame photometry, although not with the simple instrument used by us.However, it is doubtful if any colorimetric method for phosphorus is so sensitive, and the limit can be lowered by a factor of 15 by increasing the period of activation to 1 week. In practice, it is seldom required to work in the millimicrogram range with these common elements, and the sensitivity is further limited by the magnitude of the blank value. Even after thorough washing, the clean polythene tubes yielded about 0.6 rnpg of sodium, 3.5 mpg of potassium and 10mpg of phosphorus, and for work of the highest sensitivity this blank correction would have to be decreased. The accuracy of the technique is determined partly by the homogeneity of the neutron flux (&2 per cent.in BEPO) and by errors in weighing the final precipitates and in the trans- ference of standards by pipette; counting errors can be decreased to 1 per cent. by counting for 10,000 counts. The total error is probably less than +_5 per cent.August, 19611 PHOSPHORUS IN BIOLOGICAL MATERIAL BY RADIOACTIVATION 51 1 TESTING THE RADIOCHEMICAL FROCEDURES- The chemical procedures described above were tested with aliquots of radiochemically pure sodium-24, phosphorus-32, sulphur-35, potassium-42 and manganese-54. Sulphur is a major element in biological material, although it is not activated to a great extent, whereas manganese is much less abundant, but has a very high cross-section for thermal neutrons.ll The results of the tests are shown in Table 111, from which it can be seen that contamination is so small as to be negligible in practice.Chlorine-38 was not used in this test because it is volatilised in the preliminary ashing step and because it has decayed almost completely after 8 hours. Rubidium-86 was not tested either; it would be expected to follow potassium closely, but under our conditions it should not contribute more than 0.2 per cent. to the measured activity for potassium. TABLE I11 CONTAMINATION OF PRECIPITATES BY VARIOUS ELEMENTS Content of nuclide found in precipitate of- ammonium sodium potassium magnesium Nuclide tested chloride, perchlorate, phosphate, % % % Phosphorus-32 . . .. . . (0.05 < 0.05 90 Sulphur-35 . . .. .. - 1.0 Sodium-24 . . . . .. 40 ~ 0 . 1 4 < 0.07 - Potassium-42 .. . . . . <0*04 40 < 0-07 Manganese-54 . . . . . . 0.27 0.1 1 0.35 INTERFERING NUCLEAR REACTIONS- Several nuclear reactions could interfere with the determinations described above, but all could be eliminated by carrying out the activation in a thermal-neutron column having no fast-neutron contaminants. Fast neutrons can give rise to interference from the reactions listed below- (;) 24Mg (ng) 24Na (ii) 27Al (%,a) 24Na (v) 42Ca (rt,p) 42K ( v i ) 4 5 S ~ (%,a) 42K (iii) 32s (n,p) 32P (iv) 35ci (n,a) 3 2 ~ Reactions (ii) and (vi) were of no importance in this work, as the amounts of aluminium and scandium present in biological material are extremely low. The cross-sections for the rest of the reactions are not well known, but appear to be greatest for reactions (i) and (iiz).The magnitude of the interference can only be calculated for specific samples in which the contents of the interfering elements are known. For example, the seeds studied by us contained 0.6 mg of sulphur per g. This would give rise to an amount of phosphorus-32 corresponding to 0.033 mg of phosphorus per g of the original seeds, which is only 0.4 per cent. of the observed value. The magnesium content of the seeds was 0.3 mg per g, but when 0-3 mg of Specpure magnesium was activated, it yielded an amount of sodium-24 corre- sponding to 0.42 pg of sodium per g of seeds (0.2 per cent. of the observed value). We con- clude that these interferences are negligible. RESULTS- Tomato seeds were collected under clean conditions from glasshouse plants grown in sand culture on Long Ashton complete nutrient solution.They were found to contain, per gram, 0-19mg of sodium, 6-96mg of potassium and 7-75mg of phosphorus. These results are the means of four replicate determinations and are similar to the values 0-21, 7.0 and 7.0 mg, respectively, found by other methods.1° CONCLUSIONS The neutron-activation method described is a remarkably sensitive technique for deter- mining sodium, potassium and phosphorus. I t should be particularly useful for determining traces of phosphorus in biological material, as flame photometry is already established as512 KAKABADSE AND MAXOHIN RAPID MICRO-DETERMINATION [Vol. 86 a satisfactory method for determining the alkali metals on the micro scale. As far as the biologist is concerned, however, the method is not sufficiently sensitive for the determination of these elements in most individual cells or parts of cells. For example, a single yeast cells weighs 5 x 10-10 g, and its potassium content is of the order of 5 x 10-l2 g. Even if fluxes of lOI4 neutrons per sq. cm per second were used for activation, this amount of potassium would not be detectable, much less determinable. It is therefore evident that the analytical challenge of biological research is not being fully met. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. REFERENCES Keynes, R. D., and Lewis, P. R., Nature, 1950, 165, 809; J . Physiol., 1951, 114, 151. Reiffel, L., and Stone, C. A., J . Lab. Clin. Med., 1957, 49, 286. Spencer, R. P., Mitchell, T. G., and King, E. R., Ibid., 1957, 50, 646. Druyan, R., Mitchell, T. G., and King, E. R., Ibid., 1958, 52, 384. Hutchinson, W. P., U.K. Atomic Energy Research Establishment Report Med/R2317, Harwell, Salmon, L., U. K. Atomic Energy Research Establishment Report C/M323, Harwell, 1957. Bowen, H. J. M., Biochem. J., 1959, 73, 381. Born, H. J., and Stark, H., Atomkernenergie, 1959, 4, 286. Cripps, F. H., D.S.I.R. Report CRL/AE49, Teddington, 1950. Bowen, H. J. M., and Cawse, P. A., U.K. Atomic Energy Research Establishment Report R2925, Bowen, H. J. M., J . Nucleav Energy, 1966, 3, 18. Received February 3rd, 1961 1960. Harwell, 1959.