33P: A superior radiotracer for phosphorus?

International Journal of Applied Radiation and Isotopes, 1969, Vol. 20 ,pp. 531-540. Pergamon Press. Printed in Northern Ireland

"P: A Superior Radiotracer for Phosphorus? J. R . R O B I N S O N Research Institute, Canada Department of Agriculture, University Sub. P.O., London, Ontario, Canada*

(Received 16 September 1968) Phosphorus-33 has half-life and energy properties which should render it superior to s2p as a radiotracer for phosphorus in most biological applications. It should be particularly useful in autoradiography, in long-term experiments, in applications where sensitivity to radiation is high and in dual labelling of intermediates containing more than one phosphorus atom. Advantages and limitations in the use of sap are reviewed with a consideration of price, half-life, energy, radioisotopic purity and methods of measurement. Determination of the 8~p content in sap is used to demonstrate Geiger, liquid scintillation and Cerenkov techniques for counting these radioisotopes. sap: U N RADIOINDIC,A T E U R SUPI~RIEUR P O U R LE PHOSPI-IORE Le phosphore-33 poss~de une demi-p6riode et des propri6t6s 6nergiques qui devraient le rendre sup6rieur au s2p comme radioindieateur pour le phosphore dam la plupart des applications biologiques. I1 devrait ~tre utile surtout dans l'autoradiographie, dans les expdrienees de longue dur6e, dans les applications oh la sensibilit6 au rayonnement est 61ev6e et dam le marquage double des interm6diaires contenant plus d'un atome de phosphore. O n passe en revue les avantages et les limitations de l'emploi du sap avee use considdration du coot, de la demi-pdriode, de l'6nergie, de la puret6 radioisotopique et des rn6thodes de mesure. Le dosage du contenu de s2p dans le sap sert it d6montrer les techniques Geiger, de scintillation liquide et Cerenkov pour compter ces radioisotopes. 3aP--HAI/IJIVqIIII/IlY[ PA~I/IOAHTI/IBHbllYI HH~I/IHATOP ~ O C ~ O P A "(I)oc~op nMeeT nepno~ nonypacna~a H TaaHe CBO~CTBa oHeprnH, qTO, Hall pa~HoaKT/HBHI)I~HHR~II~aTOp /~oc~opa B 6ont,mei~ qaCT~I6Ho~orHqeCaHX npHMeI-Ie/HH/~,OH CTOHTnbIIne 32~)oc~opa. On Oco6eHHO ~eHett B paJInoaBTorpaqbHH, B npo~onmnTeJir, aL[x ~KcnepHMeHTaX, B cayuaax, Ror~a nMeeTerl BHcoHa~I '~yBCTBHTe~HOCTbH o6ny,~enam H n ~BOfiHOMMeqeHnH npoMemyTo,m~,[x coe~nHeaHi~, co~epmamax 6o~ee oaHoro aTOMa q~oc~opa. ~aercH o6aop npeHMynIeCTB~I orpaHH,~emti~ B HpnMeHeBHH *a~ e yqeToM ~4eH~, nep/¢io~a no~ypacnaaa, oHeprHH, paj~nOHaOTonHqecKoIttIHCTOT/aIH MeTo~oB HaMepeHH/~. Onpe~eztenHe co~epmaHna a~l) B asp cJ~yH{HT 8~ecb ~JIH ~eMOHCTpHpOBaHHH CqOTHOI~ TeXHHHH HaMepeHH/~ aI~THBHOCTH9THX pa~HOHaOTOnOB CH0co6aMH Fefirepa, qepeHRo~a n MeTO~OMm/cI~HOCTttOl~CnHHTHJI~IfII~HH. asp: EIN V O R Z U G L I C H E R I N D I K A T O R F U R P H O S P H O R sap hat Halbwertzeiten und Energieeigenschaften die ihn besser als a~p ftir die rneisten biologischen Zwecke als radioaktiven Indikator auf Phosphor erscheinen lasses. Er k6nnte besonders ntitzlich werden in der Autoradiographie, in Langzeitversuchen, fiir Zwecke wo die Strahlungsempfindlichkeit hoch ist, und bei der Doppelmarkierung yon Zwischenprodukten mit mehr als einem Phosphoratom. Die Vorziige und die Beschr~inkungen in der Verwendung yon asp werden besprochen ira Hinblick auf Preis, t-Ialbwertzeit, Energie, radioisotopische Reinheit und Messmethoden. Die Bestimrnung des a~p-Gehaltes in sap wird zur Vorftihrung yon Geiger-Fltissigszintillations- und Cerenkov-Methoden zum Ziihlen dieser Radioisotopen verwendet. INTRODUCTION PHOSPHORUS, a most i m p o r t a n t e l e m e n t i n biological systems, has b e e n represented almost exclusively b y a2p as a r a d i o t r a c e r isotope. Although economically produced in acceptable * Contribution No. 391 531

radioisotopic purity, this n u c l i d e is n o t ideal i n m a n y biological studies. Its half-life (14.3 days) is sufficiently short t h a t i n e x t e n d e d a n i m a l or field experiments little r a d i o a c t i v i t y r e m a i n s unless v e r y h i g h initial doses are a d m i n i s t e r e d . Its beta-particle m a x i m u m energy (Emax = 1-71 m i l l i o n electron volts, M e V ) is

532

.I.R. Robinson

such that these high initial doses may significantly influence the behaviour of the system under study; ~1-3) it similarly limits the amount which may be administered for therapeutic purposes and it can present considerable radiation hazard ~4) in the performance of multimillicurie synthesis operations. In autoradiographic applications, it causes considerable loss in resolution when attempting to examine fine-structure. Phosphorus-33 does not suffer these disadvantages in that its half-life is nearly double, while its beta-energy is only about one-seventh, that of 3zp. Beginning with equal activities, after a 100-day growing season there will be about 8 times as much residual ~ P as s2p; in a 6-month experiment the residue ratio is 44:1 and the beta-dose delivered to the subject in the interval is much lower, permitting higher initial applications. Both isotopes are pure beta emitters. Although known and characterized since 1951,~ 5-s) exploitation of the favorable properties of this radionuclide has had to await technical improvements allowing production at acceptable purity and price levels. Among others, ~9-1~) LEwis at Oak Ridge (13"14) has reported on the production of ssp from enriched sulfur and chlorine targets and the product has recently been offered commerically.* Price considerations, greater ease of detection and lack of any specific need to change will doubtless result in continued use of 3~p but, in some cases, ssp may now become the radioisotope of choice. T h e development of liquid scintillation spectrometry eliminates earlier problems in counting of its soft beta radiation and, further, will encourage projects utilizing both isotopes as a dual label for the single element.

DECAY FACTORS For the convenience of users, Table 1 lists appropriate factors for calculating ~ P decay. Values are given for 6-hr intervals over one half-life period and may be combined with those indicated for complete half-lives to determine the decay over longer periods.

RADIOISOTOPIC PURITY Use of a radioactive isotope as an element tracer requires certainty that the labelled material is "radiochemically pure", i.e. contains radioactive atoms of one element only. In addition it may be necessary to specify "radioisotopic purity" where all radioactivity present is due to a single radioisotope of the element. I f the latter is not essential it may be necessary, at least, to know the approximate content of the contaminant. Inherent in ~ P production methods is the simultaneous production of 32p.{13,14) T h e desired nuclear processes szS(n,p)aaP and z6Cl(n,a)33P are always accompanied by the reactions 3~S(n,p)s~P and zsCl(n,~t)s~P because the target elements, although enriched, still contain abundant amounts of the naturally predominant isotopes. Hence, freshly prepared sap will contain relatively large amounts of 3zp which are removed only by decay. Storage for 4½ months will attenuate the sap to 0.1 per cent of its initial activity, simultaneously reducing the 33p content by 96 per cent. Obviously the costs of target enrichment, ageing, and attendant loss of 3zp account for the higher price of this radioisotope. Experiments using z2p are little affected if a few per cent of saP contamination is present and this is an us-noticed common occurrence; HALF-LIFE at one half-life commercial 32p m a y contain Several half-life values (T1/2) have been 2-3 per cent, but at 10 half-lives, 38 per cent of reported for 8sp(5-9.15) in the range 25-4-1 the residual activity may be due to 3zp~5). In days, the most recently determined value being contrast, contamination of aap by the higher25-3 q- 0.05 days for radioisotopically purified energy isotope will not go un-noticed. In material cle). For practical use, particularly of counting planchet samples, the efficiency of the commercial grade m a t e r i a l containing up to counting system is much lower for aap than for 5 per cent of asp, a figure of 25 days is both 3~p, and a small amount of the latter, because of convenient and more accurate. this and of backscatter, will introduce a disproportionate error; in dual-label experiments * Isotopes Development Center, Oak Ridge National Laboratory, P.O. Box X, Oak Ridge, Tenn. with ~*P and 3~p, the degree of cross contamin37830, U.S.A., and New England Nuclear Corp., ation will have to be known; a 5 per cent content of SZP 'shortens' the half-life of SSp by about 575 Albany Street, Boston, Mass. 02118, U.S.A.

~P : A superior radiotracerfor phosphorus?

533

TABLE 1. Decay factors for phosphorus-33 (TI/2 = 25 days) Hours

Days 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

0

6

12

18

~

Hours

0

6

12

18

Days

1-0000 0"9727 0.9461 0.9202 0.8950 0.8706 0"8467 0-8236 0.8011 0.7792 0.7579 0.7371 0.7170 0.6974 0.6783

0.9931 0.9659 0.9395 0.9138 0-8888 0-8645 0.8409 0-8179 0.7955 0-7738 0.7526 0-7320 0.7120 0.6926 0.6736

0.9862 0-9593 0.9330 0.9075 0.8827 0.8586 0.8351 0.8123 0.7900 0-7684 0.7474 0.7270 0.7071 0-6878 0-6690

0"9794 0.9526 0-9266 0-9013 0.8766 0.8526 0.8293 0-8066 0.7846 0-7631 0.7423 0.7220 0.7022 0.6830 0-6643

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

0.6598 0.6417 0.6242 0.6071 0.5905 0.5743 0.5586 0.5434 0.5285 0.5141 0.5000 0.4863 0.4730 0.4601 0-4475

0-6552 0-6373 0.6199 0-6029 0-5864 0-5704 0.5548 0-5396 0.5249 0.5105 0.4965 0.4830 0.4698 0.4569 0.~I~4

0.6507 0.6329 0-6156 0-5987 0.5824 0.5664 0"5510 0.5359 0-5212 0.5070 0.4931 0.4796 0.4665 0.4538 0.4414

0-6462 0-6285 0-6113 0,5946 0,5783 0-5625 0"5471 0"5322 0.5176 0-5035 0'4897 0.4763 0.4633 0-4506 0.4383

Factorsfor complete half-lives 2 0-2500 3 0.1250 4 0.0625

5 0.0312 6 0.0156 7 0.0078

one day and it is, of course, changing continuously. I n liquid scintillation counting, the efficiency for , , u p samples" keeps shifting if the szp content is more than 3-4 per cent. These effects are easily dealt with but it is essential to recognize their presence and suppliers' literature m a y not specify the degree of radioisotopic purity. BETA

ENERGY

(a) Maximum energy (Era,x) A knowledge of Emax is useful in calculations for discrimination, shielding, and Cerenkov application. For ~P, reported values lie in the range of 0.238-0.26 M e V and 0.25 M e V appears to be the practical value. Based upon this, the maximum penetration or range (R) in an absorber material will be about 47 mg/cm s. * This m a y be compared to a m a x i m u m range of 870 m g / c m ~ for 8~p. Thus if a "radiochemically pure" ~ P sample on a planchet exhibits a count when covered by an absorber of thickness more than 47 m g / c m ~, the net count above this absorber will be due to sap and will decay with a • Maximum range, in g/cms, R -----0"37Eraax117).

8 0.0039 9 0.002 10 0-001

half-life of 14 days. Similarly, the lower discriminator of a liquid scintillation counter can be raised to exclude all pulses resulting from beta particles of energy below 0.25 MeV, allowing the z2p content to be counted. Another discrimination process is the Cerenkov threshold. (b) Mean energy (1~) The mean energy is that value which would be possessed by all particles were the total energy distributed equally. It is, therefore, the effective energy in all considerations involving the delivery or dissipation of energy within a subject medium and hence is used in calculating the number of silver grains likely to be exposed in an autoradiogram in a given time, in tissue doserate estimation, in ion current generation and similar applications. It is a matter of observation that the beta spectra of most isotopes are of about the same shape and so a relationship exists between maximum and mean energy values in which /~a approximates 35-40 per cent of Emax ~xs>. From this, the mean energy of phosphorus-33 is about 100 thousand electron volts (keV), as compared to <19>5-5 for tritium, 49 for sulfur-35,

534

J. R. Robinson

50 for carbon-14, 100 for calcium-45 and 700 for phosphorus-32.

500

AUTORADIOGRAPHY

42K

CAgO has reviewed the relationship between beta particle energy and autoradiographic resolution and has shown (s°) with electron microscopic autoradiographs that tritium (Emax ~-18 keV) provides resolution of 0.1 /z compared to 0.3 F given by s2p (Emax ~- 1700 keV). U n d e r identical experimental conditions ssp should provide a resolution of about 0.13-0.15/~. MAYg has used autoradiography to estimate the ssp content of s~pC~Xa) and has reported a significant improvement in resolution when using ~pc21b). Increasing resolution with decreasing beta particle energy is to be expected since a lower-energy isotope expends all its energy in a track extending only a few microns from the source and, equally important, that track is entirely within the emulsion and can be followed back to a point source. Particles from a higherenergy isotope largely escape entirely from the emulsion and there are few exposures of silver grains in the vicinity of the point of origin.

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For a number of years we have used our liquid scintillation spectrometer as a nonfocusing Cerenkov counter ~2-~) for assay of 82p in water solution. More recently, other reports (26-~8) have discussed this technique which requires no phosphors, allows easy sample preparation, and permits complete recovery of unadulterated. sample. M a n y beta emitters can be assayed in this way although the biologically important SH, 14C, ssS, ssp and 45Ca may not, as shown in Fig. 1. Also implied in Fig. 1 is a quick and easy means of estimating the s~p content of ssp. In water, beta particles must possess about 0"265 MeV kinetic energy in order to induce measurable Cerenkov emission (~s) and therefore any counts arising from an aqueous ,,ssp sample" will be due to s2p. A combination of Cerenkov and scintillation counting would find many applications in the assay of dual-labelled samples, one isotope of which lies above, and the other below, the Cerenkov energy threshold.

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FIo. 1. The relative Cerenkov intensity of a number of commonly used beta-emitting radioisotopes in terms of their maximum beta energy, illustrating the discriminating Cerenkov threshold. T h e Cerenkov system is excellent for standardizing a2p because it is so little influenced by sample composition, in contrast to liquid scintillation counting. Figure 2 illustrates the absence of any important chemical quench effect due to p H variations in a series of acids and bases, except for nitric acid; Fig. 3 shows the stability of the Cerenkov count-rate in the presence of a number of commonly encountered ions; again, nitrates (as well as nitrites) exhibit large quenching action, confirming that it is the anion, and not the p H of these nitrogen acid solutions which is responsible for quenching. Spectrophotometric measurements on these

s3p : A superior radiotracer for phosphorus?

extinction coefficient (e) of about 9 whereas the e for nitrites is approximately 24 and the absorption peak, although maximum at 350 mF, is broad and extends well across the Cerenkov emission region. The latter ranges from 300700 m/z <2~>. Figure 4 demonstrates the effect upon countrate during transition from water to pure ethyl alcohol as solvent. Any miscible, non-quenching solvent m a y be used with water; immiscible solvents may be used alone. The Cerenkov effect, when present, also contributes to the total count of a regular liquid scintillation system where soluble phosphors are used; Fig. 5 shows a plot of the extent of this contribution as a function of the beta energy (Emax) of the radioisotope (24). Predictably, materials producing colored solutions (e.g. indicators, Cu ~+, Cr~O~=, methylene blue, phosphomolybdenum blue, etc.) affect the Cerenkov count by color quenching. I f such samples can be bleached by a p H change, oxidation, reduction, complexing, or other type of reaction, the residual colorless solution usually can be counted free of serious quenching. The degree of color quenching, also, may be related

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536

J . R . Robinson TABLE 2. Cerenkov count of

20(

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190

Cone. KC1 (Molar)

Count-rate (cpm) of a 12 ml sample

0 0.1 0.4 0.8

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Fro. 4. The effect of solvent composition on Cerenkov count in a water-ethanol system; 12 ml of each solution was treated with 0"1 ml of an Ha32PO 4 solution. to optical density and so Cerenkov counting can be used to determine the concentration of a colored but non-radioactive solute by measuring the depression in count-rate of an added asp standard tu~. T h e same type of calibration can be made for colorless solutions of nitrites and nitrates over restricted ranges of concentration. T h e Cerenkov counting efficiency for a~p in water is increased by 25 per cent when glass vials are replaced by polyethylene. T h e natural 4°K content (0.0119 per cent abundance) of all potassium compounds increases the background and may need to be considered if potassium salts are used as buffers. (Table 2). T h e introduction of glass fibre filters or strips causes a marked increase, proportional to weight, in the Cerenkov count. As in liquid scintillation counting, samples to be compared should contain equal volumes. Predictably, larger sample volumes--at constant concentration--, as well as increasing concent r a t i o n s - a t a constant volume--, both produce higher count rates in a linear manner; but, taking a given quantity of radioactivity and diluting it continuously from 1 ml to 20 ml, i.e. changing both volume and concentration simultaneously, will show an optimum volume

with a peak count-rate at 11-12 ml. As higher dilution of the radioactive species occurs the Cerenkov emission is attenuated. CLAUSEN (2s) has shown this same effect in the presence of 10 m Molar KHaPO4 carrier. These effects are illustrated in Fig. 6. EXPERIMENTAL Materials

Aqueous solutions of NaasaPO4 and Nas sspo4 (1.0 ml each; 0.001 Molar; 0.1 m Ci/ml) were purchased* and suppliers' specifications state "radiometric purity, 99 + per cent" and " m i n i m u m radiochemical purity of 99 per cent"; radioisotopic purity is not stated. " T F C " (Toluene-fluor-cellosolve scintillation fluid): A solution of 4 g PPO(2,5-diphenyloxazole) plus 0.05 g MSB (p-b/s-(0-methylstyryl)-benzene) per 1. of toluene, 10 volumes diluted with 8 volumes of methyl cellosolve (ethylene glycol mono-methyl ether). T e n ml of this mixture, conveniently made from the regular toluene-fluor combination, will accommodate at least 0.5 ml of water at 0°C and provides fair counting efficiency for t4C as well as higher energy isotopes. Instruments

For standardization of a~p, a Model R-12A phosphorus-32 simulated reference source with calibrated aluminum absorbers, a T y p e TGG-2 (1"2 rag/era ~) end-window geiger tube in a shielded sample holder with reproducible geometry and a T y p e SC-71 Compumatic scaler were used.~" * New England Nuclear Corp., Boston, Mass. t Geiger counting instruments from Tracerlab Inc., Boston, Mass.

raP: A superiorradiotracerfor phosphorus? For liquid scintillation and Cerenkov counting, we used a Model 6860 beta scintillation spectrometer.* Keferring to this instrument, in our terminology "gain" is the attenuator setting; "open window" is a 0.5 to 9.9 V pulseheight span between lower and upper discriminators with differential (L-U) counting; "balance point" is the optimal gain setting which provides maximum count-rate for a given sample in a specified window.

o ~~

3. Cerenkov counting of 32P Aliquots (10pl) of the NasS2po4 stock were dissolved in 12 ml of distilled water and counted in the scintillation counter at balance-point gain (B-5"0) in an open window. T h e cpm value obtained (6.32 × 10 n) indicates a Cerenkov counting efficiency for sap of 29 per cent; a value of this order is predictable from Fig. 5. 4. Estimation of n2p in mP by Cerenkov counting Aliquots (10 F1) of the Na33apo4 were counted, as above, in water and the dpm value, * Nuclear--Chicago Corp., MKL

/

~- 80

1. Standardization of 32p Aliquots (10 #1 each) of the Nassapo4 solution were evaporated on copper planchets and standardized through a 130 mg/cm 2 absorber against the 32p simulated standard. T h e absolute disintegration rate of the Na3 saPO4 was then calculated to a 'zero-time' reference value; the d p m for 10/A was 2.18 × l0 G, equivalent to 98.2 gCi/ml. 2. Estimation of 3ap in ~P by planchet counting Aliquots (10 /A) of the Na3~PO4 solution were evaporated on copper planchets and counted under the same conditions as the asp. Since the absorber will pass only beta particles having energy in excess of 0.5 MeV (from R = 0"37 E 31a) any counts registering will not be due to 8np. Under these standard conditions the efficiency for 32p is known and therefore the mP content of the ~ P can be calculated. T h e value found was 7-38 × 104 dpm, corresponding to 3-3 FCi/ml. Subsequent countings confirmed this activity to be decaying with a half-life of 14 days. Lacking a standard, the G3p component cannot be determined in this way.

537

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Fzo. 5. The effect of beta energy on the Cerenkov contribution to liquid scintillation samples (Data from HABEPJ~R(24)). after efficiency and decay corrections, was 6.10 × 104 per 10#1, or 2"7 #Ci/ml. T h e Cerenkov count-rate was observed to decay with a half-life of 14 days and the balance-point gain setting remained stable at B-5.0, identical (Conc'n constant)

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538

d. R. Robinson

to that required for a known asp sample, indicating the same energy spectrum. Thus the 82p content of the sample is established; but if a ssp/mp ratio is required, a value for the 33p component must be obtained by other means.

5. Liquid scintillation counting of asp Aliquots (100 /tl) of the supplier's solutions were diluted to 1.0 ml for scintillation counting purposes. T e n / A of the Na3 3sPO4 solution in 12 ml of T F G were counted at balance point gain (G---3.0) in an open window, giving 1.77 × 105 cpm, an efficiency of 81 per cent. This somewhat low figure (for asp) is due to the "diluter" characteristics of methyl cellosolve which is both an alcohol and an ether (sg). Addition of more P P O fluor has no effect. 6. Scintillation counting of 38p As indicated (in 2 and 4 above) the zzp content of the Na333PO4 was approximately 3 per cent on delivery. Aliquots (10/tl) of the diluted Nas~PO4 were counted in 12 ml of T F C giving a count-rate of 1.72 X 105 cpm. These samples balanced in an open window at gain setting D-0.0 but at this high amplification the 32p component is far out of balance and the simple application of a 3 per cent correction factor for s2p is not possible. However the scintillation efficiency at this gain setting for a known asp sample can be measured and a separate Cerenkov count of the mixed zg.-ssp sample provides the absolute 3~p content; the true ssp value in the scintillation sample can then be estimated by difference, e.g. In this instance, a known 32p sample counts with 81 per cent efficiency (1.77 × 105 cpm) at G-3.0 gain, but at only 27 per cent efficiency (5.9 × 104 cpm) at D-0.0. Cerenkov counting indicated 6.1 × 104 d p m of 3zp per 10 /zl of stock Nas~PO4 and, in a liquid scintillation sample (diluted tenfold and counting 32p at 27 per cent efficiency) this activity will contribute only 1.65 × 108 cpm to the total count at D-0.0, and may be neglected, being less than I per cent of the total. When the content of higher energy isotope is significant, its contribution is subtracted.

Lacking a pure = P standard, the exact counting efficiency for this isotope cannot be determined but, assuming the delivered Na3 ~PO4 to be 97 per cent ~ P and to contain 100 ~ 5 ktCi of total radioactivity, then the scintillation counting efficiency for 33p is about 80 per cent.

7. Estimation of S~P in ~P by scintillation counting A high-activity ~ P sample was balanced in an open window at gain G-3-0, thus positioning the pulse-height spectrum across the 0.5-9.9 V span. Keeping the same gain (to maintain the shape of the spectrum) the discriminators were used to effect differential counting of 94 windows, each 0.1 V wide, right across the pulse-height span. This is, in effect, a manual 94-channel pulse-height analyser, and the count-rate found in each of these channels is plotted in Fig. 7. Since the amplifier is linear and the pulse-heights are proportional to primary beta-energies, the 1.71 MeV energy scale for asp may be superimposed on the 0.59.9 V pulse-height scale. T h e setting required to discriminate against beta particles of any given energy, for this isotope, at gain G-3.0, is at once apparent. Selecting a window of 3.0-9.9 V, gain G-3.0, will allow counting of only 0.5-1.7 M e V betas; it was observed too, that counting asp standard samples at these settings gives a count-rate exactly one-half that obtained in an open window.* T h e efficiency then is 40.5 per cent (½ of 81 per cent) for asp and is zero for 33p. Aliquots (10 /A) of the diluted Na83spo4 solution were counted to good statistics at these settings and after correction for efficiency and decay, gave 5.2 × 108 dpm of SSP in the sample or 2"3 #Ci/ml. Subsequent countings showed this activity to have a stable balance-point and a 14 day half-life. CONCLUSIONS T h e three experimental techniques for estimating the asp content of 33p are in good * This implies the median energy for 3s]? is about 0-5 Meg. This is a quite different property to the modal energy at 0.33 Meg (from Fig. 7), the mean energy (0.7 MeV) and the maximum energy (1.71 MeV).

~P: A superior radiotracerfor phosphorus?

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FIO. 7. The a2p spectrum at balance point; relationship between pulse-height (V) and primary beta energy (MeV). agreement and the average of the values is 2"8-4-0.5 per cent, indicating both liquid scintillation spectrometry and Cerenkov counting to be suitable for measuring the degree of 8~p contamination. 38p has two main advantages as a radiotracer for phosphorus in biological studies, lower radiation energy and longer half-life. I n specialized applications requiring less than mCi amounts of radioactivity, it m a y find considerable use, particularly to gain higher resolution in electron microscopic autoradiographs, or to permit dual labelling of phosphorus; lower cost will probably support the dominant position of 32p as the phosphorus tracer of choice in the immediate future, particularly in high specific-actlvity synthesis of organic molecules. Experiments in which the radiation is to be determined remotely such as within seeds, insects, woody tissue, or through glass by Geiger counting of solutions will still require the use of the higher-energy istope or perhaps even substitution by a suitable g a m m a emitter ~s0). Acknowledgements--The author is grateful to H. J. MURPHY and J. I. PULLEN for technical assistance, to C. H. UmqlN and N. L. JERRY for the illustrations, to Dr. D. M. MILLER for a Fortran program to prepare the decay factors and to Dr. R. E. LEWISof Oak Ridge for information regarding the production of phosphorus-33.

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