Liquid scintillator beta counting of iron-59 in clear and colored systems

International Journal ot Applied Radiation and Isotopes, 1962, Vol, 13, pp. 75-86. Pergamon Prom Ltd. Printed in Northern Ireland

Liquid Scintillator Beta Counting of Iron-59 in Clear and Colored Systems* T . P. L E F F I N G W E L L , ~

R . W . R I E S S + a n d G. S. M E L V I L L E , JR.~"

(First received6 July 1961 and in final form 16 November 1961) A semimicro technic has been developed which, in either physical or biologic systems, permits essentially simultaneous assessment of stable and radioiron. Increases in quantitative sensitivity and resolution are inherent. To fully evaluate the method, a U.S. National Bureau of Standards Fe59C18 standard was employed in model systems to quanfitate liquid scintillator beta counting of Fe 69. In systems which contain a colored iron-orthophenanthroline chelate, decline in counting efficiency is a linear function of color density for the concentration gradient investigated. However, presence of the colored chelate causes an increased photon yield relative to the corresponding visibly colorless sample. The enhanced photon yield elevates the entire differential coincidence counting efficiency curve by approximately 6 per cent. The enhancement phenomenon is (1) chelate concentration dependent, (2) beta-particle energy dependent, (3) at its maximum at the emission peak of the scintillators in the counting system employed, (4) effected only in the presence of the scintillators and (5) gamma-ray independent. C O M P T A G E A S C I N T I L L A T I O N S BETA DE Fe ~s EN SYSTEMES CLAIRS ET COLORES Nous avons mis au point une m~thode semi-microscoplque qui permet, essentlellement, de connaltre la rdpartition simuhan~e du fer stable et du fer radioactif dans les pr6parations physiques et blologiques. Cette m~thode est bien sensible et aussi tr~s exacte. Pour l'~valuer pleinement, nous avons employd un dchantillon de Fe59C13 standard du "U.S. National Bureau of Standards" dans un compteur ~ scintillation b~ta. Dans les preparations qui contlennent un combin~ colord de fer orthoph~nanthroline, la balsse d'activitd du compteur est une foncfion lindaire de l'intensitd de la couleur pour le degrd de concentration recherche. Cependant, la prdsence du combin6 colord provoque une importante ~mission de photons en rapport avec la visible ddcoloration de l'~nchantillon. L'~mission de photons dl~ve, par enti~re coincidence, la courbe d'efficacltd du compteur d'environ 6 pour cent. Ce phenom~ne ddpend (1) de la concentration du comblnd, (2) de l'~nergie des parficules b~ta, (3) ~t son maximum au sommet de l'dmission des scinfillateurs dans le syst~me de mesure employ6, (4) s'effectue seulement en pr~ence des scintillateurs et (5) n'est pas influencd par les rayons gamma. I43MEPEHIIE BETA-AI~TIIBHOCTtl F e 59 B IIPOSPAtIHBIX H OKPAIIIEHHBIX CIICTEMAX I I P t l IIOMOII~tl H(II)~I~I4X CI~HHTHJIJIHTOPOB B ~ a a paspa6oTaHa CeMH-MHKpoMeTO~HI~a, HO3BOJIHmmaH onpe~e~ATI~ O~HOBpe~eHHO yCTO~HHBOe H pa~IIOaHTHBHOe a~eJie3o a ~}HaHtleCRHX H (~HOJIOFIItIeCI~HXCHCTeMax. ~)Ta MeTO~H~a o ~ a ~ a e T IIOBI~IIIIeHHO~qyBCTBHTOJILHOCTI~IOH TOHHOCTbIO. ~JIH oI~eH~H ~ e T o ~ a 5LIgIa ii3MepeHa, iipH I~OMOIKHa~H~I~OrO CI~HHTHJIYlfII~HOHHOPO CHeTHIIRa~ ~eTa*aI~TIIBHOCTb ~'~e 59 B CTaH~apTHOM o S p a ~ e Fe~gC1~ Ha~HoHa~IbaOrO Bxopo CTaH~apToB. B CHCTeMax, * This work was conducted at the Radioblological Laboratory of the University of Texas and the United States Air Force and supported (in part) by Contrac$ A F 41 (657)-149 and the School of Aerospace Medicine. Radiobiology Branch, Austin Section, School of Aerospace Medlcine~ USAF, Brooks Air Force Base, Texas, U.S.A. University of Texas, Austin, Texas, U.S.A. 75

76

7". P. Leffmgwell, R. W. Riess and G. S. Melville, Jr.

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BETA SZINTILLATIONSZ)i,HLUNG VON Fe~9 IN GEF/~RBTER UND UNGEF/X,RBTER ANORDNUNG Eine Semimikrotechnik ftir physikalische oder biologische Systeme ist entwickelt worden, die eine praktisch gleichzeitige Erfassung des stabilen und des radioaktiven Eisens erm~glicht. H6here Empfindlichkeit und Aufl6sungs-verm6gen sind hiermit verbunden. Um diese Methode voll auszuwerten wurde eine "National Bureau of Standards" Fe59Cls Standardprobe in Modell-Anordnungen benutzt um die Beta Z~hlung des Fe59 in einer szintillierenden Fltissigkeit quantitativ zu erfassen. In Systemen welche ein geffirbes Eisen-orthophenanthrolin Chelat enthalten ist die Abnahme einer erfassbaren Ziihlung eine lineare Funktion der Farbdichte ffir das untcrsuchte Konzentrationsgef~ille. Jedoch bewirkt die Gegenwart des gef~irbten Chelats einen Anstieg an Photoenausbeute in Vergleich zu dcr entsprechenden Probe. Die verst~rkte Photonenaubeute hebt die ganze Koinzidcnzziihlkurve um ungei~ihr 6 pro zent. DiesVerstiirkungsphenomen (1) hiingt von der Chelatkonzentration ab, (2) h~ngt ab von der Betateilchenenergie, (3) erreicht sein Maximum in der Emissionsspitze der szintillierenden Substanz in dem bentitzten Ziihlsystem, (4) wird hervorgerufen nur in der Gegenwart der szintillierenden Substanz, und (5) ist unabh~ngig von Gamma Strahlen. IRON-59 decays to stable cobalt-59. G a m m a photons of five distinct wavelengths plus a weak X-ray, and beta particles of four energy continuums plus conversion electrons of two energies, have been reported for the process of the unstable iron attaining a ground state. O n the basis of relative frequency, the 0.271 MeV (max) beta-2 and 1.29 MeV gamma-5 pair, plus the 0.462 MeV (max) beta-3 and 1.099 MeV gamma-4 pair, combine to account for nearly all of the decay particles and photons ~1). This probably has been the basis for earlier reports that a 1:1 beta-gamma decay scheme obtained for this radioisotope (2). The substantially true 1:1 beta-gamma ratio sometimes has been employed in radioassays of iron-59, with thin window G.M. and gas-flow counting for beta determinations, 13,4) and several methods for gamma estimations. ~5,n) I n tracer studies of iron metabolism in experimental animals in this laboratory ~,s), a crystal scintillator gamma estimation technic has been used. In small animals, however, it became desirable to reduce the volume of biologic materials subject to assay. Since a concomitant reduction in total iron also was involved, the requirement arose for a radio-

assay method more sensitive than that previously employed. A technic for selectively chelating and extracting microgram quantities of iron from biologic materials was devised. The process-a modification of the basic technic of PETERSON{9) --involved formation of a colored iron chelate which allowed spectrophotometric determination of total iron. When introduced into a liquid scintillator solution the beta decay component of iron-59 in the chelate could be counted with an efficiency of 35 to 40 per cent (10). This paper describes the physical determinations required to evaluate beta counting of iron-59 in liquid scintillator solutions.

MATERIALS AND METHODS The original semimicro technic involved chelation of all tissue iron. Methods for preparing biologic materials, preparation of counting samples, counting equipment* and settings, and indices of the potential and limitation of the technic have been reported (1°~. Since * Packard "Tri-Carb" Liquid Scintillation Spec: trometer, Model 314, Serial D-5.

Liquid scintillator beta countingof iron-59 in clear and coloredsystems

77

tissue iron concentrations may vary, color of 11.0 ml of a stock toluene-PPO-POPOP densities of samples derived from biologic scintillator solutionCl0). Constant activity "point" source. Commercial materials were varied. Therefore, a color gradient was investigated in the model solutions of Fe59C13/FeCI8 in 0.1 normal HCI were diluted with low C14-ethanol in the systems. Initial model system determinations were manner previously described. Aliquots of the made using commercial FeS~C13 solutions which, ethanol solution were pipetted onto a recaccording to the supplier, had a calibration tangular piece of Mylar film (0.98 rag/era z) precision of ± $ per cent. T o quantitate such and air-dried. The film was folded once, which information, dilutions made from a National gave a monolayer of the film surrounding the Bureau of Standards gamma-calibrated Fe69C13 deposited radioiron compound. Edges of the standard were placed into counting samples Mylar packet were double-folded and sealed completely analogous to those used for com- with a thermosetting epoxy resin bonding mercial radioiron measurements. All NBS agent. The envelope thus formed was susdilutions and sample preparations were in pended from the plastic cap of a counting vial triplicate. with platinum wire. This constant activity T h e colored iron-orthophenanthrolinechelate source was employed to determine the precision was prepared from stock iron solutions; the with which counting sample replication could solutions were processed by the method for be effected, and to relate the counts derived biologic iron ~t°). All counting samples were from colorless and colored systems. routinely flushed with argon to eliminate the The same Mylar envelope was later covered oxygen effect which sometimes was apparent with aluminum foil (about 105 rag/era 2) calcuin early phases of the work. This oxygen effect lated to stop all of the beta particles emitted by appears to be due to the presence of trace the iron-59 <~). The foil-covered envelope was amounts of oxidizable inpurities in the com- used to indicate the contribution of gamma mercial reagents used for preparing counting decay to the count. samples. Although such envelopes were of constant activity, they presented altered geometry, optics, and particle attenuation with respect to Fe59C13 counting samples the homogeneous systems. T o minimize optical Both homogeneously distributed and constant differences, the envelope was always oriented activity "point" sources of iron-59 were em- to present the minimum surface area to the ployed. Preparation of samples was as follows. opposing phototubes. In either form, the Homogeneous samples. A National Bureau of envelope was introduced into a continuum of Standards gamma-calibrated Fe59C13 standard samples which were completely analogous to (in carrier solution of 2 g/1 stable FeCls.6H20 the homogeneous series, with the exception of in 1 normal HC1) was made into stock solutions radioiron content. Between transfers the enby pipetting aliquots into low C14-ethanol. velope was washed in low C14-toluene and Dilutions were calculated so that 7.0 ml of the air-drled. ethanol would contain the desired number of disintegrations per unit time. These 7.0 ml Homogeneous C 14 counting samples aliquots of Fe59Cls-ethanol solution were then A National Bureau of Standards C14-benzoic placed in a counting vial, to which 2.0 ml of acid standard was also employed in an analeither low C14-ethanol, isoamyl alcohol (IAA), ogous counting sample series. Using this 0.156 0.5 per cent solution of orthophenanthroline in MeV (max) pure beta emitter ~1) permitted the IAA, or freshly prepared solutions of desired reference evaluation of the Fe59C18 beta countconcentrations of the iron-orthophenanthro- ing system. T h e Cl~-toluene solution was line chelate were added. Introduction of the pipetted directly into the toluene-scintillation ferric iron caused no measurable alteration in solution. Other components were introduced color density of the sample. Counting sample as in the FeS~Cln method for homogeneous volume was brought to 20.0 ml by the addition samples.

78

T. P. Le•ngweU, R. W. Riess and G. S. Melville, Jr.

Pulse height analyses Relative photon yield as a function of discriminator voltage settings was determined by "scanning" individual model systems with a sweeping 10V "window", as well as by setting a lower gate but no upper gate. The procedures employed thus were analogous--by definition--to obtaining differential and integral pulse amplitude spectra by both noncoincidence and coincidence counting methods. The precise procedure for effecting these measures on a "Tri-Carb" have been detailed~XS). On the basis of the pulse amplitude spectra and the signal:noise ratio of homogeneous ethanol samples and the blanks, final coincidence count rate settings were selected. , For the liquid scindliatlon spectrometer in use, these settings were (for both monitor and analyzer circuits) 6.5 and 70.0 V, thus giving a maximum coincidence count "window" of 63.5 V. A photomultiplier voltage of 1050 V was found to give maximum response for both Fe so and C 14. Counting e~ency is defined in this paper as the ratio of the net wide-window differential coincidence count to the activity calculated for the National Bureau of Standards Fe 69 and C 14 standards which had beenplaced in the counting samples. The capacity of any individual system to record one event per nuclear disintegration was determined by making single-channel (non-coincident) integral counts on either the monitor or analyzer channels as a function of lower discriminator voltage on attenuations 1 and 2 (amplifier g a i n = 3,000 and 1,500 respectively). The net counts were plotted on semilog paper, and lines were fitted visually. The lines were then extrapolated back to the ordinate. Comparison of the intercept with the activity calculated for the sample permitted evaluation of the effect of adding each component to the counting system. Optical measurements Absorption spectra of all components of the system--separately and in all combinations-were determined on a Beckman DU spectrophotometer. Absorption by the colored compound as a function of concentration was

determined by reading the solution against an appropriate blank at the absorption peak of the iron-orthophenanthroline chelate--512 m/~. RESULTS For homogeneous ethanol solutions of NBS FeSgCln, semi-logarithmic plots of net singlechannel (non-coincident) integral counts do not extrapolate to the calculated activity for the system (ordinate: net count; abscissa: lower discriminator voltage setting), but are low by about 10 per cent. C14-benzoic acid in an analogous system extrapolates to the calculated activity within 2 per cent. Representative data are shown in Figs. 1 and 2. Differential pulse amplitude spectra for representative clear and colored samples of equivalent radioisotopic activity are shown in Figs. 3 and 4. Whereas the C 14 in ethanol (Fig. 4) has a rather flat curve, that of the corresponding Fe 59 sample (Fig. 3) is peaked. Shapes of the curves for the two isotopes are similar in samples which contain 0.5 per cent orthophenanthroline in IAA, although differences exist in slope and in displacement on the voltage axis. In counting samples which contain 0.4 #g Fe/ml counting solution (in the chelated form), shape of the curve resembles that for the corresponding colorless sample. However, amplitude of the Fe s9 curve in the colored system is greater than for the corresponding clear sample. This enhancement is not apparent in any of the C 14 samples. The enhancement phenomenon noted for iron-59, which is manifested when either differential or integral coincidence counting is employed, has been reproduced more than forty times. As is apparent from the differential curves (Fig. 3), amplitude of enhancement is somewhat voltage-dependent: maximum enhancement occurs at the emission peak of the total system. At this maximum, integral pulse enhancement averages about 22 per cent. Table 1 depicts representative wide-window differential coincidence counting data obtained from either radioisotope in samples containing only ethanol-toluene-PPO-POPOP. It further reflects the decline in counting efficiency which occurs in systems containing the visibly colorless orthophena;~throline-IAA solution, and

Liquid scintillator beta counting of iron-59 in clear and coloredsystems

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FIG. 1. Net non-coincident integral counts for NBS FeS~C13- ~ ethanol solution in a toluenePPO-POPOP liquid scintillation counting solution. Open circles--attenuation I (gain = 3,000). Solid circles-- 2: attenuation 2 (gain = 1,500). The extrapolated intercept is lower than the calculated ~" activity by approximately 10 per cent.

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then compares the counts recorded for the gradient o f colored systems. Figures 5 and 6 express these data in terms o f the wide-window differential coincidence counting efficiencies (see definition) which m a y be expected from either Fe 59 or C 14 in comparable systems. Consistent with the e n h a n c e m e n t p h e n o m -

enon s h o w n for the Fe n9 in the presence of the colored chelate, efficiency with which C 1~ betas m a y be counted is more markedly affected by system components than is that for the Fe 59. T h a t counting efficiency is dependent on betaparticle energy is clearly illustrated; the e n h a n c e m e n t p h e n o m e n o n a n d continued high

80

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Fzo. 4. Differential pulse amplitude spectra as a function of lower discriminator voltage for samples of equivalent NBS C14-benzoic acid activity (10V window). Counting solutions same as for samples in Fig. 3. Differences lie in that C14-benzoic acid was introduced into solution via toluene, whereas Fe~aCls was introduced via its ethanol solution. Note differences between the spectra as a function of counting sample composition.

Liquid scintillator beta counting of iron-59 in clear and coloredsystems TABLE

81

Representative net wide-window differential coincidence counts recorded for samples* prepared from National Bureau of Standards materials

1.

Fe59C13 series Type of sample t

CZLbenzoic acid series +

Net coincidence count (counts]min)

Ratio to IAA sample

Net coincidence count (counts/min)

Ratio to IAA sample

2,579 2,340 2,471 2,483 2,428 2,321 2,316 2,284 2,156 2,166 2,054 2,090

1.10 1.00 1.055 1.06 1.04 0.99 0.99 0.97 0.92 0.92 0.88 0.89

3,218 1,767 1,425 1,310 I, 182 1,177 983 909 887 ----

1.82 1.00 0.80 0.74 0.66 0.66 0.55 0.51 0.50

Ethanol IAA 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

* Calculated sample activities: FeS~Cla = 5,100 d.p.m. : CXa--benzoic acid = 4,930 d.p.m. t Ethanol--7.0 ml stock Fe59Cla---ethanol solution +2.0 ml ethanol + 11.0 ml stock scintillator ~olution. IAA--7.0 ml stock FesgCla--ethanol solution +2.0 ml 0.5 per cent orthophenanthroline in isoamyl alcohol + I 1.0 ml stock scintillator solution. 0.1 to 1.0--Same as IAA, except that the solutions contain a concentration gradient of the iron-orthophenanthroline chelate. Numerical designation reflects the concentration of iron (in chelate form) in #g/ml of counting solution. ++C 14 series--same as FeSOCLa series, except that CZ4--benzoic acid was dissolved in toluene and introduced directly into a toluene-scintillator solution; 11.0 ml thus contained both C 14 and scintillators at the desired concentrations.

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Fio. 5. Counting efficiencies determined for samples containing 7.0 ml aliquots of a stock NBS Fe59Cls--ethanol solution. Counting samples constituted as described in Table 1; counting efficiencies calculated from data given in Table 1.

82

T. P. LeffingweU, R. W. Riess and G. S. Melville, Jr.

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Fzo. 6. Counting efficiencies determined for samples containing stock solution of NBS CZ4-benzoic acid of equivalent activity. Counting samples are constituted as described in Table 1; efficiencies calculated from data given in Table 1. efficiency of counting in the presence of the colored materials are evidenced only in the higher energy Fe 59 series. Coincidence counting data from the constant activity Mylar envelope evidenced departure from the shape of the curves for homogeneous samples only in the concentration region of 0.1 through 0.4/~g Fe/ml counting solution. However, lower efficiencies of counting were uniformly apparent. Since this envelope was prepared from commercial radioiron, and because of the modified optics, geometry, and possible particle attenuation, further quantitation has not been attempted. Although the background count for colored samples varies somewhat with color density, a mean value of 45 counts/re_in would be representative for the entire color gradient. In contrast to this mean background of 45 counts• rain the mean count for the foil-covered envelope in the same samples is about 54 counts/min. The envelope contained an activity in excess of 5,000 d.p.m, as based on calibration against the National Bureau of Standards Fe~gC13. When allowances are made for errors in calibration of the commercial radioiron, calculations on necessary foil thickness, and the several means by which t h e

gamma decay component might transmit degradational energy to the scintillator system, it appears that gamma contribution to counting is essentially undetectable. T h e optical density of various concentrations of the iron-orthophenanthroline chelate, at the absorption peak o f 512 m/~, is shown in Fig. 7. Linearity was established by reading multiple samples at each concentration. The line shown is a least squares fit of the data. Transmittance thus appears to obey Beer's law over the concentration gradient shown. O f the components in the homogeneous counting samples, only orthophenanthroline and the iron-orthophenanthroline chelate absorb in the region in which the scintillators emit. Figure 8 depicts the absorption spectra of these two components at approximately the maximum concentration in which they exist in the counting samples, and compares them to idealized emission spectra (Fig. 9) for the two scintillators which have been reported by HAYEs eta/. (nJ~l DISCUSSION Beta counting of National Bureau of Standards gamma-calibrated Fe59Cla, introduced by means of its ethanol solution into a liquid scintillator system, may be accomplished with

83

Liquid scintillator beta counting of iron-59 in clear and colored systems

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FIo. 7. Optical density as a function of the iron-orthophenanthroline chelate concentration on a Beckman DU spectrometer. The line represents a least squares fit of data derived from multiple readings at different concentrations.

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Fro. 8. Absorption spectra for 0.5 per cent orthophenanthroline in isoamyl alcohol solution and the iron-orthophenanthroline chelate in isoamyl alcohol (excess of orthophenanthroline). Amplitude of the chelate absorption spectrum corresponds to the maximum concentration at which it exists in the counting samples. These two components are the only ones in the counting system which absorb in the region in which the scintillators emit. Determinations made on a Beckman DU spectrophotometer. an efficiency exceeding 50 per cent on the Packard " T r i - C a r b " liquid scintillation spectrometer (Model 314, Serial D-5). I n the presence of a colored iron-orthophenanthroline chelate, a high Fe 69 counting efficiency can be maintained. Decline in counting efficiency in the presence of the chelate is essentially a linear function of in-

creasing color density. However, introduction of the colored compound into the scintillator system results in an increase in the signal with respect to a corresponding "clear" sample (one containing ethanol, 0.5 per cent solution orthophenanthroline in isoamyl alcohol, and 11.0 ml of a stock toluene-PPO-POPOP solution). Thus, a sample which contains 0.4 # g Fe]ml

84

T. P. Leffingwell, R. W. Riess and G. S. Melville, Jr.

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FIO. 9. Idealized emission spectra for 2,5 diphenyl-oxazole and 1,4-di(5phenyl-2-oxazolyl)-benzene as reported by HAYESet al. (u) Data published by permission of authors. counting solution (in the chelate form), reflects an increased signal which averages about 22 per cent on integral counting and still permits wide-window differential coincidence counting to be effected with an efficiency approximating unity with a comparable clear sample. At concentrations lower than 0.4 #g Fe/ml, efficiency of wide-window differential coincidence counting almost approximates that for the "best" ethanol-radioiron-liquid scintillator counting sample, and exceeds the efficiency with which the corresponding "colorless" sample may be counted. Counting efficiency is directly related to the enhanced differential signal: if it were to be assumed that decline in counting efficiency is inversely related to increasing color density in the absence of the enhancement phenomenon, it then appears that the enhancement effect elevates the entire wide-window differential coincidence counting efficiency curve by approximately 6 per cent. T h e enhanced photon yield appears to be (i) chelate concentration dependent, (ii) betaparticle energy dependent, (iii) at its maximum at the emission peak of the scintillators in the complex counting samples, (iv) effected only in the presence of the scintillators and (v) essentially gamma-ray independent. T h e nature of the enhancement may be related to the probable physical nature of the iron-orthophenanthroline chelate and to the absorption spectra of both this chelate and the orthophenanthroline-isoamyl alcohol solvent system. In their discussion of the bonding of transitional elements with donor groups,

MARTELL a n d CALVIN(ls) point out that, in general, such bonding increases the oxidation potential and hence increases the relative stability of the higher valence state. However, they further state the ferrous-ferric-orthophenanthroline couple to be the exception which "proves the rule". T h e ferrous complex is diamagnetic, involving covalent bonding with possible resonance contributions to the Fe-N double-bond character. T h e ferric chelate, also covalent, has a magnetic moment corresponding to that of a single electron. The relative stability of the ferrous state in this instance is due to the fact that the divalent form has completely filled the 3d, 4s and 4p orbitals, giving it the favorable xenon electronic configuration. Oxidation to the ferric state would alter this favorable structure by removing one electron. T h e y further state that because of steric hindrances a 3:1 ferric-orthophenanthroline chelate is not formed directly, but may arise through oxidation of the corresponding ferrous compound. T h e ferric state would normally be expected to form a 2:1 chelate, or alternately, a binuclear structure. Thus it appears that the 3:1 ferrous-orthophenanthroline chelate is a more stable form. In this work we have found the color complex to maintain the same absorption spectrum and amplitude after several weeks of storage. Moreover, in forming the colored compound, the iron has been reduced to the ferrous state prior to chelation. It consequently appears that the 3 : 1 ferrous chelate is the form present in the counting solutions, and that the characteristics ascribed for it by MARTELL and CALVIN

Liquid scintillator beta counting of iron-59 in clear and colored systems

would obtain. Because of the characteristics of this electronic configuration and possible resonance of the Fe-N double bond, the chelate may couple with the other resonating components of the system. The orthophenanthroline-IAA near-ultraviolet absorption spectrum spans the 295 to 340 m/~ region at concentrations roughly comparable to those found in the counting samples. Under these same conditions the iron-orthophenanthroline chelate appears to contribute a slight additional absorption at approximately 310 m/~. This spectral region coincides with portions of both the absorption and emission spectra of at least the 2, 5 diphenyl-oxazole scintillator.m,12) When introduced into a counting sample the "colorless" orthophenanthroline-IAA effects a reduction in the recordable count; when low concentrations of the colored chelate are introduced, the enhancement effect is noted. T h e visible wavelength absorption spectrum of the chelate extends to encompass the emission spectrum of the other scintillator- 1,4-di (5 phenyl-2-oxazolyl)-benzene. Either a direct (radiationless) resonance energy translation or a fluorescence by the chelate might be involved. I f the former were the case and the chelate effected uniform energy transfer steps (possibly with conservation of lower-energy meta-stable states) which involved the entire solvent/solute system, then one might expect an enhanced signal such as that observed. However, if the latter were to obtain, energy absorbed by the chelate would be emitted as photons of defined wavelengths. These photons would have to be perceivable by the phototubes or would have to be absorbed and re-emitted by the primary scintillators. No photon yield enhancement can be detected by the phototubes unless the scintillators are present; neither white light nor ultraviolet (Woods lamp) excitation produces visible fluorescence of the chelate-orthophenanthroline-IAA solutions. Thus, if a fluorescence is involved, it is not detectable with the equipment currently available to us except when the primary scintillators are present to re-emit the fluoresced light. Decline in counting efficiency in the colored complex does not occur at the same rate at which optical density (at 512 m/~) increases.

85

From the absorption and emission spectra, it would be expected that the decline in counting efficiency would be related to the portion of the curves wherein the absorption and emission spectra overlap (and not at the 512 m/t absorption peak). Although these wavelengths have not been specifically tested, the data suggest that the decline of counting efficiency may be attributed to absorption at wavelengths shorter than 512 m/~. The authors offer only a partial explanation for the differences in the ethanol-scintillator photon yield curves for the Fe ~9 and C 1~ samples and the higher wide-window differential coincidence counting efficiency found in these systems for the lower-energy C 14. At least three contributory factors might b e involved : (1) relative chemical purity of the samples (the NBS Fe ss standard was in a sealed ampoule designed for gamma calibration whereas the crystalline Cl*-benzoic acid was chemically pure); (2) modes of introducing the isotopes into the counting solution differed (the C 1~ standard, a resonating-type compound was actually soluble in the toluene, as were the scintillators, whereas the ionic Fe 59 was mainmined in the counting sample through its ethanol solution); (3) the relative energy distribution of the particles differs appreciably more than might be anticipated from differences immediately apparent when only the maximum particle energies are considered.~l, 14) It is the chemical format of the isotope in the counting sample, plus the differences in the beta spectra, which combine to offer a more plausible explanation--the resonating radiocarbon compound probably can translate resonance energy directly throughout the solute/ solvent system, whereas the ionic iron cannot effect the same degree of energy communication. In the chelate form, in which the Fe 5~ apparently can effect resonance energy translations with greater efficiency, both the altered efficacy and the contribution of the higher mean particulate energy are manifested. The efficiency of counting C 14, with a mean particulate energy of 0.050MeV per disintegration, is markedly affected by the presence of system components such as isoamyl alcohol and the iron-orthophenanthroline combination. On the other hand, Fe 59, which had a mean

86

7'. P. LeOingwell, R. IV. Riess and G. S. Melville, Jr.

particulate energy of 0.118 MeV per disintegration, is less affected by the presence of the components which " q u e n c h " the C 14 systems u). This does not yet fully explain why extrapolation of non-coincident integral counts back to the ordinate falls below the calculated activity for the NBS Fe 59 when the NBS C 1~ extrapolation agrees with the calculated activity. O f interest here is the fact that the NBS Fe s9 standard is calibrated in gammas per second, but the NBS radiocarbon sample is calibrated in microcuries--e.g, by definition, disintegrations per second. T h e two are not exactly synonymous in the case of the Fe 59, since there are four beta continuums to five g a m m a photons, plus the two conversion electrons to a weak X-ray. Whereas one might not ordinarily detect these small differences, neither would one necessarily expect unity for the photon to particle ratio in the rather complex liquid scintillator system. In addition, the efficacy with which energy communication is effected in the ethanol systems should be considered. Thus, it appears that the beta-counting extrapolation method perceives approximately 90 per cent of the photon activity of the sample; this 90 per cent represents the algebraic sum of the beta particles--and possibly, the conversion elect r o n s ~ i n a rigidly defined counting system. Differential coincidence counting efficiencies described herein are based on the gamma activity of the sample, and thus take any anomalies of the beta-counting method into account. Biologic data are derived by this differential coincidence count method, and the efficiency with which the counting is effected is determined by relating the unknown samples to simultaneously determined NBS Fe ~ Standard differential coincidence counting effÉciency curves on the basis of optical densities (at 512 m/~) and the knowledge that absorption spectra show the samples to be "clean". Thus, failure to achieve the calculated photon activity using extrapolated non-

coincidence integral values does not influence the quality of determinations made on unknowns from biologic sources. Rather, the extrapolation method is one of the several methods which we employ for the re-assay of radioiron preparations from commercial suppliers and for the assay of dilutions of these commercial solutions before injection into experimental animals.

REFERENCES 1. SLACKL. and WAY K. Radiations from Radioactive Atoms in Frequent Use. U.S. Atomic Energy Commission (1959). 2. KINSMANS. (Ed.) Radiological Health Handbook. Washington: U.S. Department of Health, Education, and Welfare (1957). 3. REnlsnE J. H., PALMER R. F. and KJ~INEJ. F. Analyt. Chem. 27, 849 (1955). 4. WASSERMANL. R. J. din. Invest. 31~ 32 (1952). 5. HUFF R. L., E-MLmO,~ P. J., GARCXAJ. F., ODA J. M., COCKWELLM. C. and LAWRENCE J. H. J. din. Invest. 30, 1512 (1951). 6. WEST H. D., HAHN P. F., CLARK W. F. and CHAPPELE E. W. Amer. J. Physiol. 169~ 194 (1952). 7. HARTWm Q. L., LEFFmGW~LL T. P., You~o R. J. and MELVXLLEG. S., JR. J. Lab. din. Med. 51, 410 (1958). 8. HARTWXGQ. L., MELVXLLEG. S., JR., LE~HNGW~LL T. P. and YOUNGR. J. Amer. J. Physiol. 196, 156 (1959). 9. PETERSONR. E. Analyt. Chem. 25, 1337 (1953). 10. LEFFINGWELLT. P., MRLVILLE G. S., JR. and Rmss R. W. J. Lab. din. Med. 53, 622 (1959). 11. HAYESF. N., ROCERSB. S., SANDERSP., SCHUCH R. L. and WmLXAMSD. L. Los Alamos Scientific Laboratory Report No. 1693 (1953). 12. Oar D. G., HAYES F. N., HANSBUR~ E. and KERR V. N. J. Amer. chem. Soc. 79, 5448 (1957). 13. MARTELLA. E. and CALWN M. Chemistry of the Metal Chelates Prentice-Hall (1953). 14. MARSHALLJ.H. Nucleonics 13, 34 (1955). 15. Operation Manual~Packard "Tri-Carb" Liquid Scintillation Spectrometer, Model 314, Packard Instrument Company, Inc., La Grange, Illinois (1958).