yangl and Edward K. C. Lee University of California Irvine, California 92664
1
I
Liquid Scintillation Counting Singlet-singlet energy transfer processes
1~ .q u. dscmtillatlon . . .
counting is commonly used for obtaining an assay of radioactive, tracer-labeled, organic substances containing tritium (3H or T), carbon-14 (I4C), sulfur-35 (%) or phosphorus-32 often it is almost indispensable for the assay of weak beta-emitters such as tritium. I t s widespread use in modern tracer-oriented biomedical researches as well as in varieties of chemical researches demands that students of biology, chemistry, and medicine be familiar with the theory and the practice of scintillation counting. Onc is surprised to find, however, that no undergraduate-level textbook on instrumental methods of analysis gives an adequate treatment of the theory useful for up-to-date methods of practice. Therefore, students are given no choice but t o read highly technical reference books or numerous literature articles in ordcr t o gain insight int,o a working theory on liquid scintillation processes. Yet in the brief period of time available t,o them, typical students are not well enough prepared t o follow easily the authoritative book written by Rirks (1). As far as the experimental procedures are concerned, students can obtain sufficient information through instruction manuals provided by the instrument makers. Thus, it scems t,o us that ready availabilit,y of a pedagogical article on the scintillation process might be of use. Our chief aim in writing this article is to show students the bare bones of the physical processes involved in liquid scintillation counting, simplificd for easy understanding, but still capable of revealing some new and very interesting complex phenomena. The scintillation processes will be regarded as a series of simple intermolecular and intramolecular processes involving electronic energy changcs. Since varieties of photochemical transformations involve electronic energy transfer processes similar to those observed in the scintillation processes, this article may stimulate the curios it,^ of the students who are interested in areas of excited-state chemistry such as photochemistry, radiation chemistry, hot-atom chemistry, photobiology, etc. We aim to emphasize how essential a knowledge of the molecular spectroscopy and the dynamics of intramolecular relaxation processes is for the detailed underst,anding of the kinetics of the intermolecular energy transfer processes.
Wavelength
2"
22
(81
*"
Wove Number ( 1 0 ' c m ~ ' l Figure 1. Absorption ond Rvorercence emiwion rpectm of toluene. IBerlmon (31, with permirrion of Acodemic Pre%)
-
2300-2700 A to a j r s t exited singlet state (8,) of toluene. The absorption spectrum corresponding to this So S1electronic transition and the fluovescence spectrum corresponding to the reverse So S, electronic transition are shown in Figure 1 (9). These spectra contain diffuse vibrational structures. The odistinct mirror~ymrnet~ry about 37,000 em-' (2700 A) indicates that the electronic energy gap between thc Soand & states is about 106 lccal/mole. The long wavelength limit (3300 A) as well as the short wavclcngth limit (2700 A) of the toluene fluorescence emission are indicated in the energy level diagram shown in Figure 2. The quantum yield of the toluenefluorescene emission (%) in dilute cyclohexane solution has been reported +
Spectroscopic Transitions of Toluene2
A grouncl singlet state (So) of toluene is promoted by the absorption of a photon of wavelength between
' An undergradoate research participant.
Any standard physical ehemist,ry textbook can serve as a convenient referonce to the photochemical termimlogy used in this article. See, for example, reference (B).
Figure 2.
Energy level diagram for excited rtoter.
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to br 0.17 aud fluorescence decau time ( T F )to ~ be 34 X 10-Qsec (8). The "missing" quantum yield of 0.83 must bc due to vadia(ion1ess processes such as inlerS1 - 3 T,transition) and system crossing yield (hc; internal conversion yield (arc,So f S,), ruling out the photochemical decomposition. If a quencher molecule ( Q ) is added to the solution, a bimolecular quenching process mill compete with the fluorescence, intersystem crossing, and internal conversion processes. As a result, the quantum yields for these processes will be proportionally reduced. The lozvest triplet slate (TI) of toluene is located a t 83 kcal/mole above the So level but considerably below the S, level (106 lical/ mole). The phosphorescence emission from this triplet state is not observed in solution a t room temperature because the extremely rapid radiationless processes from this state compete with the slow emission p r o c e ~ s . ~ Since this triplet state appears not to participate directly in the liquid scintillation processes under normal conditions, the fate of the triplet state is considered inconsequential for the purpose of the present discussion and will be i g n ~ r e d . ~
-
Spectroscopic Transitions of PPO (2,S-diphenyloxozole) and POPOP (1.4-bis-2-(5-phenyloxazo1e)-benzene)
The structural formulas of two scintillator solutes commonly used in combination, PPO and POPOP, are shown in Figure 3. The absorption and fluorescence
Figure 3. Structures of 2.5-diphenylorarole IPPOI and 1.4-his-2-15. phanyloxazoielbenrene IPOPOPI.
emission spectra of PPO and POPOP are shown in Figure 4. As in the toluene spectra, the mirrorsymmetry relationship holds true for these molecules. The first excited singlet energy levels deduced from the spectral data are shown in Figure 2. It is interesting to note a red-shift of about 3500 cm-' of the POPOP spectra compared to the PPO spectra; this lowering of about 10 kcal/mole of the SI state of POPOP compared to the S1state of PPO can be attributed to the higher degree of chromophore conjugation in POPOP than in PPO. It is also shown that the maximum molar extiuctioncoeficient (r,..) is 18,000 l/mole/cm a t A, = 3000 A for PPO, and 47,000 l/mole/cm a t , .X. = 3500 A for POPOP. The fluorescence emission quantum yield (Q.,) is 1.00 for PPO and 0.93 for POPOP, while the fluorescence decay time (rr) is 1.4 X loW9sec for PPO and -1.7 X loW9see for POPOP (3). High quantum yields of fluorescence and short decay times are important, desirable characteristics possessed by scintillator solutes for efficient and low coincidence-loss counting a t high count rate, and PPO and POPOP certainly have such qualifications. Toluene Exeimer Formation
It has been known for sometime that an excited dimer (excimer) can be formed bet,ween an electronically excited organic species and the parent ground state molecule (6, 7). The spectroscopic and kinetic properties of a toluene singlet excimer produced by ultraviolet irradiation of pure toluene and in dilute solution have been investigated recently (8). The existence of a toluene singlet excimer is established through the observation of enhanced fluorescence emission of a new hand (about 5000 cm-I red-shifted from the toluene monomer fluorescence emission) a t high concentrations of toluene and a t low temperature, suggesting the following equilibrium and non-equilibrium processes +CHdSr)
+ 6CHdSo) * 6CHz.+CHdS,)
-
2 +CHa(Sd
+ hv
The excimer fluorescence emission profile is shown with open circles denoted "DF" for comparison with the toluene monomer emission profile with filled circles denoted "MF" in Figure 4a. The excimer fluorescence emission quantum yield is about one-half of the monomer fluorescence quantum yield in pure toluene a t room temperature, and it increases by an order of magnitude a t -76'C. The fluorescence decay time of the excimer in hexane solution is 16 nanosec sec) as compared to 24 nanosec for the monomer decay time (7). Tberefore, most of the fluorescence a t room temperature, under uv excitation, is due to the monomer singlet in dilute hexane solution.
Figure 4. la) Upper; absorption and fluorescence emiuion spectra of PPO IBerlmon 131, with permission of Academic Press) and fluorescence emission proflles of toluene monomer IMFI and toluene excimer IDFI. (b) Lower; absorption and fluorescence emission rpectro of POPOP 1 Berlmm (31, with permision of Academic Prersl.
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"he intensity of the fluorescence emission ( I ) decays exponentially with time (t), as in radioactive decays (first order), and the decay time (m) is determined by experimental measure ment using the following relationship: I = I a exp (-t/m) where 10is the intensity at t = 0. 'The phosphorescence emission from toluene can be observed at low temperature (liquid air) where the r a t e of radiationlesr processes are reduced. Read the classical paper of Lewis and Kasha ( A ) . ~ r i p i i states t are, however, very important indeed in photw chemistry. See reference (6).
A somewhat different situation arises under pulsed electron bombardment of pure toluene (9). It appears that only the toluene excimer is produced in this case, since the monomer fluorescence component is almost absent. The short wavelength limit of the emission is near 2830 A, and therefore the lowest singlet excimer energy level may be located nearly 100 kcal/mole above the ground state monomer level. Excitation of Solvent Molecules by Beta Decay
Let us take the beta decay of tritium for the purpose of illustration. The half-life of tritium is 12.3 yr arid the amount of energy liberated in the decay is 17,600 electron volts (eV). Among the three decay products shown below, the electron and the anti-neutrino remove most of the excess energy in the form of kinetic energy, since they are much lighter than the helium-3 ion.
Therefore, the kinetic energy of 0-particles emerging from the trit,ium decays will have a characteristic distribution below 18.6 keV, the maximum energy of the 0-particle (EBlilrx). Suppose that these beta decays have taken place in liquid toluene whose lowest ionization potential is about 9 eV. The average 0-particle of 10 keV energy is capable of ionizing a t least a few hundred toluene molecules before it loses most of its recoil kinetic energy. For the sake of brevity, let us be satisfied with a simplified ionization and neutralization mechanism that is consistent with representative observations as shown below P*" CHI -> (&Ha)+ + P* + e -
+
where two asterisks are used to indicate that the 0particle has a higher kinetic energy before ionization of the toluene than after. We shall ignore other radiation induced processes and consider the most important end-product of the interaction between the @-particle and the solvent toluene molecule to be the toluene singlet excimer (D*) rather than the excited singlet monomer (M*), since the former is the predominant species observed in the 56-keV electron bombardment study (9). Under high energy electron bombardment, there will be excited ionic and excited neutral species in addition to the first excited singlet species, and thus one observes additional processes leading to the excimer formation. If uv excitation of the solvent were considered, however, the logical choice of the singlet excited species would be the toluene monomer singlet species. As far as the formalism used for the following kinetic considerations is concerned, it makes no difference which singlet excited species is chosen.
available (10, 11). The process of interest is D*(S,)
+ A (So)
-
D (So)
+ A* (S,)
where D is the donov and A is the acceptov. Vor this process to occur, the energy level of D* (8,) must be higher than t,he energy level of A* (8,). If the donor is the toluene singlet excimer and thc acceptor is 1'1'0, the process is about 15 kcal/mole exothermic. However, if the donor is the toluene singlet monomer and the acceptor is PPO, then the process is about 21 kcal/ mole exot,hermie. The evidence for the singlet-singlet energy t,ransfer is provided by the observation of the sensitized Jluorescmce emission from singlet exeked 3'1'0 produced by t,he expected ~adiulionlessenergy tvansjer from t,oluene to PPO, when toluene containing a small amount of PPO is directly excited by either 0-particles or photons. The fluorescence spectrum is like a fingerprint: If a wavelength sensitive phot,on detect,or is used, the direct fluorescence emission from the excited toluene monomer can easily be distinguished from the sensitized fluorescence emission arising from t,he excited l'PO, since their emission profiles (intensity versus wavelength) do not overlap (see Fig. 4 a ) . The toluene excimer fluorescence, however, does show some overlap wit,h the PPO fluorescence. Another important point must be made. The fluorescence quantum yield of PPO (8,) is 1.00 and that of the singlet toluene monomer (SJ i q 0.17. Therefore, if the S-S energy transfer were I ,.?plete, the luminescence from the sensitized system (to . m e PPO) would be 6 times brighter than the luminescence from pure toluene. Use of a sensitizer can thus result in an amplification of t,he photon in tens it,^.
+
Kinetics of Sensitized Fluorescence
Let us examine an idealized liquid scintillation counting system consisting of four components: toluene as solvent, PPO and POPOP as scintillating solutes, and O2 as non-fluorescent quencher. There are four major energy t,ransfer events: (1) excitation of solvent following the radioactive decay, (2) S-S energy transfer from the excited solvent species (let this be the toluene singlet excimer = D*) t,o the primary solute, PPO, (3) sensitized fluorescence emission from t.he excited PPO, and finally (4) "trivial" energy transfer via absorption of the PPO-fluorescence by the secondary solute, POPOP, and reemission of fluorescence by the excited POPOP. This energy transfer model is shown below Solvent Excitation: 2114CHa
-
+ @*
n
m
+ e-
(1)
Singlet Energy Transfer:
Singlet-Singlet Energy Transfer Processes and Sensitized Luminescence
Intermolecular electronic energy transfer processes, particularly the singlet-singlet (8-8)transfer between two different organic molecules in solution, have been studied for many years, and recent review articles are
Trivial Transfer: ( h u h
+ POPOP(So)
- IPOPOP' (&)/+
POPOP(Sd
(hv)ssr (4)
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The slcnrlpslate solutionG for thc cmit,ted photon intrnsity of the sensitized fluorescence (ISF')is
where a is thc stcndy-statc rate of radioactive decay, (I'I'O*)., is the steady-state concentration of PPO*(S1), and the li's are individual rate coefficients as shown above. It. is convenient to rearrange thc above equation to obtain a Stern-Volmer equation as shown below
The above equation can be simplified further in the absence of the O2 quencher, and the photon intensity (Isr) becomes dependent upon the concentration of 1'PO alone if a and n remain constant. I n actual liquid scintillat,ion counting experiments, a count rate (R) is measured where R = f Isr and f is an operational counting efficiency factor. In the absence of O2and POPOP, count rates resulting from a fixed amount of tritium activity with varying amounts of PPO have been measured, and they are shown in Figure 5. As expected from the above intensity equation, the value of R increases with increasing (PPO) and reaches a saturation limit a t 5 mM PPO concentration. A Stern-Volmer plot of 1/R versus l/(PPO) is shown in Figure 6, and the PPO quenching constant, lcs/(ka kro km), can be obtained by dividing the intercept by the slope. The quenching
+
+
Figure I Count rote dependence on the PPO concentration in tho 0 3 - f r e e toluene rolvtion containing a constant amount of tritivm activity.
Figure 6. Kinetic I y i of the lineor Stern-Volmer relationship for tho ringlet-ringlet energy transfer fmm toluene to PPO, showing the plot of the reciprocal count rote versus the reciprocal PPO concentrotion.
constant thus obtained is ( 2 1 1) X lo3l/mole, and its reciprocal value is the half-value concentvation of PPO, (PPO),, S 0.5 X 10W3 moles/l.' At the halfconcentrat,ion of PPO, the rate of the S-S transfer from the toluene singlet excimer (D*) competes equally with the other rates combined (kr klo klsc). The following calculation of the rate coefficient is possible on the assumption that fluorescence decay time of D * (m), 1/m = kr klC km, and the halfvalue concentration of 1'PO are known. For (Pro),, = 0.5 X 10W3molcs/l (our measurement) and T~ = 16 X sec (7), ks = l / [ ( P P o ) h 7 ~ ]= 1 X 10'' l/mole/sec is obtained as the bimolecular rate coefficient for the S-S energy transfer from D* to PPO. If the 8-S energy transfer rate coefficient were calculated on thc basis that the process involves the toluene excited singlet monomer (>I*), = 31.7 X sec in pure toluene (13) and (PPO),, = 0.5 X lo-% moles/l (our measurement), ks is 6 X 101° l/mole/sec. This calculated monomer rate coefficient is not much
+ +
+ +
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different from the excimer coefficient calculated above. Furthermore, it is interesting to note that the above monomer rate coefficient (6 X 10'0 l/mole/sec) is only twice the S-S energy transfer rate coefficients (3 4 X 101° l/mole/sec) ohtained in the solution photochemical experiments a t dilute donor concentrations where It* = benzene; toluene; o-, m-, and p-xylenes; and ethyl benzene; and A = biacetyl (10). The energies of the lowest excited singlet states of these aromatic substances are close to the singlet energy of toluene, and the lowest excited singlet level of biacetyl is located a t 63 kcal/mole above its ground state (14). Therefore, the monomer energy transfers are definitely exothermic. It has been suggested that the energy transfer in the photochemical system involves a collisional enevgy tvansfev mechanism, since the observed rate coefficients are close to the limits of the diffusion contdled vate coeficients. Since the S-S energy transfer rate coefficients obtained in our scintillation experiments are not much different from those obtained in the photochemical experiments, the S-S energy transfer from the toluene excimer to PPO might be explained satisfactorily by the collisional energy transfer mechanism. The expected and unique role of POPOP in the liquid scintillation solution is to undergo a trivial energy transfer by an efficient absorption-reemission process. Since the POPOP absorption spectrum overlaps well with the PPO emission spectrum and since POPOP has very large molar extinction coefficients (>lo5) in the overlap region, only a very small amount of POPOP is necessary for nearly complete absorption of the PPO fluorescence. At the commonly used concentration of POPOP (0.14 mM), only a few mm thickness of the solution will absorb completely 3600 A radiation. Furthermore, POPOP is a useful wavelength-shifter for the intensity maximum of the PPQ fluorescence (3600 A), since its emission is about 500 A red-shifted and its emission profile overlaps well with the photocathode sensitivity profile of the popular photomuJtiplier tubes (maximum detection efficiency a t 4100 A). Therefore, the use of POPOP as a wavelength-shifter and a matching photomultiplier tube will enhance the quantum efficiency of scintillation counting. Since the concentration of POPOP is about 1/100 of the concentration of PPO, the fraction of the S-S transfer process from the toluene excimer to POPOP should be negligibly small compared to the transfer to PPO.
-
Molecular Efficiency of Quenchers
Quenching substances are those which lower the quantum efficiency of the overall scintillation process. 'The steady-state can only he maintained at extremely high rates of the radioactive decay, whereas the liquid scintillation method is not usually employed to measure such high rates. However, the above solution provides an answer that is useful, since one can suppose the "steady-state" to he valid within a. time scale that is short compared to the duration of the "smgle train" of scintillation events following a. radioactive decay. The quenching constant ohtained by a pulse height measurement using I3'Cs internal-conversion electron line is 1.0 X loa l/male (ref. 18) and is in essential agreement with the shove value, although the methods of measurement employed are quite different and direct comparison is not simple. In any case, these values are good enough for the present discussion.
Claasificotion of Quenching Substances Strong Quenchers RCOR RCIiO 13JN RSH RI RN02
Mild Quenehers
RCOOH RCH=CHR RNH2 RSR RBr
. ..
Ouenched
Diluters
1
RCOOR ROH ROR (ROhPO KC1 RF RH
Figure 7. D i a g r a m showing the energy migration among the rcintillotor components in the sample vial and the procewing of t h e e l e ~ t r o n ri i~g n d .
Operationally, quenching ability has been divided into three broad categories: (1) st,rong, (2) mild, and (3) dilut,ers. The tahle shows this classification for commonly encountered organic functional groups (15). Quenchers may interfere with the scintillation process cither by absorbing light quanta or by "chemical int,eractionn with components in the system. The fact that O2 quenches some excited singlet states (particularly aromatic molecules) is not widely recognized, although thc fact t.hat O1 quenches varieties of triplet stat,es and scavenges radicals is generally well known. There is enough O2 dissolved in typical scinbillat,ion solutions a t room temperat,ure and a t a n at,mospheric pressure to quench a significant fract,ion of the toluenc monomer fluoresccncc. For example, the fluoresccncc intensit,y of the dilute solution containing dissolved air is of the intensity of t,he air-free solution (5). However, the Orquenching mcchanism is not well undcrst,ood. I n order to minimize t,he O r quenching effect which will vary with varying amounts of t,hc dissolved air, a great excess of PPO (18 mAf) is normally used in t,hc scint,illation counting. Since t,he half-valuc concentration of PPO is 0.5 mM, the mean lifetime of thc t,olucrie excimer will he short,ened by a factor of 37 a t (PPO) = 18 mM (or 4 g/l) in the airfree toluenc solution, when t,he Stern-Volmer equation is examined. Therefore, the ext,ent of the 02-quenching of the toluene excimer will aL3o he grcat,ly diminished in the toluene solut,ion containing 4 g PPO/I. In fact, in the air-saturated, normal scintillat,ion solution only 18y0 diminntion of the fluoresccnce int,cnsit,y has beet] observed (I). Other quenchers may interferc by competing wit,h PPO for the S-S energy t,ransfer process; these usually exhibit ult,raviolet absorption in the 2500-4000 A region as PPO does. "Color quenchers" which absorb light quant,a in the visible region interfcre by competing with POPOP for the trivial energy transfer process. Compounds which absorb only far ultraviolet radiation merely lower the concentration of excited toluene molecules by competing for the 8particle energy, and lower the spccific activity of the sample by dilution; hence the term "diluter." Any substance undergoihg a chemical reaction with either the solvent or the primary or secondary solute is likely to seriously lower the overall luminescence yield. Energy Flow and Signal Processing
We summarize the essential features of the enercv migration among the scintillator components in the samplc vial and thc clcctronic signal processing in Figure 7. I n general, the instruction manual accompanying an clectronic counter for liquid scintil-
latiou counting offers a sufficiently detailed explanation of the theory of operation, and therefore only a brief summary will he attempted here. As mentioned earlier, a blue-sensitive photomultiplier tube will act as a wavelength discriminating detector of the POPOP fluorescence. The scintillation photons will be emitted as a short-burst following the initial ionization of toluene molecules by the energetic (3particle from the radioactive decay, and the duration see, since the of this burst may be as short as entire sequence of the energy flows in the liquid scint,illation system may take only sec. The number of emitted photons per typical beta decay event may be as many as 10%within this time period, and thc photon burst is converted to an electronic pulse by the photomultiplier with a quantum efficiency of about lo6. This pulse is further amplified by the pre-amplifier and the amplifier section. A sketch of a typical pulse size distribution (or pulse height distribution) is shown in Figure 8. The shape of the pulse height distribution resembles the P-particle energy distribution that is characteristic Nld I ~wmr of the P-emitter. The I energy spread (pulse height) I I is also proportional to the PYIS. HFI(JM I V O I I ~ energy of the 8-particle, since the number of the ~i~~~~ 8. pulse height bution in t h e unquenched lrolid toluene excimers formed is line) and quenched (dotted line) proportional to the amount liquid rcintillotion rolvtiont containing beto-emi~terr. of energy deposited by a given decay. Therefore it is possible to discriminate, on the hasis of t,he observed pulse height, the tritium dccay (EBm.. = 0.0186 MeV) from the "C decay (Earn,, = 0.15.5 MeV) but not the '4C decay from the 3 S decay (EBmnr = 0.167 MeV). The instrument,al pulse height rliscrinzinatinn is handled by an encrgy analyzer spcct,rometer. I t discards the signals of pulse height less than I,8(thermal noise) and those higher than Ls (high encrgy cosmic ray events or high energy backgrounds). Howeveri it feeds the signals with pulse height between L3 and L4 (lou3er channel) to a scaler for registering the number of such events, while it feeds the signals with pulse height between Ln and Ls (upper channel) to another scaler for registering these latter events. An elaborate coincidence circuit is commonly used to lower the background count rates and the details are found in the manuals.
1~
I\
U"
ERiciency
Chgnnel Ratio
The upper-to-lower channel ratio, (I,, - L,)/(L, L4), is an important parameter in liquid scintillation Volume 46, Number 5, Moy 7 969
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counting. Statistically large numbers of beta decay events from a given radioisotope will give rise t,o a characteristic pulse height distribution in an unquenched scintillation liquid as shown by the solid line in Figure S, but the distribution in a quenched scintillation liquid is skewed to the left as shown by the broken line. The degree of the lowering of the pulse height distribution as well as the lowering of the observed count rate becomes severe as the efficiency and the quantity of t,he quencher increase, and a t the same time the channel rat,io decreases accordingly. Since the degree of quenching varies from sample to sample depending on the quencher composition, it is necessary to evaluate the degree of the quenching in each individual sample. This analytical task is accomplished for a given radioisotope by measuring the channel ratio in each sample vial and by comparing it with a channel ratio calibration obtained for a series of que?lcherlstandard veferences. If the quenched st,andard references contain exactly known and equal amounts of the radioactivity and known varying amounts of quenchers, not only is i t possible to calibrate the channel ratio versus the fraction quenched, but it is also possible to calibrate the counting efficiency versus the channel ratio. Therefore, using a set of quenched standard references, the counting efficiency (cpm/dpm) can be calibrated against the channel ratio. (Typical results are shown in Figs. 9 and 10.) The variation of the channel ratio
Figure 9. Channel ratio variation d u e to the increasing omounts of cyclohexonone quencher added to the toluene-PPO-POPOP solulion I15 ml) containing o mnrtont omount of tritium activity.
Figure 10. Calibration curve obtained for the absolute counting efficiency of tritivm ver3us channel rmtio by u3e of a set of "quenched
standards."
versus the variation of the cyclohexanone quencher added to 15 ml of a standard scintillation solution (toluene, PPO, and POPOI') contairiirig a fixed amount of tritium activity is show~iin Figure 9. The calibration of the count,ing efficiency versus t,hc channel ratio in a set of quenched standard references co~itaining tritium activity is shown in Figure 10. I t is typical that about 30% counting efficiency is obtained for tritium samples with minimum amounts of quencher present for reasons of limitation in counter geomet,ry and efficiency of signal recovery. For more energetic beta decay isotopes, much higher counting efficiencies will be observed. Common Liquid Scintillators
R'onpolar solvents (toluene, xylene, etc.) as well as polar solvents (p-dioxane, glycol ethers, etc.) are used, and they have good energy transfer characteristics and varying solubility properties. Typical primary solutes used are I'PO, p-terphenyl and FBI? [2-phenyl-5-(4-biphenyl)-1,3,4oxadiazole],and typical secondary solutes are POPOP and bis-RISB[p-bis-(omethylstyryl)henzene]. 282
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Journal of Chemical Edumtion
Student Experiments
Two simple experiments which can be completed in a single 3-hr laboratory period are suggested: (1) An experiment in which the radioactivity content of tritium in an unknown sample is determined and (2) an experiment in which the sensitized fluorescence quenching is studied by the addition of varying amounts of an organic quenchcr. A stock solution for preparing unknown samples can be made by dissolving small amounts of tritiumlabeled benzoic acid in the toluene-PPO-POPOP scintillator solution. A small package containing about 5 millicurie of the ring-labeled benzoic acid-t with a specific activity of about 50 mCi/mill is available commercially a t extremely moderat,e cost, and enough stock solution to produce several hundred unknowns can be made with it. Since benzoic acid is a quencher, it is advised that the stock solution be made without additional benzoic acid as carvier, and it is convenient to have the specific activit,y of the stock solution a t a level of (1-5) X 106 dpm/ml. Students may need to dilute this solution by a factor of 5 for their experiments. First, students are expect,ed to calibrate the counting efficiency versus channel ratio for tritium counting as shown in Figure 10 by using a set of quenched standard references. They then make three independent activity determinations of their urilcriown solut,ion after diluting it in three different proportions. I n order to avoid significant coincidence-loss a t high count rates, the count rates should remain below the 2 X lo5 34 Y cpm level. The activity '0 8 - 0 of the sample should be ml of Cyslehaxoncm, Added/l5ml reported as a specific actiFigure 11. A lineor Stern-Volmer relation.hip for the reciprocal vity of tritium in units of count rote venur the concen- dpm/ml. tration of the cyclohexonone Secondly, students can quencher (as expressed in ml of .dded quemher pet 1 5 4 of choose an optimum count the toluene-PPO-POPOP solution rate a t which an accurcontaining o constant amount of ate and convenient quentritium activity) derived from a . student on the quench. c h ~ n g experiment can ing tinetic.. be done. The instructor can choose either.a solution or solid quencher for the quenching experiment. Many liquid carbonyl compounds have roughly equal quenching efficiencies and are convenient to use. Figure 11 provides an example of such an experiment carried out a t room temperature. Students may add 50-~1portions of cyclohexanone to a 15-ml toluene-PPO-POPOP-sample after each count rate measurement. The Stern-Volmer plot of 1/R versus molarity of the cyclohexanone should yield a straight line as in Figure 11. With the knowledge of the rate coefficients discussed earlier and the observed quenching constant, students can calculate the rate coefficient of the S-S energy transfer from the toluene excimer to cyclohexanone. The efficiency of the quenchers can be compared to the efficiency of PPO. Of course, one should remember that there is a constant amount of quenching by 0%in the sample. One further exercise is to check the activity measurements in each quenched sample by measuring the counting efficiency
?$pI
via a channel ratio measurement: Since the activity thus calculated remains constant, st,udent,s should be able to demonstrat,e to their own satisfaction t,hat the above described method really worlcs. I t might also he worthwhile to show the drastic effect,s of color quenching by adding 5.~1 of nitrobenzene to an unquenched sample (15 ml). The exact dctails of the experiment can be worked out by an iustructor depending upon iudividual needs and taste. The experiment can also be carried out with other radioisotopes. We believe the above suggested experimends are useful in teaching the technique of liquid scintillation counting and also in teaching something of the kinetics of energy transfer processes in solution. The singlet-singlet energy transfer processes also occur in the gas phase hetweeo aromatic compounds and ketones (16-18) and thus they are quite prevalent in photochemical systems. We believe that the experiments described provide a starting point for many interesting experiments on electronic energy transfer phenomena which may be readily carried out using a liquid scintillation ~ o u n t e r . ~ Acknowledgment
For the purpose of illustration, Figure 11 was taken from the report prepared by Mr. William Dimpfl for his laboratory course a t Irvine. The many valuable suggestions made by Dr. George Miller for the manuscript are much appreciated. Partial support from the Petroleum Research Fund of the American Chemical Society is gratefully acknowledged. Several commercial liquid scintillation counting set,ups for single-sample counting at room temperature sre available a t relat,ively low cost (
