added to 50-ml. aliquots of Sample KO. 3663. Beta and alpha counts obtained immediately after separation of lead from samples which were high in radioactivity but low in lead-210 content have given no indication of measurable contamination from other radionuclides. Although smaller amounts can be detected in low-background counters, about 1 ppc. is required to establish by means of ingrowth curves that the measured activity is due to lead-210.
ACKNOWLEDGMENT
The authors are indebted to D. E. Rushing for suggestions and advice and to Boyd C. Sorenson for assistance in performing the analyses. LITERATURE CITED
(1) Bambach, K., Burkey, R. K., IND.
EKG.CHEM.,ANAL.ED. 14, 904 (1942). ( 2 ) Frank, Adrian, $m. Ind. H y g . Assoc. J . 23, 424 (1962). (3) Gibson, W. XI., "The Radiochemistry of Lead," Kational Academy of Sci-
ences, National Research Council, Nuclear Science Series, KAS-KS 3040, Office of Technical Services. IT. 8. DP~ a r t m e n t of Commerce, kashington, D. c., lg6'. (4) Kirby, H. W., AKAL.CHEM.26, 1063 114.54) ,-"--,.
(5) McCord, W. M . , Zemp, J. W., Ibid., 27, 1171 (1955). (6) Millard, H. T., Jr., Ibid., 35, 1017 (1963) ( 7 ) West, P. W., Carlton, J. K., Anal. Chim. Acta 6, 406 (1952). RECEIVED for review February 8, 1965. Accepted March 30, 1965.
Measurement of Low Energy Beta-Emitters in Aqueous Solution by Liquid Scintillation Counting of Emulsions MICHAEL S. PATTERSON and RONALD C. GREENE Radioisotope Service, Veterans Administration Rospital and the Department of Biochemistry, Duke University School of Medicine, Durham, N. C. Triton X-100 has been found to be an acceptable agent for the formation of stable emulsions of water and toluene for liquid scintillation counting. The properties of three counting mixes a r e described. These are: toluene: Triton, 2 : 1 , which can b e mixed with 23% water and give counting efficiencies of 10% for H3 and 68% for C I 4 ; toluene: Triton : ethanol, 8 : 4 : 3, which can hold 4 3 % water with counting efficiencies as high as 58% for C I 4 ; toluene:triton, 7:6, which forms a fluid emulsion with 43% water that sets to a rigid gel on cooling in which CI4 can be counted with 5770 efficiency.
I
N R E e m T YEARS liquid scintillation
counting has become the method of choice for measurement of low energy @-emitters such as C14, P5, or H3. Unfortunately, solvents like the alkylated benzenes which give the highest yield of photons per p particle do not dissolve significant quantities of many biological materials, while conversely, some of these biological compounds and water are pot'ent quenching agents. Thus an increase in sample size generally results in a decrease in efficiency, and specimens with low concentrations of radioactivit,y are difficult' to measure with accuracy. Many techniques have been developed, some simple and some complex, some general and some specific, to overcome the problems of counting polar, water soluble compounds. The specific procedures, which have been developed by many workers, are too numerous to be individually cited here but they are well covered in the recently published book by Schram ( 3 ) and in a comprehensive review by Rapkin ( 2 ) . 854
ANALYTICAL CHEMISTRY
I n spite of the wide variety of techniques which have been developed, the counting properties of emulsions containing high percentages of water have not been evaluated; although on theoretical grounds their properties would be expected to be highly superior to most of the previously described mixtures. We have tested a number of emulsifiers for this purpose. Of those tested, which satisfactorily emulsify toluene and water, only Triton X-100 (Rohm & Haas, Inc.) has acceptable counting properties, in that it does not quench light emission from the scintillators, and it has low concentration of phosphorescent contaminants which can easily be removed by treatment with silica gel. Emulsification with toluene solutions of PPO (2,5-diphenyloxazole) and POPOP [1,4-bis-2-(5 phenyloxazoly1)-benzene] has been found to allow counting of large volumes of aqueous solutions with high efficiency. After completion of the Rork reported here, a paper by Meade and Stiglitz ( 1 ) was called to our attention. These workers used a mixture of Triton X-100 and a toluene solution of PPO and POPOP to suspend small quantities (10 to 100 mg.) of tissue for counting, but did not investigate emulsification of aqueous solutions. EXPERIMENTAL
Scintillators, radioactive standards (benzoic acid C1*, tritiated water and toluene, calibrated in d.p.m.), and counting vials were obtained from the Packard Instrument Company, La Grange, Ill. All other chemicals, except Triton X-100 were reagent grade and were used nithout further purification. Triton X-100 contained small quantities of phosphorescent materials and
had to be purified by the following procedure before it was suitable for use in scintillation counting: one-tenth part of silica gel (6-16 mesh) is added to the Triton X-100 and the mixture is vigorously mechanically stirred (Eberbach Con-Torque stirrer) for 15 to 20 minutes. The mixture is allowed to sit for 15 minutes, whereupon most of the silica gel settles out. The supernatant Triton X-100 is then decanted through a thin layer of glass wool spread across a Buchner funnel. A second similar filtration is often required to remove the last traces of silica gel. Material prepared in this manner can be stored in the laboratory without special precautions for several months. The counting and emulsification properties of a given batch of Triton X-100 remain constant on storage but considerable vbriation has been observed between different batches of Triton. Therefore when a new lot of Triton is used, it is advisable to redetermine the optimal proportions of the counting mixes. All s'amples were counted in 20-ml. screw-cap glass vials in a Model 314 EX-2 Packard TriCarb liquid scintillation spectrometer. Throughout this work, constant discriminator settings were used (red channel 80-1000, green channel 400-1000) and optimal counting rates for each mixture were obtained by adjustment of the phototube dynode voltage. The properties of the various emulsions are dependent on temperature. As the emulsions used here are cooled, each clears at a temperature (between 25" C. and 0' C.) characteristic of the particular emulsion. T o assure formation of adequate emulsions, the mixtures must be agitated after they have been cooled below their respective clearing points. Evaluation of Counting Mixtures. I n order to allow ready comparison of the counting properties of Triton X-100 emulsions with other mixes,
we have calculated two figures of merit. 'rhe first is the product of the perc4rntage water content of the mix and the per(-entage counting efficiency. This figure has been widely used by other workers, but its usefulness is limited by the fact that counting efficiency is a function of the instrument. used to count the samples as well as t,he properties of the count'ing mix. The second figure of merit, is the product of the percentage water content and the counting efficiency of the mix divided by the counting efficiency of the same isotope counted in a standard solution (toluene containing 4 grams per liter of PPO and 100 mg. per liter of POPOP), when each solution is counted under optimal conditions. This figure has a built-in calibration of t'he counting equilment which should remedy the inherent defect of the first figure. Keither of these figures includes the background because it is so dependent on the instrumentat'ionused in counting. Rather, counting rates of blank vials of the same composition, counted under the same conditions as the sample are simply reported so they may be compared with that. of a n unquenched blank, containing only the st'andard toluene solution of PPO (4 grams per liter) and POPOP (100 mg. per Mer). The background counting rate of the model of liquid scint'illation count'er used in this work is high. Alore recent designs of counting equipment have succeeded in markedly lowering the background while achieving counting efficiencies slightly higher than those obtained with our instrument. RESULTS
Effect of Triton X-100 on Counting Efficiency. Table I shows t h e effect of addition of successive increments of Triton X-100 on the counting efficiency of C14 and H3 in the absence of other quenching agents. Addition of 5 ml. of Triton to 10 ml. of the t,oluene solution of phosphor caused no significant decrease in C1* counting efficiency and about a 2501, reduction in the counting efficiency of H3. Counting Mixes. Of t h e many workable solutions, we have selected three mixtures whose compositions and characteristics are given below. Each has particularly advantageous properties under some sets of counting conditions, b u t each mixture has limitations and none is preferred for all counting conditions. Data is given for t h e counting properties of emulsions containing specific quantities of aqueous phase (generally the maximum amount, possible). I t is possible to reduce the proportion of aqueous phase and in some cases att,ain a higher counting efficiency, especially with tritium. Or, the proportion of aqueous phase may be kept the same, and the total volume of water and counting mix could be reduced to achieve a lowering of background by diminishing the radiosensitive volume.
Table 1.
Effect of Triton X-100 on Counting Efficiency
Volume of
Triton added, ml.
Dynode voltage
0.0 1.0 2.0 3.0 4.0 5.0
1060 1080 1080 1080 1080 1080
Isotopic compounda Toluene-H3 Toluene-H3 Toluene-H3 Toluene-H3 Toluene-H Toluene-H3
Added activity, d.p.m.
Observed activity, c.p.m.
Efficiency,
2 . 4 4 X lo6 2 . 4 4 x 105 2 . 4 4 x 105 2 . 4 4 X 1W 2 . 4 4 x 105 2 . 4 4 X lo6
6 . 1 5 X lo4 5 . 7 1 X lo4 5 . 2 2 x 104 4.91 x 104 4 . 6 0 x 104 4 . 3 7 x 104
25 23 21 20 19 18
%
1021 Benzoic acid-C14 1512 840 Benzoic acid-C14 1512 1014 840 Benzoic acid-C14 1512 1030 840 Benzoic acid-C14 1512 1042 840 Benzoic acid-C14 1512 1015 840 1512 997 Benzoic acid-C14 840 a Aliquots (0.1 ml.) of standard toluene-H3 or benzoic acid-C" in toluene were to 10 ml. of PPO (0.4y0), POPOP (0.0170) in toluene. 0 0 1 0 2 0 3 0 4.0 5.0
Table II.
Aqueous phase 0 . 1 HCI ~ 1 , O N HC1 0.1,V KaOH 1 .ON XaOH 0 . 1 M pH 6 . 8
K phosphate
0 2MpH6 8
K phosphate
0 . 4 M DH 6 . 8
K phosphate
68 67 68 69 67 66
added
Counting Efficiency of tT 21 Emulsions"
Added activity, d.p.m. i , 4 x 106-~3 1 . 4 x 105-~3 1 . 4 X 105-H3 1 . 4 x 105-~3 1.4 x 106-~3 1 . 4 X 105-H3 1.41 X 105-H3 1 . 4 x 105-~3
Efficiency,
Observed activity, c.p.m.
%
Figure
of merit #2b 230 207 161 207 184
Figure
of merit
#ac
1.34 x 1.24 x 0.95 x 1.25 x 1.08 X
104 104 10' 10' 10'
10 9 7 9 8
x 1.39 x
104
10
230
7.9
104
10
230
7.9
0 . 9 6 X 10' 52
7
161
5.5
...
...
...
1.36
7.9 7.1 5.5 7.1 6.3
H20
None
HzO 0 . 1 N HCl 1 .ON HC1 0 . 1 N NaOH 1 . ON NaOH 0 1M DH 6 . 8
2035-C" 2035-C" 2035-C" 2035-C" 2035-C l 4
1377 1207 1252 1317 1194
68 59 62 65 59
1564 1357 1426 1495 1357
22.4 19.7 20.7 21.6 19.7
K phosphate
2 0 3 5 4l 4
1249
61
1403
20.3
K phosphate
2035-C"
1263
62
1426
20.7
0 . 2 M pH 6 . 8 0 . 4 M pH 6 . 8
1055 52 1196 17.3 2035-C" K phosphate 66 None . . . KO a hIixture contains 5 ml. of aqueous phase and 17 ml. of t T 21 (toluene:Triton, 2: 1 ) . Labelled compounds are tritiated water or C14 benzoic acid. Dynode voltage is 1155 for H3 or 940 for C14. b Figure of merit #1 = 7"aqueous phase X Yo counting efficiency. c Figure of merit #2 = Figure of merit #l/yocounting efficiency of standard (20 ml. 0.47, PPO and 0.017, POPOP in toluene). Counting data of standard solutions: Toluene-H3, dynode voltage 1060, efficiency 297,, background 68 c.p.m. ; benzoic acid-Cl4 dynode voltage 840, effiriency 69%, background 42 c.p.rn.
tT 21. The most generally useful solution for counting C14 or H3 in the presence of reasonably high concentrations of aqueous phase (UII to 23%) is a mixture of two volumes of toluene (containing 0.40/, PPO and O . O l ~ o POPOP) and one volume of Triton X-100. The counting efficiencies of these isotopes in the presence of a wide variety of different aqueous phases are given in Table 11. The counting properties of the emulsions are little
affected by alteration of the composition of the aqueous phase, which ranges from strong acid to strong alkali and relatively concentrated ~)hosphate buffer. These solutions are representative of most of the types encountered in biochemical work. The efficiency of tritium counting varies with different batches of Triton X-100, but remains quite constant in different vials prepared with the same batch. Thus, as long as the batch of Triton i i not VOL. 37, NO. 7 , JUNE 1965
855
changed within an experiment, counting rates of vials having similar aqueous phases are directly comparable. tT 7 6 . The counting solut,ion, obtained by mixing seven volumes of phosphor in toluene with six volumes of r, , 1riton X-100, forms emulsions with high water contents (up to 4 3 7 9 , which have adequate counting efficiencies and desirable physical properties, Table 111. F h e n a vial containing 13 ml. of mix and 10 ml. of aqueous phase is vigorously mixed a t or slightly above room temperature a translucent emulsion is obtained. On cooling to temperatures below 10' C., a rigid transparent gel is formed, which is stable at' low temperatures (to -5" C. or below), but which can be reliquefied by warming. This gel is not only useful because of its high wat,er content, but' also because of its ability to hold solid materials in suspension in somewhat the same manner as Cab-OSi1 (colloidal silica manufactured by Godfrey L. Cabot,, Inc.). This gel is preferred to that obtained with Cab0-Si1 because the Triton X-100 facilitates dispersion of the liquid and solid phases, and because the increased
fluidity at' higher temperatures allows easier mixing. Relatively high water contents (-35y0) are required for rigid so care must. be taken to proportion in preparing the mixes. The counting efficiencies, obtained with tritium and water, and with C i 4 in the presence of different aqueous phases, is given in Table 11. The Ci4 efficiencies are high enough to make this mix useful when counting of large samples is desired. The mix is not recommended for t'ritium counting unless counting in a rigid medium is necessary. One particularly useful application of this mixture is the counting of whole tissue homogenates. I n one experiment, S35 was count,ed with approximately 35% efficiency in a vial containing 13 ml. of t T 76 and 8 ml. of brain homogenate (50 mg. tissue per much larger amount' of tissue ml.). ( 5 x as much) can be suspended in the emulsion, but the resultant gel is less clear and more highly colored, and thus would be expected t,o have a lower counting efficiency. tTe 843. The counting propert'ies of emulsions of a mixture of eight parts of A\
Counting Efficiency of tT 76 Emulsions"
Table 111.
Added activity, d.p.m. 1.41 X 106-H3
Observed activity,
Efficiency,
Figure,
of merit
Figure
of merit #2b
Aqueous phase c.p.m. 7c #1b H2O 6939 5 215 H?O 2035-C" 1119 55 2365 0.1-V HC1 2035-C l 4 1065 52 2236 1 .OSHC1 2035-C '* 876 43 1849 0 . 1 s XaOH 2035-C l 4 1112 55 2365 1 ,O S S a O H 2035-C l 4 1127 55 2365 0.lM H 6 8 K plosphate 2035-C1* 1157 57 2451 0.251 pH 6 . 8 K phosphate 2035-C14 1164 57 2451 0.4J1pH 6 . 8 K phosphate 2035-C14 1134 56 2408 H,O Sone 62 , . . ... a Coiinting mixtiire contains 10 ml. of aqueous phase and 13 ml. of tT 76 Triton 7 :6). Labelled compounds used are tritiated water or C14 benzoic acid. voltage is 1155 for €I3 or 1020 for CL4. b See Table I1 for definition of Figures of merit.
7.4 34.3
32.4 27 0 34.3 34.3 35.6 35.6 34.9 ... (toluene: Dynode
~
Table IV.
Aqueous phase H2O
Counting Efficiency of tTe 843 Emulsions"
Added Observed activity, activity, d.p.m. c.p.m. 5837 I 41 x 1 0 6 - ~ 3 1045 2035-C14 996 2O35-Cl4 687 2035-C14 1059 2035-C14 1096 2035-C l 4
Figure
Figure
Efficiency,
of merit
of merit
%
$2b
54
$Ib 172 2193 2107 1462 2236 2322
4 51 49 34 52
5 9 31 8 30 5 21 1 32 4 33 6
K phosphate
2035-CI4
1020
50
2150
31 1
K Dhomhate
2035-cl4
1102
54
2322
33 7
0 2M pH 6 8 0 4'11 pH'6 8
2494 36 2 iia8 58 K phojphate 2C135-C'~ H?O Sone 47 a Coiinting mixtiire contains 10 ml. of aqueous phase and 13 ml. of tTe 843 (toluene: Triton:ethanol: R:4:3 ) . Labelled compoiinds are tritiated water or C14 benzoic acid. Dynode voltage is 1155 for H3 or 1020 for C14. h See Table I1 for definition of Figures of merit.
856
ANALYTICAL CHEMISTRY
phosphor solution in toluene, four parts of Triton X-100, and three parts of absolute ethanol with various aqueous phases is given in Table IV. These emulsions are useful for counting p particles as energetic as those emitted by C14 when it, is desirable to use a large proportion of aqueous phase and to have a fluid emulsion, but, they are not recommended for tritium counting. The counting efficiencies of t>heseemulsions are somewhat more sensitive to the nature of t'he aqueous phase than those using the other mixtures. The solutions with high sodium hydroxide or high phosphat'e concentration form clearer emulsions which are stable a t the low temperatures (-5' C.) used in this work. Stability of the emulsions containing water or solutions with low salt concentration a t low temperatures varies with the particular batch of Triton X-100. The problem of emulsion instability can be avoided by diminishing the concentration of aqueous phase to 7 to 8 ml. per 13 ml. of tTe 843, rather than the 10 ml. used in these experiments. DISCUSSION
One major factor which limits the sensitivity of liquid scintillat'ion counting is the quenching of light emission by materials in the sample (3). When potent quenching agents, such as water and most' polar materials, are counted, addit,ion of successive increments of sample causes progressively great'er quenching. In most systems presently used for counting aqueous solutions, a point of diminishing returns is met a t rather low sample concent'rations when t'he increase in quenching on furt'her sample addit'ion equals or exceeds the addit'ional radioactivity. In homogeneous counting media, the dominant cause for this reduction of light emission is a group of phenomena which have been called chemical quenching. This category includes all effects which cause nonradiative deactivation of excited molecules of solvent or scintillator that might have ot'herwise dissipated their energy by photon production. By use of heterogeneous systems, where the water and polar matmerials are physically separated from the scintillat,ion phase, quenching by these mechanisms is greatly reduced and, under theoretically ideal conditions, could be eliminated. Two quenching phenomena which affect both homogeneous and heterogeneous count,ing systems are color quenching and dilution quenching. Color quenching is the absorption of emitted photons by colored materials in the sample. With the higher concentrations of sample, which can be handled by emulsion counting techniques, this effect becomes more important and in some sit,uations mav limit
the sample size. Dilution quenching is the reduction of light emission due to dilution of the scintillation phase with saniple. Sinc,e most of the materials which would partition in the aqueous ]]has(>of these eniulsions have very poor energy transfer properties, it is expected that very few or none of the sample molcculw, which are excited by primary interaction with p particles, will transfer their escitation energy to phosphor moleculcs. Thus, light emission will be reduced in proportion to the decrease in 1)otentially productive primary excitations. The limitations imposed by thcw quenching phenomena of course become greater as isotopes with weaker p's are count,ed,but in the case where the avei'age path lrngth of the p particle is not large compared to the droplet size of the dispersed sanii)lr, self absorption becomes a further limiting factor. Under these conditions, a dispro. large fraction of the energy lost in the droplet in which they originate. This effect can be niinimized by adequate dispersion of samlile and scintillation phases, which is morr easily achieved by preparation of chemical1~-stabilized emulsions of liquid phases, than by other heterogeneous counting techniques. Even under ideal conditionb of sample dispersion and chemical quenching elimination, these phenomena will ultimately limit the proportion of aqueous phase that can be counted with reasonable efficirncy. As espected, our results show that the counting efficiency of H 3 is reduced to a
much greater extent by dilution than i!: that of CI4, since a much greater proportion of the H3 D's produces barely detectable numbers of photons in undiluted scintillation phase. But, in spite of this reductmion.tritium can be counted with reasonable efficiency (10i27c) in mixes containing a much higher percentage of aqueous phase than is possible with any previously described syPtem. Carbon-14 and other isotopes which emit more energetic p particles, can be counted with much higher efficiency in mises which contain even greater percentages of aqueous phase. Though no observations have been made, it is assumed that the emulsions described in this paper are of the water in oil type, since the organic phase is always present in greater quantity than t,he aqueous phase and since the viscosity of each emulsion increases n-it'h the amount of water. Recause of such increases in viscosity, it is not practical t o increase the water content any higher than described here, while using toluene, Triton X-100 water emulsions. Only a small number of emulsifiers were tested before Triton X-100 was found to have desirable counting properties and the search was stopped. h systematic evaluation of the many commercially available emulsifie hould yield several systems which are scintillation counting, including oil water emulsions. Emulsions of this type would allow the susp relatively small amounts of sc phase in aqueous solution, thus further
increasing the quantity of water which can be counted in a standard vial. As discussed above, the results which we have obtained suggest that, with presently used scintillators and counting equipment, it is not pract,ical to increase the aqueous phase for tritium counting, but some increase in water content is apparently feasible for counting kot.opes with more energetic betas. Since light emission by the scintillators, and photon detection by the photomultipliers, are both relatively inefficient (-10% of theoretical), an increase in the ratio of aqueous phase to scintillation phase should be possible if the efficiency of either or both of these processes \wre significantly improved. Thus, under theoretically ideal conditions, emulsion counting should be preferable to most other methods of counting aqueous solutions because it allows the incor1)oration of a high percentage of aqueous phase with reasonably high counting efficiency, and, except for colored materials, the efficien relatively unaffected by quenche the aqueous phase. LITERATURE CITED
(, 1,) IIeade. R. C.. Ht,iditz R.. Intern. J . A p p l . Rddintion'lsot&es' 13,' 11 (1962). (2) Rapkin, E., l b i d . , 1 5 , 69 (1964). ( 3 ) Schram, E.) "?rganic* Scintillation
Detectors," Elsevier, Amsterdam-London-Sew Tork, 1063. RECEIVED for review October 9, 1964. Accepted April 16, 1965. This work war slipported in part by Grant Yo. (>SI 10317 from the Tationd Institutes of Health .
Gas Analysis by Geiger Pulse Attenuation FREDERICK W . WILLIAMS and ARTHUR F. FlNDElS School o f Chemistry, University o f Alabama, University, Ala.
b Nucleonics counting gases possess electronic characteristics which are markedly altered by the admixture of additional components. Alteration of the composition of the common counting gas, 1.3% butane-98.7% helium, results in a change in gas amplification. The presence of all gases, except argon, investigated in this work resulted in a decrease in the pulse amplitude when the counter is operated in the Geiger region of gas amplification. The decrease in pulse amplitude is a linear function of the amount of gas injected in the stream for many gases over wide ranges of sample injections. In most cases the pulse amplitude variations are additive. Linear calibration curves were obtained for many binary mixtures. In those cases where one of the components had a high electron attach-
ment probability, nonlinear but reproducible data were obtained. The gases investigated were ammonia, nitrogen, oxygen, argon, ethylene oxide, carbon dioxide, hydrogen, sulfur dioxide, low molecular weight hydrocarbons, and binary mixtures of the above.
T
are many ways of detecting gases, one being by ionization of a gas. .i gas may be detected if ionized by bombardment with high-speed charged particles, y- or x-rays, ultraviolet photoionization, or by direct application of a sufficiently high electric potential. Deal et al. (I) descr ibea detector which depends on the differences in the ionization cross section of different molecular species for its action. .1 10-pc. source of stronHERE
tium-90-ytt'rium-90 was used in this detector. Harley and Pretorius (2) designed a glow discharge detector which will detect mole per second of a gas. A sensit,ive ionization gauge detector developed by Ryce and 13ryce ( 8 ) makes use of the fact that the ionization potential of the helium carrier gas (24.5 volts) is very much greater than that of most other volatile substances. A sensitive detector whose method of operation is based on the uniqudonization properties of ai'yon was developed by Lovelock (4). Lovelock and Lilisky ( 5 ) developed an electron absoiytion detector. -4 stream of inert gas is passed through the chamber and the i~otentialis adjusted so as to collect all of the electrons liberated from the gas hy ionizing radiation. -\ new, simple, and highly VOL. 37, N O . 7, JUNE 1965
a
857
