Oxygen f'lask Method for the Assay of Tritium-, Carbon-14-, and Sulfur-35-Labeled Compounds HORACE E.
DOBBS
United Kingdom Atomic Energy Research Establishment, Wantage, Berkshire, England
b Solid samples weighed onto filter paper, and liquid samples injected into gelatin capsules, are burned in an oxygen-filled, round-bottomed, threenecked flask fitted witha rubber vaccine serum cap. After the combustion, which i s triggered by an electric discharge, a liquid scintillator i s injected into the flask. A liquid phosphor containing water i s (equilibrated with the radioactive water produced by burning tritiated materials. The gas produced by the combustion of carbon14 and sulfur-35 compounds i s absorbed in liquid sciniillator containing phenethylamine. Fhhing the liquid phosphors with nitrogen gas, prior to counting, increases the counting efficiency for all thr'se isotopes, and improves the reproducibility of the method for tritiated materials. The counting efficiency of the liquid phosphors i s similar after the combustion of most compounds, but reduced efficiencies are observed when azo compounds are burned.
T
method (9) for the assay of labeled compounds consists of burning the sample in a sealed flask containing pure oxygen and counting the rac'ioactive combustion products. -1variety of methods for collecting and counting the products have been reported ( 2 , 4-7, IO). Kelly et al. (7) froze out tritiated water, HE OXYGEN FLASK
COM0USTlON
HEAD
\ ,TUNGSTEN
LEADS
described, in which the combustion flask is sealed before ignition of the sample and not opened until preparation of the counting sample is completed, thereby preventing possible errors due to loss by escape or exchange of the active sample. The activity of the sample is measured by liquid scintillation counting. The over-all number of operations is reduced by premixing the liquid phosphor with diluent (for tritium) or absorbent (for carbon-14 and sulfur-35).
PLUG OF SOFT GELATIN C O Z Y N D
HARD GELATIN
CAPSULE-
EXPERIMENTAL
Figure 2. Combustion platform and gelatin capsule for liquid samples
dissolved it in a liquid phosphor containing ethyl alcohol, and counted the solution in a liquid scintillation counter. Gotte, Kretz, and Baddenhausen ( 4 ) reacted the carbon dioxide produced by burning labeled organic compounds with barium hydroxide, and counted the barium carbonate. Others used phenethylamine (IO)and the quaternary ammonium hydroxides Hyamine 1OX ( 7 ) , tetramethylammonium hydroxide (I),and benzyltrimethylammonium hydroxide (2) to trap the combustion products of carbon-14-labeled compounds. The solutions were subsequently dissolved in a liquid phosphor and counted in a liquid scintillation counter. Ethanolamine has been used in a similar manner for both carbon-14 and sulfur-35 compounds (6). Some have used hydrogen peroxide to trap sulfur-35 combustion products, which were subsequently counted in the form of solid barium sulfate (2, 5 ) . iz simple and rapid method is now
Apparatus. The flask and combustion heads used for the experiments are shown in Figures 1 and 2. The 500-ml., round-bottomed flask has three standard ground-glass joints (B14/23, B19/26, and B29/32). To facilitate introduction and removal of scintillator by syringe the B14/32 socket was fitted with a skirted rubber vaccine cap of the type used with Clinbritic vaccine bottles. All-glass syringes fitted with stainless steel Luer needles were used to introduce and withdraw scintillator from the flask. Two types of platform were used in the experiments. The flat open platform illustrated in Figure 1 was used to support solid samples. It was constructed from platinum mire approximately 0.4 mm. in diameter, spotwelded to a tungsten lead 1 mm. in diameter. The cradle-shaped support shown in Figure 2 was used for the gelatin capsules containing liquid samples. This was covered with fine platinum gauze to prevent the molten gelatin dropping to the bottom of the flask during the combustion. The tungsten leads were coated with a thin layer of glass over most of their length. The ground-glass joints were securely held together with elastic bands to orevent their coming apart during the transient
SERUM
FLOWMETER
TO WATER PUMP
MANOMETER
Figure 1. Combustion head and flask for burning active solid samples
RESERVOIR
Figure 3. Gas line for flushing with oxygen and partially evacuating combustion flask VOL. 35, NO, 7, JUNE 1963
a
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pressure rises inside the flask when large samples were burned. T l e flasks were connected to a simple gas line as shown in Figure 3. The vacuum reservoir provided a convenient method of adjusting the pressure inside the flask prior to each combustion. The combustible containers for liquid samples shown in Figure 2 were prepared from Parke Davis empty gelatin capsules, size 5. A capsule was opened and one drop of a hot molten gelatin mixture (prepared by dissolving 1 gram of gelatin in 6 ml. of glycerol plus 2 ml. of water) was placed in the smaller of the two components. The capsule was immediately reassembled and allonTed to cool. When the gelatin mixture sets, it seals the two capsule components permanently together and provides a rubberlike plug through which a hppodermic needle easily passes. The plug seals hermetically when the needle is withdrawn. The liquid scintillation samples were placed in disposable glass vials and counted in a coincidence liquid scintillation counter (1). The optimum volume of liquid scintillator for the counting equipment used in the experiments wai 9.0 ml. Tritium Phosphor. The tritiated water produced from the combustion was equilibrated with 10 ml. of liquid scintillator containing 6% water. The composition of the liquid scintillator, based on a formula prescribed by Kinard (8)for the assay of aqueous samples, was: 5 parts by volume of xylene; 5 parts by volume of 1.4-dioxane; 3 parts by volume of ethyl alcohol; 100 grams per liter of naphthalene; 60 grams per liter of water; 5 grams per liter of PPO (2, 5-diphenyloxaeole) ; and 0.05 gram per liter of POPOP { 1,hbis [Z(&phenyloxazoyl) ] benzene). All liquid components were redistilled, and the dioxane was purified by refluxing over sodium. Carbon-14 and Sulfur-35 Phosphor. Phenethylamine (11) was used for the absorption of the carbon-14 and sulfur35 gaseous combustion products. It was incorporated in a liquid scintillator having the following composition: 1 part by volume of phenethylamine (2phenylethylamine), 1 part by volume of methanol, 1 part by volume of toluene, 100 grams per liter of naphthalene, 4 gram per liter of PPO, and 0.05 gram per liter of POPOP. All liquids were dried and redistilled (the phenethylamine in a stream of nitrogen). The liquid scintillator solutions were stored in subdued light in two-necked flasks fitted with rubber serum caps. The flask containing the phenethylamine was also fitted with a carbon dioxide trap, as shown in Figure 4. Procedure for Tritium. The active solid sample is weighed onto a piece of filter paper (2 cm. square) which has been left in the balance case for several hours to equilibrate its moisture content with the atmosphere. The paper is then folded t o form a tight package (approximately 1 cm. square) completely encasing the sample. 784
ANALYTICAL CHEMISTRY
is sealed. The sample is then counted
SERUM CAP
w-
LIQUID SCINTILLATOR
~
Figure 4. Flask for storing and dispensing liquid scintillator for carbon-1 4 and sulfur-35 estimations Before sample i s withdrawn, flask is orientated sa that solution covers the serum cap
The package containing the active sample is placed on the flat platform, and the combustion head is inserted in the flask. A liquid sample is injected with a fine hypodermic needle into a gelatin capsule, which is placed on the cradle-shaped platform. A small strip of filter paper, placed alongside the capsule and ignited electrically, initiates the combustion of the capsule. The head is secured with elastic bands, and the flask is flushed thoroughly with oxygen. When this is complete, the gas flow is stopped. The pressure inside the flask is reduced from atmospheric pressure by 5 cm. of mercury in order to facilitate introduction of the liquid phosphor, and to reduce the possibility of leakage of active gas after the combustion. The sttopcock on the combustion head is closed and the platform is connected to ground. The combustion of the sample is initiated by applying a laboratory Tesla discharge to the ignition electrode. Ignition is usually instantaneous and the sample burns to completion in a few seconds. When the flask is cool, it is removed from the gas line and 10 ml. of liquid scintillator is injected by syringe into the flask through the serum cap. The amount injected is determined from the weights of the syringe before and after injection. The liquid is swirled around the flask for a few seconds, and then the flask is vigorously shaken. The flask is left for 15 minutes with occasional shaking. A 9-ml. aliquot of the now active scintillator is withdrawn with a syringe, and introduced into a counting vial of known weight. The counting vial is reweighed. A stream of nitrogen is bubbled through the scintillator a t 0.05 liter per minute for 3 minutes, and the counting vial
a t the optimum voltage and amplifier settings ( I ) of the scintillation counter. After the combustion of liquid samples contained in gelatin capsules and of solid samples containing nitrogen, a n hour is allowed to elapse between injecting the phosphor into the combustion flask and measuring its activity. Procedure for Carbon-14 and Sulfur-35. The procedure for carbon14 and sulfur-35 is essentially the same as for tritium, except t h a t the scintillator containing phenethylamine is injected into the flask after the combustion. For routine analysis the nitrogen purge is omitted. The samples are counted at the optimum voltage and amplifier settings for carbon-14, which are different from those used for tritium. All samples can be counted immediately after withdrawal of the aliquot of active phosphor from the combustion flask. RESULTS
The rate of equilibration of the tritiated water formed during combustion with liquid scintillator was determined by withdrawing small aliquots a t different time intervals after injection, and measuring their activity. The same method was adopted for measuring the rate of uptake of the combustion products from carbon-14 and sulfur-35 samples. Equilibration for all three isotopes was complete in 5 minutes. The rate of these reactions, however, is partially dependent upon the degree and time of shaking. In routine analyses, 15 minutes are allowed to elapse between injection of the scintillator and removal of a counting sample, to prevent errors due to incomplete equilibration. The mist, which, sometimes forms when the phosphor is introduced after the combustion of sulfur samples, slowly disperses when the flask is shaken. I n preliminary experiments with large tritiated solid samples, the results had a considerable spread if the nitrogen purge was omitted. This was probably due to variable amounts of dissolved oxygen, which is a strong quenching agent, in the liquid scintillator. The effect of dissolved oxygen on the counting efficiency of the liquid phosphor used for tritium was measured (Table I). Flushing with nitrogen removes the oxygen that dissolves in the liquid scintillator when it is in the combustion flask, and the counting efficiency reaches a constant and reproducible level. The loss of counts due to removal of tritiated water and methanol from the liquid scintillator in the gas stream was measured by passing the flushing gas through a trap cooled in an acetone-C02 bath. The condensate was dissolved in liquid scintillator and counted. The results show that losses due to flushing are negligible. Seventy counts per
60
I
-x-
a-
/ /" %$/
COUNTED AFTER N2 PURGE COUNTED BEFORE N 2 PURGE
w 40
q f
30
--
L
b 8 WEIGHT
IO
12
14
OF SAMPLE
Ib
18
2 0 22
24
(MILLIGRAMS)
26
Figure 5. Effect on count rate of passing nitrogen gas through liquid scintillator solutions used to absorb combustion products from different weights of sulfur-354abeled benzidine sulfate
minute were lost from a sample with a count rate of 18.500 counts per minute flushed n i t h nitrogen for 3 minutes a t a flow rate of 0.05 lite1 per minute. The quenching effect of dis-olved oxygen on the counting efficiency of the liquid phosphor used for carbon-14 and sulfur-35 was also measured (Table I). The effect of purging the liquid scintillator with nitrogen after the combustion of a n u r b e r of sulfur-35 compounds is illustrat,ed in Figure 5 . Similar results were obtained with carbon-14 samples. Flushing with nitrogen was omitted from the routine analysis of sulfur-35 and carbon-14 conipounds, however, as reproducible results were obtained if it was omitted. Tritiated liquid samples contained in gelatin capsules, c r tritiated solid samples containing nitrogen, gave an abnormally high count rate if the aliquot of liquid phosphor n a s counted immediately after its withdrawal from the combustion flask. Yhe count rate gradually decayed to a constant level over a period of half a n hour from injection of the phosphor into the conlbustion flask. A possible explanation for this phenomenon i 3 that there is a chemiluminescent reaction beta-een, or catalyzed by, the oxides of nitrogen formed during the combustion and a component of the liquid phosphor. A similar, though much more shortlived, effect was produced by heating the inactive liquid Fhosphor. After the combustion of tritiated nitrogenous materials, therefore, ;he aliquot was allowed to stand for 1 hour a t a constant temperature before it was counted. In this way reproducible results were obtained. Similar effects were not observed after the combustion of carbon-14 and sulfur-35 compo inds containing nitrogen, which are coiinted in a liquid phosphor of different composition. T h e quantity of liquid phosphor introduced into the flask after the combustion of large tritium samples
was increased to 15 ml. if the laboratory temperature was below 18" C. Below this temperature the small additional quantity of water produced during the combustion is sufficient t o cause 10 ml. of liquid phosphor to separate into two phases, resulting in a change in counting efficiency. The reproducibility of the method for single compounds of all three isotopes is demonstrated by the linearity of the calibration curves shown in Figures 5 and 6. These were prepared by burning different weights of the same material with all of the combustions performed under standard conditions. The counting efficiencies were measured by burning inactive 15-mg. samples, and injecting 10 ml. of the appropriate liquid scintillator into the flask, which was then left for 15 minutes with occasional shaking. Small amounts of non-quenching standards 13 ere added to 9-ml. aliquots of scintillator !+ithdrawn from the flask. Eexadecane nas used for tritium. Carbon-14labeled toluene was utilized for both carbon-14 and sulfur-35 measurements because of the similarity of thpir p emissions ( 3 ) . The results are listed in Table 11. To determine the usefulness of the method for a variety of substances, 10 mg. each of a range of compounds TT ith different chemical groupings mere burned nith 15 mg. of a radioactive standard (Table 111). Compounds that possessed strong quenching characteristics were chosen, so that any unburned material, or undesirable combustion products, would combine to accentuate diminution of the expected count rate.
Table 11.
4E
GQ
44
2
4 6 0 IO 12 14 16 IE 2 0 22 2 4 2 6 WEIGHT OF 54MPLE COMBUSTED @ I L L I G R I M $
28
30
Figure 6. Variation of count rate after combustion of different weights of tritiated and carbon-1 4-labeled compounds Scintillator containing tritium was subiected to a nitrogen purge; that containing carbon-1 A was no?
DISCUSSION
The use of liquid scintillation counting for assaying radioisotopes is continuously rising. The technique is not suitable for substances which phosphoresce strongly, are insoluble in
Table 1. Counting Efficiencies of Two Phosphors before and after Saturation with Nitrogen and Oxygen
Phosphor Tritium Cl4andSZ
Counting efficiencies, % Satd. Satd. with with Normal nitrogen oxygen 8.7
3.8 39.7
10.3 51.0
48.2
Background and Counting Efficiency Values Measured after Combustion of 15 M g . of Inactive Material
Inactive substance burned Background, counts per min. Counting efficiency, yo Before nitrogen flush After nitrogen flush
Table 111.
/
52
Tritium Stearic acid 75.7 6.0 9.6
Carbon-14 Palmitic acid
Sulfur-3.5 Benzidine sulfate
69.1
69.1
46.8 50.3
46.5 50.5
Count Rates Obtained b y Burning 10 Mg. Each of a Variety of Quenching Agents with 15 Mg. of Standard
Substance burned with standard Control (standard only) 2,4-Dinitrophenylhydrazine
Anthraquinone 2-sulfonic acid sodium salt 2,4-Diaminophenol hydrochloride Benzoic acid Succinic acid Azobenzene p-Phenyl azobenzoyl chloride
Counts per minute per milligram Tritium (stearic acid) Carbon-I4 (palmitic acid) Before After Before After nitrogen nitrogen nitrogen nitrogen flush flush flush flush 644 566
so0 708
1463 1449
1570 1555
615
747
1477
1600
510 505 575 160
732
1450
695 810
232
1320 1470 124
257
955
204
VOL. 35, NO. 7, JUNE 1963
1553
1420
1535 222 1105
785
liquid scintillator, or are quenching agents. These problems can be resolved, however, if the substance can be burned r>ntirely to coinrnon combustion products that can then be dissolved in liquid scintillators of suitable composition and counted with a reproducible counting efficiency. This enables the activity of compounds of different chernical composition to be compared directly, removing the need for a separate calibration for each substance. Use of a coincidence liquid scintillation system enables relatively low backgrounds to be obtained with very weak p emitters-Le., tritium-thereby permitting an accurate assay of relatively small amounts of the isotope. The use of 2-phenylethylamine, instead of the more widely used Hyamine lox, reduces the cost of assaying carbon-14 and sulfur-35 without adversely affecting the counting efficiency. The flask combustion method can be used for the assay of biological samples (6, 7 ) , or for the determination of the distribution of activities on chromatograms, which can be cut into strips and burned. Larger samples that do not burn readily, or burn with a sooty flame, can be mixed with cellulose
powder, prior to ignition, to assist the combustion. The linearity of the calibration curves (Figures 5 and 6) indicates that the iiiethod described is reproducible for different weights of the same substance. The results for ~1 range of different cheiiiicd compounds (Table IV) also show reasonable agreement nith the expected value, with the exception of the azobenzenes which gave low count rates. Because the azo compounds burned vigorously without a trace of ash, and the aliquots of liquid phosphor showed no discoloration, one can exclude the possibility that the low count rates were due to unburned starting materials. A plausible explanation is that the combustion of this class of compounds produces a unique product with very strong quenching properties. These results indicate it is necessary to determine the counting efficiency after the combustion of different classes of compounds, to ascertain if any pronounced quenching agents are produced. This can be achieved by spiking the aliquot after it has been counted, with a small known volume of a nonquenching standard, and recounting. The reproducibility of the method, however, elimi-
nates the need for repeated efficiency measurements when similar weight. of the same clnsi of compoiinrl m’ to hr asisyed. LITERATURE CITED
( I ) Dobbs, H. ,E., Atoriiic Energy Research Establisliiiirnt (Gt. Brit.), R e p t . M1075 (1962). ( 2 ) Eastham, J. F., Westbrook, H. L., Gonzales, D., Proc. I.A.E.A. Sym-
posium on Detection and Use of Tritium in the Physical and Biological Sciences, Vienna, 1961, Vol. 1, p. 203. (3) Glendenin, L. E., Solomon, 4 . K.,
Phys. Rev. 74, 700 (1948). (4) Gotte, H., Kretz, R., Baddenhausen, H., Angeu. Chem. 69, 651 (1957). (5) Habersbergerovfi-JeniEkovB, h.,Cffka, J., Collection Czechoslov. Chem. Commzcn. 24, 3777 (1959). (6) Kalberer F., Rutschmann, J., Helv. Chim. Acta 44, 1956 (1961). ( 7 ) Kelly, R. G., Peets, E . A , , Gordon, S.,Buyske, A , , Anal. Biochenz. 2, 267 (1961). (8) Kinard, F. E., Rev. Sei. Instr. 28, 293 (1957). (9) , , Macdonald. A . M. G.. Analust 86. 3 (1961). (10) Oliverio, V. T., Denham, C., Davidson, J. D., Anal. Biochem. 4 , 188 (1962). (11) Woeller, F. H., f b i d . , 2, 508 (1961).
RECEIVEDfor review October 15, 1962. Accepted February 21, 1963.
Statistical Aspects of Liquid Scintillation Counting by Internal Standard Technique Single Isotope R. J. HERBERG ,!illy Research laboratories, lndianapolis 6, Ind. Equations are given by which the percentage error in a calculated rate, corrected for counting efficiency, can b e determined. These equations are evaluated for a range of counting efficiencies, activities of internal standard, and counting times before and after internal standard addition. For routine counting the addition of 50,000 d.p.m. of carbon-14 and 150,000 d.p.m. of tritium as internal standard activity, and a 1-minute counting time after addition of internal standard, are adequate.
T
HE ease of sample preparation and the relatively high counting efficiencies attainable have made liquid scintillation counting a common technique. In general, samples prepared for this method of counting are quenched
786
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ANALYTICAL CHEMISTRY
to various degrees by a solvent component of the counting solution, by color produced by the sample in the counting solution, by a colorless sample itself, or by a combination of these three. For determining the counting efficiency of such quenched solutions, the internal standard method is much used. In this procedure one counts a sample, adds a known amount of activity of the same or other appropriate isotope, and recounts the sample. The increase in count rate resulting from addition of the internal standard is a measure of the counting efficiency of the solution. I n the counting procedure as outlined, seven quantities must be determined for each sample to permit calculation of its absolute disintegration rate: (1) gross count of the sample, (2) sample counting time, (3) gross count of the sample after addition of the internal standard]
(4) counting time of the sample after internal standard addition, (5) background count, (6) background count time, and (7) activity of the added internal standard. The optimum values, or even good values, of some of these quantities are not immediately obvious. The simple case of a sample and its background is discussed for counting systems in general in any book dealing with radioactive counting procedures. Whisman, Eccleston, and Armstrong (9) discuss errors relating to liquid scintillation counting. However, for the situation as a whole-Le., considering the seven quantities mentioned-no complete analysis is available. One merely has available such qualitative information as that as counting time for a sample is increased, the fractional error of a rate decreases, or that the
