Liquid Scintillation Technique for Measuring Carbon-14-Dioxide Activity JOHN M. PASSMANN, NORMAN S. RADIN, and JOHN A. D. COOPER Veterans Administration Research Hospital and Northwestern Un’iversity Medical School, Chicago, 111. A method is described for the determination of carbon14 in large amounts of carbon dioxide using a liquid scintillation counter. The carbon dioxide is dissolved in a toluene-methanol counting medium by diffusing it into a solution of a high molecular weight quaternary ammonium hydroxide. The efficiency of counting is independent of the amount of carbon dioxide, but does depend on the aniouiit of amine. The efficiency ranges from 33Yo to greater than 679’0.
L
IQUID scintillation counting is useful with p-emitting iso-
topes because of its high efficiency and relative freedom from loss of activity by self-absorption. Very large samples can be analyzed compared to those which can be used with Geiger counters, thus facilitating the practice of isolation with nonradioactive carriers and permitting the use of low levels of activity when a large sample is available. The latter is particularly useful for work with humans. Unfortunately, the composition of the solvent which may be uscd practically in scintillation systems is restricted to a small number of organic compounds. Toluene appears to be the preferred solvent, but there are many substances i t cannot dissolve. The solubility problem is further aggravated by the need to operate the instrument below room temperature. I n work with carbon-14, it is frequently necessary to measure the activity in carbon dioxide, a substance of distinctly limited solubility in toluene. The use of high pressure and very low temperatures (9) is not practical for routine work. It has been found that carbon dioxide can be dissolved in toluene by combining it with a high molecular weight quaternary ammoniuni hydroxide. Samples as large as 5 mmoles. can be counted with good efficiency using a simple technique. Other toluene-insoluble acids caii be dissolved similarly. APPARATUS
A commercially available liquid scintillation counter incorpo-
rating a coincidence circuit and a 2-channel pulse height analyzer is used (Tri-Carb Counter, Model 314, Packard Instrument Co., LaGrange, Ill.). The samples and photomultiplier tubes are contained in a freezer maintained a t about 5” C. Samples are counted in 21/z-ounce cylindrical jars (HazelAtlas, KO. 3296 from Crown Glass Corp., Chicago) fitted with black metal screw caps having “pulp and vinylite” liners. A roughly cut disk of tin foil, 0.001 inch thick and a little larger than the cap, is placed over the mouth of the jar before screwing on the cap. This prevents extraction from the cap of material which affects counting efficiency. Samples are readily labeled by writing or scratching on the cap. All-glass veighing bottles, used in early work, resulted in great breakage losses, difficulty in opening the bottles, and evaporation on storage. The use of silicone oil as a light pipe between the photomultiplier tubes and sample jar was abandoned when it was found that the oil was rapidly contaminated with light-absorbing materials, which resulted in lower and variable counting efficiencies. With carbon-14 and stronger p-emitters there is little loss in efficiency when the oil is omitted. The data reported here were obtained without oil. A simple diffusion flask is used t o transfer the carbon dioxide of each sample into the organic base. The flask is made (Delmar Scientific Laboratories, Chicago) from two 50-ml. Erlenmeyer flasks joined by a short tube of 16-mm. outside diameter a t a point near the necks. To minimize the danger of spray carryover, the tube is made in the shape of an inverted S T . The seals are made EO that the two flasks sit firmly on any flat surface. One of the flasks has a side arm a t right angles to the connecting tube, also near the neck. Three glass hooks, together with the side
ann, permit the use of lubber bands to hold down the two rubber stoppers. For small scale work, a typical Warburg flask with enlarged center well can be used. T o speed the diffusion a shaker is used, such as a platform rotator (No. 16L14824, A. S. Aloe Co., St. Louis, Jfo.). MATERIALS
Silver oxide is prepared from equal volumes of 1N silver nitrate and I N sodium hydroxide. The precipitate is washed twelve times by decantation with carbon dioxide-free water and then four times with carbon dioxide-free absolute methanol (redistilled ACS reagent grade), then dried in vacuo over calciuni chloride. Mallinclrrodt silver oxide may also be used. The quaternary ammonium hydroxide is prepared froin p-(diisobutylcresoxyethoxyethyl) dimethylbenzylammoniuni chloride (Hyamine crystals 10-X, Rohm & Haas, Philadelphia), which is first recrystallized from 4 volumes of toluene (J. T. Balier, ACS reagent grade). The chloride is converted to the free base by dissolving 252 grams (500 inmoles.) of the chloride in 750 nil. of absolute methanol, and adding 9 ml. of water and 63.3 grams of silver oxide. The suspension is swirled vigorously with the platform rotator for 30 minutes, then centrifuged, and the supernatant is transferred to a bottle. Storage of the colorless solution usually results in yellowing and formation of a small amount of insoluble material, evidently derived from soluble silver complexes. Therefore, it is necessary to age the solution. This process is accelerated by exposing the bottle to a bright light for about 3 days. The supernatant is drawn off when further exposure produces no more deposition. This is most readily detected by tipping the bottle a little after each observation of the inner mall. For carbon dioxide samples of about 1 niniole. or less, the mcthanolic solution can be used. However, methanol decreases the pulse sizes and for greater efficiency of counting a t a given background rate, it is necessary to reduce the methanol content of the scintillation system. Evaporating the amine to dryness and dissolving in toluene produce a dark solution. A satisfactory solntion (about 0 . 5 M ) can be obtained by titrating an aliquot with aqueous hydrochloric acid (using phenolphthalein), then evaporating off enough methanol to give approxiinately 1M base and adding an equal volume of toluene. The evaporation is done under vacuum in a mater bath a t room temperature using a “swirler” (6). At this point the solution may turn yellow within 1 day and will then s l o d y decolorize, depositing a gray precipitate. This is presumably due t o an additional silver complex, but the color appears to have no effect on the counting efficiency. The hydroxide solution is stored in a bottle fitted with a Teflonglass stopcock and a soda-lime protection tube. The bottle is made by sealing an Ultramax double-port valve (Fischer and Porter Co., Hatboro, Pa.) a t right angles to the outlet of an aspirator bottle (Corning Glass Works, Catalog S o . 1220). PROCEDURE
The required amount of hydroxide (at least 1.1 moles pel mole of carbon dioxide) is placed in the flask without the side arm. ‘The carbon dioxide, as carbonate in sodium hydroxide, an Imzymic digest, or a slurry of barium carbonate, is then placed in ,he other flask. Excess sulfuric acid is next placed in the side :trm with a curved pipet, the vessel is closed, the acid is tipped in, :md the flask is shaken for 90 minutes. After diffusion is completed, the amine-carbon dioxide solution is transferred to the counting jar by pipet, using two rinses of toluene t o effect quantitative transfer. Toluene is added to bring the solution t o 30 ml., then 5 ml. of a 2.1% solution of 2,5-diphenyloxazole in toluene ( 4 )is added. The scintillator is obtained from Tracerlab, Boston. The jar is then capped and cooled in the counting freezer a t least 45 minutes before counting. This delay is not imperative, but samples give a slightly lower count r i t e while warm. Because the samples are not phosphorescent il is not necessary to transfer the jars t o the counting position in t l e dark. A blank for background measurement is prepared by using the same amount of hydroxide and a solution of nonradioactive 4 84
485
V O L U M E 28, NO. 4, A P R I L 1 9 5 6 T a b l e I. Time Required for C o n i p l e k Transfer of Carhon Dioxide i n Diffusion Flasksa Time, Minutes
Observed Activity, C.P.hl .
30
Av.
14,363 14,003 14,633
45
15,648
GO Ax-.
16,088 16,343 16,216
75
16,418
90
16,608 16,448
Av. 16,528 a Each flask contained 1 ml. of Hyarnine solution and 1 ml. of sodium carbonate solution; activities have been corrected for background.
carbonate, with enough carbonate to neutralize most of the amine. RESULTS 4ND DISCUSSION
Stability Experiments. Hydroxide solutions tested by titration showed a decrease in baaicity of about 6% in 1 month, but the rounting efficiency was unchanged. Radioactive and background samples gave the same activity readings over a period of 2 months or mole, although fluctuations of a fern per cent occurred, due to instrumental variation. Diffusion Time. A series of similar diffusion flasks was shaken for varying times and the resultant amine solutions mere counted (Table I). The observed activity levels off a t about 75 minutes; hon ever, 90 minutes is the recommended time. The samples shoiild bc n-ithdran n from the diffusion flask fairly soon after this time, as prolonged diffusion might cause sufficient diffusion of methanol and r a t e r to affect the counting efficiency of the Hyamine solution.
9
necessary for an accurate count. Moreover, reagents need not be carbon dioxide-free if sufficient excess of hydroxide is used. Figure 1 s h o m that there is no need for a dead-time correction even a t counting rates of 90,000 counts per minute. Efficiency and Sensitivity of Counting. The efficiency of the counting technique decreases with increasing amounts of quaternary amine, as shown in Table 11. A solution of known carbon-14 content, from the National Bureau of Standards, was used to convert activities to efficiencies. The tn.0 columns of efficiency data mere obtained for pulse heights of LO to 50 volts and 10 volts to infinity. At the lower levels of amine, the data on the 10 volt to infinity range are usually used; a t the upper levels, 10 to 50 volts. The data in Table I1 also show that the counter efficiency increases as wider ranges of pulse heights are accepted. This effect arises from the distribution of pulse heights resulting from the spectrum of beta energies. Because the background also increases with increased pulse height range, the maximum sensitivity is obtained by a conipromise. In the system used here, the activity of a 0.9-mmole. sample of carbon dioxide is equal to background when the specific activity of the carbon is 1.3 X 10-6 pc. per milligram (background is 157 c.p.m. a t 10 volts to infinity). Similarly, with 4.65 mmoles. of hydroxide and 4.2 mmoles. of carbon dioxide, the activity equals background Then the specific activity is 2.5 X 10-6 pc, per milligram (background is 105 a t 10 to 50 volts). Nine mmoles of carbon dioxide can be counted also, but only a small increase in sensitivity is obtained. For other instruments or even different models of the same instrument, the efficiencies and optimum range of pulse heights may vary from those presented heie.
Table 11. C o u n t i n g Efficiency LIS F u n c t i o n of Amount of Q u a t e r n a r y Aniin'e Efficiencya
/4
c
J-I
a
b
1
MMOLES COz
Figure 1. Relation between observed activity and amount of carbon-14-dioxide Proportionality of Counts to Sample Size. The observed activity is independent of the amount of carbon dioxide in the sample. This is illustrated in Figure 1, which was derived by analyzing varying aliquots of a stock solution of radioactive sodium carbonate, using 2 mmoles. of hydroxide. This situation, which corresponds in Geiger counting to absence of a self-absorption correction and limit, means that exact control or knowledge of the nctnal amount of carbon dioxide present in the system is not
hIillimoles of Base
10 t o 50b,
0.468 0.936 1.40 1.87 2.81 3.74 4.68
40 ~. 41 44 41.5 38 36.5 33
%
10 t o
rn
b,
%
67
60 .~
68 53 47 42.5 38
100 X observed activity/absolute activity. Range of pulse heights counted, in volts.
Addition of 2.3 mg. of 2-( l-naphthyl)-5-phenyloxazole, a wave length shifter sometimes used in liquid scintillation counting ( I ) , gave no increase in efficiency. Counting Volume. A test with varying volumes of a solution containing a trace of carbon-14-sodium acetate in nzter-ethanoltoluene-diphenylosazole showed that the counting volume of 35 ml. is not critical. The same specific activity was given by 30 and 35 ml. of radioactive solution; with 40 ml., the specific activity was 1.7% lonTer. It is true, however, that the carbon dioxide counting system might show a little greater sensitivity to errors in measuring the toluene or amine solutions because of the resultant changes in counting efficiency (Table 11). Precision. The standard deviation found for a set of nine similar samples of high activity processed through an entire operation was 0.34y0 of the mean value. This reproducibility compares favorably with other routine methods of measuring radioactivity. Effect of Methanol. A solution of Hyamine base in methanol was prepared in the usual way; part of it was evaporated to give a 1M solution, then diluted with an equal volume of toluene. The two solutions were compared for counting efficiency a t three levels of base: 1, 5, and 10 mmoles. The ratios of activities in methanol compared to methanol-toluene were 0.905, 0.795, and
486
ANALYTICAL CHEMISTRY
0.352, respectively. Evidently there is considerable loss in counting efficiency a t high carbon dioxide levels when part of the methanolis not removed. Counting Substances Other Than Carbon Dioxide. Preliminary studies have shown that the hydroxide solution will dit+ solve large amounts of amino acids, mucic acid, hydrogen sulfide, and sulfur dioxide in toluene. This suggests the possibility of measuring radioactive sulfur by converting i t to hydrogen sulfide or sulfur dioxide. The double flask has also been used for counting carbon-14 compounds by combusting the sample with silver nitrate and potassium persulfate ( 5 ) in one flask and collecting carbon dioxide directly in quaternary amine in the other flask. Details of this procedure are t o be published. Comparison with Other Counting Techniques. Carbon dioxide is ordinarily counted in the form of barium carbonate or carbon dioxide gas (2, 7 , 8). The use of barium carbonate restricts sample sizes to a maximum of ahout 0.G mmole. of carbon because of self-absorption. A t the higher levels the efficiency is very lorn. The techniques for counting gaseous carbon dioxide result in good efficiency, even with somewhat larger samples. The liquid scintillation method described here is similar to these techniques in sensitivity and permissible sample size. I t appears to be
superior to these procedures in that (1) the sample is readily stored after counting; (2) the carbon dioxide need not be purified before counting; (3) there is no need for liquid nitrogen and high-vacuum manifolds; and (4) intercomparison with other carbon-14 compounds can readily be made vithout burning the samples. A method of scintillation counting with a sodium carbonate slurry that has been described briefly (3)may prove useful for somewhat larger amounts of carbon dioxide. LITERATURE CITED
(1) Arnold, J. R., Science 119, 155 (1954). Proc. QOC. Exptl. Biol. (2) Baker, E. M., Tolbert, 13. Il.,Rlarcus, If., ilfsd. 88, 383 (1955). (3) Bliih, O., Terentiuk, F., Nztcleonics 10, No. 9, 48 (1952). (4) Hayes, F. N., Hiebert, R. D., Schuch, R. L., Science 116. 140 (1952). (5) Kats, J . , Abraham, S., Baker, N., ANAL.CHEM.,26 1503 (1954). (6) Radin, N. S., Ibid., 28, 542 (1956). (7) Sinex, F. hl., Plasin, J., Clareus, D., Bernstein, W., Van Slyke, D. D., J . B i d . Chern. 213, 673 (1955). (8) Van Slyke, D. D., Steele, R., Plasin, J., Ibid., 192, 769 (1951). (9) Williams, D. L., U. S. Atomic Energy Commission LA-1484, 3-14 (1952).
RECEIVED for review October 21, 1955. Accepted January 6, 1956.
Particle Size Distribution and Number of Particles per Unit Mass of Some Fluorescent Powders JAN
ROSINSKI, HARVEY E. GLAESS,
and
C. ROLAND MCCULLY
Department of Chemistry and Chemical Engineering, Arntour Research Foundation, Illinois Institute o f Technology, Chicago,
Fluorescent fine particles were used as a tracer in tlie studies of washout of particulate matter by rain. The particle size distribution and number of particles per unit mass were determined by means of conventional methods, which appear to be unsatisfactory. New dilution-microscopic and dilution-photonietric methods were developed. Equations for size distributions of different tracers were calculated. T h e results of analyses were compared and discussed. Whenever collected particles must be sized and coiintccl, the dilution-microscopic method should be used to classify the tracer. If the size distribution of the sample is known, the rapid photometric method can be u s e d to obtain the total number of parlicles.
D
ISPERSIOKS of fluorescent particles present a convenient means of tracing the movement of air parcels over distances of many miles and of studying the behavior of aerosols under laboratory or field conditions. Fluorescent tracers have been used by Perkins (8) in mesometeorological research and by Brahani ( 2 ) in tracing of air particles to distances over 100 miles. I n this laboratory fluorescent tracers have been used in a study of Langmuir’s accretion theory under field conditions ( 6 ) . Knowledge of the particle size distribution and of the number of particles per unit mass of tracer material is necessary for these studies. In a study of dust washout in the free atmosphere, fluorescent particles (New Jersey Zinc, Inc., powders) mere dispersed by a low-flying aircraft over a line of collectors resembling rain gages. Washed-out particles were collected by filtration from the rain, and the number of collected particles was determined. The total number of particles n-as estimated by area measurement (photometer readings of fluorescence). This method was satis-
Ill.
factory, providing the particle size distribution of the tracer as dispersed and the particle size distribution of the collected material were known. To minimize error in the determination of particle size distribution, the same method should be used both on the original tracer and on the collected sample. The tracers used consist of a large number of single particles and a smaller number of aggregates and agglomerates. The number of particles per unit mass is obviously dependent upon a number of agglomerates originally present in a particulate matter. During experimentation some agglomerates are usually broken into single particles. The number of particles increases t o a maximum when agglomerates are no longer present in the powder. Every particle or aggregate possesses a definite irregular shape which is nearly impossible to define in geometric terms. This fact leaves only statistical methods available for interpretation of size distribution of finely divided materials. The so-called “diameter” is used for defining the single dimension of the particle. The particle will have a diff erent diameter a t different orientations with respect to the linear graticule of the microscope. The diameter recorded was the length of the distance between two tangents on opposite sides of the apparent outline of the particle parallel to an arbitrary fixcd direction a t its random orientation. This is the so-called Feret’s “statistical” diameter (4). This method requires a statistical number of measurements to eliminate the error in estimating the diameter from which the area may be derived. The shape factor was not introduced, because particles of needlelike shape or other odd shapes were not present in these tracers. This made it possible to choose the Feret’s diameter for use in this study. The results of the microscopic examination are compared, when possible, Tvith those of sedimentation methods. To have representative numbers of particles, the microscopic counting and sizing were performed as follows: Heavily covered filters mere evaluated by counting random fields; for less popu-
