water mixtures confirmed t h e applicability of the principles to liquid analysis. LITERATURE CITED
(1) Bogardus, B.J., Ritter, R. C., “ACOUS-
tic Gas Analyzer Development and Manufacture,” Union Carbide Nuclear Co., Oak Ridge Gaseous Diffusion Plant, February 4, 1959 (K-1240). ( 2 ) “Chemical Engineers Handbook,” John H. Perry, Ed., 3rd ed., pp. 387 and 403, RIcGraw-Hill, New York, 1950.
(3) 1bid.JP. 405. (4) Fribance, A. E.,“Industrial Instrumentation Fundamentals,” p. 218, fig. 9-38, McGraw-Hill, New York, 1962. ( 5 ) Lunge, G., “Technical Gas Analvsis,” pp. 191-3, Nostrand, New York, iG34. (6) ”yerson~ A* L . ~Eicher~J. H . ~ J . Am. Chem. SOC.74,2788-61 (1952). (7) PaPPW w.S.J Weber, c. pv., A N A L . CHEM.37,407-10 (1968). ( 8 ) Rodden, c. J.9 “Analytical Chemistry of the Manhattan Project,” 1st ed., p. 83, McGraw-Hill, New York, 1950.
(9) Weber, C. W.,ANAL.CHEM.32, 38791, 1960. (10) Woodall, He c., “Viscosity of Some Commercial Gases,” Engineering Data Sheet, Carbide and Carbon Chemical Company, K-25 1 7 J lg51* RECEIVEDfor review March 22, 1965. Accepted July 1, 1965. Division of Analytical Chemistry, 134th Meeting, ACS, Chicago, Ill., September, 1958. Work performed a t the Oak Ridge Gaseous Diffusion Plant operated by Union Carbide Corp. for the U. S. Atomic Energy Commission.
Liquid Scintillation Counting of Carbon-14 in Aqueous Digests of Whole Tissues RUSSELL TYE and J. DAVID ENGEL Kettering laboratory, Department o f Environmental Health, College o f Medicine, University o f Cincinnati, Cincinnati, Ohio
b The accuracy and precision of a method for the determination of carbon-14 in animal tissues and excreta has been evaluated. This method has the merit of being very general in applicability to different tissues and to different p emitters and is relatively simple in execution. Animal tissues may b e digested to near homogeneity in aqueous sodium hydroxide. Such digests are suitable for the determination of carbon-1 4 by liquid scintillation counting. Dispersions of the digests are stabilized by suspension upon Cab-0-Sil, in a scintillation medium consisting of dioxane, naphthalene, toluene, the scintillators 2,5-diphenyloxazole and 1,4-bis-2-(5-phenyloxazolyl)-benzene, and a compound to prevent freezing. In this medium urine may b e dissolved and its radioactivity counted without a gelling agent, but larger amounts may b e handled as a second phase, suspended upon Cab0-Sil. Carbon-14 in feces may b e determined by partial digestion in aqueous hydrochloric acid, mechanical homogenization, neutralization, and counting in suspension. Labeled cornpounds of widely differing polarities and characteristics of solubility have been used to show that the counting efficiency of the system is adequately independent of phase distribution or other phenomena affected by such differences.
I
THE COCRSE of studying the physiologic absorption and disposition of radiolabelled materials, it was desirable to obtain accurate analyses of whole tissues. A means of accomplishing this is to digest the tissues and to measure the radioactivity of dispersions of the digests by liquid scintillation counting. Thixotropic gels-e.g., Cab-
N
0-Sil-are convenient media for the dispersion of radioactive mixtures that are insufficiently soluble in the organic solvents used to dissolve available scintillators (3). However, uncertain distribution of radioactivity in the phases necessitates a counting system which is substantially independent of phase distribution. The problem of the influence of the distribution of unidentified compounds has been examined by comparison of the counting of cai bon-14 in three compounds of widely differing polarities. Acetic acid niay be expected t o be found distributed in significant proportions in both phases of a n organic aqueous system, and to be entirely within the aqueous phase (as the salt) a t high pH. The second compound, 4,4’ - methylenebis(2,6 - di - tert - butylphenol), abbreviated X B P , is highly insoluble in water a t any pH, but moderately soluble in organic liquids, whereas Banvel D, 2-1nethoxy-3~6-dichlorobenzoic acid, a coiiiniercial weed killer, has intermediate characteristics of solubility. EXPERIMENTAL
A11 counting was done b y a Packard Tri-Carb automatic liquid scintillation spectrometer, Model 314X, with optimum amplifier voltages and a “window” setting of 10 t o 50 volts. T h e samples reported in t h e first three tables were counted in glass vials (low potassium, Packard Instrument Co.) , and t h e remainder in polyethylene vials. Reagents. Cab-0-Si1 was supplied by Packard Instrument Co. T h e scintillation medium ( I ) , (DTS), was prepared as follows: dioxane, 1 liter; toluene, 50 ml.; naphthalene, 100 grams; 2,5-diphenyloxazole (PPO) , “Scintillation grade,” Packard Instrument Co., 16 grams; 1,4-bis-2-(5Apparatus.
phenyloxazolyl) - benzene (POPOP), “Scintillation grade,” Packard Instrument Co., 1 gram. This mixture may be expected to freeze near 0” C. and in that region an antifreeze must be present. Ordinarily, the water from the sample functions as the necessary antifreeze. However, if the sample as prepared for counting contains less than 5% of water, water or alcohol should be added to make up the difference. Procedure. Tissues were prepared for counting b y chopping weighed specimens with surgical scissors and warming with 0 . 5 N aqueous X a O H (4 inl. per gram of tissue, with 4 ml. minimum) with occasional swirling, on a 60” C. water bath. After 10 t o 20 minutes a substantially homogeneous solution, or an emulsion of f a t t y and aqueous phases, was produced. Samples mere weighed before and after warming, and any lost water mas replaced. The absence of substantial loss of water was taken as evidence that no loss of volatile metabolites had occurred. One-tenth to 0.5 ml. of the solution (or homogeneous emulsion) was added to 10 to 20 ml. of D T N in a 25-ml. polyethylene counting vial and shaken. Cab-0-Sil, in quantities sufficient to encompass the volume of DTX (-0.5 gram per 10 ml.), was added, and the sample \vas shaken vigorously for 1 minute. The emission of the sample was counted for periods of 10 minutes, several times in 24 hours, to allow for stabilization; then 0.1 nil. (activity approximately 10,000 c.p.m.) of a standardized solution of the radioactive compound in D T N was added from the 0.1-ml. pipet, the sample was again shaken thoroughly, and the emission was recounted. The quantity of radiotracer originally in the sample was then calculated on a relative basis. Urine was neutralized with HC1 and specimens ranging up to 1 ml. of urine or 1 ml. of aqueous solution of urine per 10 ml. (see discussion) were added to D T N for counting. If a n aqueous VOL. 37,
NO. 10, SEPTEMBER 1965
0
1225
phase appeared, Cab-0-Si1 was added. Counting and correction for quenching were done in the manner employed with tissues. Feces, or specimens of food, may be difficult to digest completely without
Table I.
120 120 120 103 103 103
Liver
RESULTS AND DISCUSSION
Counts per minute Sample Standard 2325 2395 2310 1761 1715 1810
9200 8430 8316 8480 8100 8099 8106
Banvel D-C14 found ( %) 99.8 104.0 98.5 105.0 94.0 98.2
a 0.27 gram peanut oil with 1.057, Banvel D-C14 (1.85 pc./gram peanut oil) mixed and let stand in 25.6 grams of finely chopped G. E. tract. b Same solution of Banvel D-C14, 0.056 gram in 5.2 grams of liver.
Table II.
Recovery of Urinary Metabolites Mixed with Liver"
(Reported as mg. of Banvel D-C") Urine (mg.)
Liver hg.)
73 73
a
b
Counts per minute Sample Standard
0 6795 0 6601 7.1 0 6275 ._ 82.5 6056 73 6057 82.5 73 5995 82.5 73 6.0 ml. of urine-C14 6.8 grams of.rat h e r . Corresponds to 20.2 mg. Banvel D in sample.
+
Table 111.
8660 8529 8375 7926 8109 7987
Banvel D-C'4 found bg.1 15.8 15.7 15.2 15.5 15.2 15.3
Counting Stability of Sodium Acetate-C14 vs. Quantity of Added NaOH, Showing Effect of Phase Distribution in Absence of Cab-0-Sil"
Normality NaOH
Counts per minute (thousands)
0 min. 20 min. 60 min. 300 min. 600 min. 39.9 39.9 39.9 39.9 O.Ob 39.9 34.7 34.7 35.0 34.7 34.7 0.0 32.6 33.5 31.0 23.0 26.7 0.05 25.8 30.8 22.3 12.6 15.4 0.10 28.2 22.5 11.4 17.8 9.3 0.15 25.3 18.7 14.7 9.3 7.8 0.20 16.0 22.1 12.3 7.5 0.25 6.4 14.3 21.1 10.8 5.5 0.30 6.5 17.1 11.9 8.9 0.35 5.4 4.6 9.9 14.2 7.7 4.9 4.0 0.40 12.1 8.7 6.8 4.4 3.7 0.45 9.1 13.1 6.8 4.0 3.2 0.50 0 Contents of samples: 0.1 ml. of aqueous sodium acetate-C" (0.05 pc./ml.), 10 ml. DTN, 0.5 ml. of water or of aqueous XaOH, no Cab-0-Sil. b Without 0.5 ml. of water or aqueous NaOH. There was risk that this sample would
freeze, but it did not.
Table IV.
Counting Stability of Samples Containing Liver Digest and Sodium Aceta te-Cl4"
Counts per minute (thousands) Stabilizer 0 min. 20 min. 60 min. 300 min. 600 min. 14.0 None 21.7 8.4 8.0 11.4 20.4 19.7 19.7 Cab-0-Si1 19.8 19.6 a Contents of averaged, duplicate samples: 0.1 ml. of aqueous sodium acetate-CI4 (0.05 ,uc./ml.), 10 ml. DTN, 100 mg. liver, 0.4 ml. aqueous 0.5N NaOH.
1226
Portions of the resulting suspensions were measured, either by weighing (-50 mg.) or pipetting (0.1 ml.), and were counted in the manner of the samples of tissue. The general method was evaluated by a study of specific factors in a series of six experiments.
Recovery of Banvel D-CI4 Added to Tissues
Wt. of tissue counted (mg.)
Type of tissue None G.ESa tract
strong oxidation (and possible loss of COz or other volatile products). These were partially digested by heating for 1 hour at 60' C. in 1N HCl (5 ml. per gram), and then homogenized with a ground-glass tube and pestle.
ANALYTICAL CHEMISTRY
The absolute efficiency of counting C14 in D T N with the settings given was approximately 50%. Tables I and I1 show that the presence of 0.5 ml. of digests, corresponding to 103 mg. of liver, or 120 mg. of alimentary tract, did not interfere with the determination of Banvel D-C14, if internal standards were used, although the samples were quenched about 10% (based on the c.p.m. of standards, with and without tissue). Other than factors arising from possible changes in the identity of the compound, there is little possibility of difference in the recovery of radiotracer added to the tissue or given to the animal, since the digests are substantially homogeneous. The possibility that metabolites of Uanvel D might be formed by the animal, and that the accuracy of analyses for these would be affected by the presence of tissue digests was examined (Table 11). The test is not conclusive; but depending upon the probability that urinary metabolites would be at least as polar, on average, as those in the tissue, metabolites of the radiotracer (if any) were measured with excellent accuracy. The presence of water, a t the level of 5% (Tables 111, and IV) did not affect the stability of counting of Banvel D-C14, acetic acid-C14, or MBP. One may presume that, in these cases, all of the water was in solution. illthough direct visual evidence did not always exist, settling or coagulation apparently occurred in specimens containing water and NaOH, since in such samples there was a continuing decrease in the count of acetic acid-Cl4 after 10 hours (Table 111). Increasing the content of S a O H increases the volume of the aqueous phase and the distribution of C14 into that phase, with a corresponding loss in count. Recounting each of these samples, after the addition of Cab-0-Sil, showed that stabilization was obtained within 20 minutes at rates ranging from 30,000 to 32,400 c.p.m., with variability unrelated to the content of NaOH. Similar behavior was observed with samples containing alkaline liver digest (averages of duplicate samples, Table
IV. Different tissues, having different colors and different compositions, may be expected to quench scintillation in varying degrees. Averages drawn from several experiments with different com-
Table V. Effect of Phase Distribution upon Quenching in Multiphase Systems; Constancy of Counting Efficiency with Changing Polarity of Compound, without and with Cab-0-Si1
Quenching materiala
Sodium acetate-C14 Water Alcohol
...
0
+ Cab-0-Si1 A + urine + Cab-0-Si1 A + liver + Cab-0-Si1 -4+ dog feces + Cab-0-Si1 A + rat feces + Cab-0-Si1
A0
11
45 85 45 77 20 78
...
...
... ...
78 77 73 78 20 60
...
... ... ...
Dog urine, 0.1 ml.; acid digest of dog or rat
* 4,4’-methylenebis(2,6-di-tert-butylphenol). c
Acetic acid-C14 Water Alcohol 100 86 89 81 85 85 86 79 81
Banvel D-C“ RIBP-C“* Water Alcohol Water Alcohol 100 100 90 90 91 88 88 89 91 92 84 84 86 89 84 81 84 78 79 ... ... 83 87 83 ... ... 83 82 82 78 60 59 77 69 64 65 60 67 62 56 59 59 54 58 57 51 54 53 39 54 51 48 37 45 feces, 0.1 ml.; alkaline digest of rat liver, 0.1 ml. (each in 15 ml. DTN).
A is either water or alcohol, added as antifreeze, 1 ml.
pounds containing C14 demonstrated quenching of approximately 10% from 100 mg. (0.5 ml. of digest in 15 ml. of DTI; Cab-0-Sil) of aninial tissues, except whole blood, which must be reduced to one tenth of this concentration to produce comparable quenching. The greater part of this difficulty may be renioyed by bleaching the prepared sample v, ith hydrogen peroxide, which does not destroy the scintillation (e). This procedure inay be applied generally to excessively colored samples. The effect of differences in phase distribution, upon counting efficiency in the DTS-Cab-0-Si1 system, is small by comparison of sodium acetate-CI4 with 31L3P-C14 (Table V), since the phase distributions of these compounds are opposite. The relatively high level of quenching in the samples containing feces yvas caused largely by unneutralized acid from the digestion process. With respect to quenching, no important difference was found between water and alcohol used as antifreeze, if the dispersions were stabilized with Cab-0-Sil. I n its absence, samples con-
+
taining alcohol have greater counting stabilities than those containing water, but the degree is dependent upon the character of the sample. Consequently, the use of water, in samples stabilized with Cab-0-Sil, appears to have a better general apvlicability, as most biological preparations are aqueous. If the concentration of salts in urine is low, up to 1 ml. of urine is soluble in 10 ml. of D T N . Ordinarily, urine (especially from rabbits) contains solids, or quenches severely, so that counting 0.1 nil., diluted to 0.5 ml. with water, provided better conditions for counting. The methods of preparation and counting which have been described have been used with moderate success with tritium, and as would be expected from the energy of the emission, with good success with sulfur-35. LITERATURE CITED
(1) Furst, M., Kallnan, H., Brown, F. €I., Nucleonics 13, 58 (1955). (2) Herberg, It. J., ANAL. CHEM. 32, 42 (1960).
(3) Ott, D.G.,Richmond, C. R., Trujillo, T. T., Foreman, €I., Nucleonics 17, 106 (1959). RECEIVEDfor review April 26, 1965. Accepted July 19, 1965.
Correct ion Determination of Small Amounts of Nitrite by Solvent Extraction and Spectrophotometry I n this article by Anthony Foris and Thomas R. Sweet [-%xAL. CHIX 37, 701 (1965)] on page 701, column 3, line 29, the sentence should read: “For procedure 11, a 2% solution of 8aminoquinoline was prepared which contained, in addition to the 5 ml. of concentrated hydrochloric acid, 50 ml. of glacial acetic acid per 100 ml. of solution.” On page 703, in Table I, the symbol Co- should be changed to C1-, the per cent error for NO3- should be -3.19 instead of -37.19 and the per cent error for SO4-* should be -4.07 instead of -4.0.
VOL. 37, NO. 10, SEPTEMBER 1965
1227
