aration and geometry can be duplicated with precision greater than the conntr ing error. The error due t o the degree of purity of the acetylated product was elitninated by purification to constant specific activity. For routine determinations the accuracy obtained by these rather extreme measures may not be necessary. The position of the labeled carbon atom in the acetic anhydride is not of great importance. The possibility of an error due to the isotope effect may be eliminated by the use of acetic anhydride-2-GI4; however, an error from this source has not heen detected. Acetylation in pyridine is probably the most widely applicable acetylation method. In the case of gossypol however, extensive decomposition was encountered. Acetylation, catalyzed
by sodium acetate, proved successful without observable decomposition. The specific activity of the acetic anhydride and the sensitivity of the available counting equipment determine the size of sample required. If acetic anhydride of 20 pc. per mmole is prepared, then only 0.5 mg. or less of acetylated material is necessary for this det.ermination, with the equipment described. If a liquid scintillation counter is available, the determination of specific activity is simplied, sample size requirements are even smaller, and counting error may be reduced because of the greater sensitivity of such instruments. This method offers several advantages over previous acetyl group methods. First, there is no hydrolysis of the derivative and, consequently, no possible
errors from unexpected decomposition or incomplete hydrolysis. Second, there is no distillation involved, eliminating the errors of loss, or errors from unexpected volatile components. Third, there is no titration, eliminating the errors due to unexpected acidic components. Finally, the sample of acetylated material may be completely recovered, unchanged, for further work. LITERATURE CITED
(1) DeWalt, C. W., 1Glenn, R. A,, ANAL. CHEM.24,1789 (1952).
J.,, "Quantitative Mibroanalysis," 4th ed., p. 161, Blakiston, Philadelphia, Pa., 1945. (3) Schwehel, A., Isbell, H. S., Moyer, .I. D.. J . Research Natl. Bur. Standards
(2) . . Preel. F.. Grant.
Org&
53, 2 2 1 4 (1954). (4) Vcriey, A,, Bolsing, F., Ber. 34, 3354 (1901). RECEIVED for review February 15, 1960. Accepted July 12, 1960.
Tritium Radioactivity Determination of Biological Materials by a Rapid Dry Combustion Technique EDWlN A. PEETS, JAMES R. FLORINI, and DONALD A. BUYSKE Pharmacological Research Department, Experimental Therapeutics Research, lederle Laborotories Division, American Cyanamid Co., Pearl River, N. Y.
b In pharmacological and biological research it i s often necessary to determine the tritium radioactivity of a large number of samples of biological materials having varied specific activities. A p,roctical method of measuring the radioactivity of large numbers of such samples i s dry combustion of the material to water and carbon dioxide and determination of the radioactivity of the products using a liquid scintillation spectrometer. Since the currently available apparatus can accommodate only single samples of relatively small size, a furnace has been designed which makes possible the rapid simultaneous combustion of three samples of up to 2 grams dry weight.
of biological materials into a solubilizing or suspending system (6, 10, 13, 14) for liquid scintillation counting has been suggested, but again sample size is limited. I n addition, most biological materials are too highly colored to allow satisfactory counting by the liquid scintillation technique. It is apparent, therefore, that complete combustion of such samples to carbon dioxide and tritium-enriched water would offer considerable advantages for this type of determination, Several sealed tube combustion techniques have been pub-
lished (1, 16, 17) but they are limited t o 5- to 25-mg. samples. The apparatus t o be described is an enlarged and modified version of the standard carbon-hydrogen apparatus. It can oxidize simultaneously three samples of up to 2 grams dry veight; the entire heating and cooling cycle requires approximately 45 minutes. Thus, 24 t o 30 samples can be prepared for counting in a normal working day. In addition t o preparing samples containing tritium for radiometric determination, this apparatus is applicable to
B .
IOLOGICAL and
pharmacological studies often necessitate the deterniination of radioactivity in both liquid and solid samples of biological origin. In studies involving tissue distribution and residue levels of drugs in plants and animals, it is not infrequent that 100 or more samples may be collected for radioactivity determinations. Due to the estremely weak energy of tritium, infinitely thick samples are less than 1 mg. per sq. em. (4). Therefore, it is not possible to make direct radiometric measurements on any except very small samples of a solid. Direct incorporation
Figure 1. Combustion furnace showing combustion tubes and one trap VOL. 32, NO. 11, OCTOBER 1960
1465
1 1 1 '2Drnm
Figure 3.
I
ZZOV AC
Figure 2.
Wiring diagram
of furnace
PC-30s. Percentage timer, 30 seconds, 1 15 v., alternating current, 60 cycles; switch rating, 1 5 amp., manufactured b y Industrial Timer Co., Newark, N. J. EM-1. Normally open mercury switch SPST; load rating, 2 5 amp. at 2 3 0 v., alternoting current, manufactured b y Ebert Electronics Corp. Motor, Bodine No. A1 2RC, Syncron super high torque, 2 r.p.m.
the preparation of samples coiitaiiling carbon-14-labeled compounds. APPARATUS
The furnace (Figure 1) is divided into a section for the sample combustion and a section for the catalyst. Each section contains an externally grooved refractory tunnel 4l/* inches wide by 3 inches high by 10 inches long (Fisher Cat. No. 10-520) through which the three combustion tubes pass. Each tunnel is wound separately and externally with Chrome1 A wire (Fisher Cat. Yo. 10-515), Cr 20%, S i 80%, in the form of a spiral. The temperatures of both sections are individually controlled by percentage timers and mercury relays connected in series with the heaters. The timers regulate the per cent of each 0.5-minute period that current passes through the heaters and can be set for a n y value from 0 to 100%. The timer switch of the catalyst section is hand set t o a fixed value of 7570 to maintain a constant temperature of 930" to 970" C. during the entire time that the furnace is in use. The sample section is gradually heated to a maximum temperature of 750" C. This is done by attaching to the timer switch a brass disk, 10 cm. in diameter, that is machined to contain 31 removable steel pegs evenly spaced along the circumference (Figures 1, 2). A cam fixed to the shaft of a 110-volt, alternating current, 2-r.p.m. motor is positioned adjacent t o the brass disk and extends between two of the pegs. The cam makes a mechanical couple with a peg with each revolution of the motor and advances the disk to the next peg. This results in a cumulative increase in the time that the switch is on and the current flows through the heater. Any given temperature can be maintained by removing the proper peg so that the cam does not advance the disk 1466
ANALYTICAL CHEMISTRY
A. B. C.
D.
once a desired position has becn reached. The maximum temperature obtainable in either section is 1100" C. The sample heater and catalyst chamber are joined as a unit in the combustion furnace housing. The heaters are encased in 3-inch thick firebrick and the two chambers separated from one another by a 2-inch thick Transite baffle. The entire unit is covered with l/rinch Transite. Three 1-inch diameter holes are drilled through the end plates and the center baffle to permit the insertion of the combustion tubes. The sample heater is divided longitudinally into upper and lorrer sections by cutting through the grooved refractory and is wound top and bottom with the resistance wire. The upper half of the section is attached to the furnace housing by a hinge a t its top surface. The heaters have an internal resistance of 18.5 ohms and are designed to operate a t 220 volts, alternating current. Furnace temperatures are monitored by Chromel-Alumel thermocouples connected to a direct reading meter. The thermocouple passes into the chambers through holes drilled into the base of the e i d plate. The combustion tubes are of thinwalled Vycor glass, 640 mm. long, with an inside diameter of 19 mm. The sample end of the tube is fitted with a standard 35/20 ground-glass socket. The conversion end of the tube is drawn down to a diameter of 8 mm. and has a t its end a 12/5 socket which receives the ball joint of the water trap (Figure 3). The tube filling (Figure 3) consists of a loose roll of about 8 layers of acidwashed nickel foil 15 mils thick and 200 mm. wide and a 20-mm. thick pad of loosely matted silver wool placed in the reduced end of the combustion tube (2,9).
6mm
Diagram of Vycor combustion tube and Pyrex radiator trap
Experiments performed in this laboratory corroborate a previous report ( 2 )
Nickel foil sleeve (catalyst) Coarse asbestos fiber p a d Silver wool Pyrex wool
that shows this type of filling to be a s efficient as the conventional copper oxide filling as a combustion catalyst. Also it has no tendency to pack and retard the gas flow as does copper oxide, causing dangerous pressures to build u p inside the combustion tube. PROCEDURE
Saniplcs to be assayed are most easily prepared by drying in a n oven at 110" C., provided the tritium-containing compounds are not volatile. After most of the water has been driven off, t h e material is ground if necessary and weighed into a disposable Combax combustion boat and covered (Fisher, Cat. S o . 7-651, 7-652). When dealing with liquids or slurries, a n aliquot may be placed directly into the boat and dried. Lyophilized samples are very difficult to combust; the powdery nature of such samples causes frequent "blox backs" and flashing, and the use of such samples should be avoided whenever possible. The temperature of the nickel catalyst section of the oven is maintained a t 930" to 970" C . , which has been determined as the optimum temperature for the catalytic conversion of pyrolytic products to water and carbon dioxide ( 2 ) . K h e n the catalyst has reached t h e proper temperature, the samples are inserted into the combustion end of the Vycor tubes and the tubes capped. The hinged arrangement of the sample heater makes i t possible to observe these operations and also the pyrolysis of the samples; this is useful in the combustion of volatile samples. The sample heater is then closed and osygen (dried first by passage through calcium chloride tubes) is passed through the system a t a flow rate of 20 to 30 cc. per minute as measured by bubble counters. Heating of the combustion chamber from room temperature to 700" C. in 30 minutes allows the combustion of large samples and even fatty substances with no loss of sample due to flashing and blow backs. The time required for a simultaneous combustion of three samples is about 45 minutes including cooling of the sample chamber to 100" C.
bc,fore additional samples are inserted. The water of combustion is collected in the borosilicate glass radiator trap imniersrd in a slurry of dry ice-carbon tetrachloride-chloroform in a Dewar flask. After the combustion is completed the tritium-water mixture is transferred to a counting vial by two successive washings of 5 ml. of polyether-611 phosphor solution (3). The sample is then counted in the liquid scintillation counter. Unless the sample weights and source materials are identical, it is necessary to determine the counting efficiency of each sample by a n internal standard (3). This is required because variations in the volumes of the water of combustion have an appreciable effect on the efficiency of counting. Also certain products of combustion such as oxides of sulfur and nitrogen, and traces of halogens not completely removed by the silver wool may affect the counting efficiencies of the vintillator solutions (8).
RESULTS AND DISCUSSION
The accuracy of the method was detcrmined in two ways: combusting s e n d samples of sucrose or filter paper t o which had been added a small amount of 7-tritio-tetracycline (tritium-labeled tetracycline) and combusting a known weight of sucrose, measuring the amount of water produced, and comparing this yield to the calculated m-ater of combustion. The recovery and reproducibility data are presented in Tahles I and 11. The average recovery of 1.953 X IO5 d.p.m. of tritium from t w l v e samples is 95.05y0 with a standard deviation of 3.03y0 and the recovery of hydrogen, determined as weight of rrsultant water, 94.9% with a standard deviation of 3.3%. Recovery experiments were also performed from blood and bone samples to which had been added known amounts of 7-tritio-tetracycline. The recovery from blood samples is 96.6070 with a standard deviation of 6.90% and t h a t from bone 93.76% with a standard deviation of 4.27y0. Recovery of tritium remains constant even a t very low isotope levels. These data demonstrate that the combustion procedure is satisfactory for the determination of residual tritium activity in solid samples which cannot be counted by conventional techniques. The method has as its particular advantnges a speedy combustion of sample nith a high degree of accuracy, and the accommodation of large samples. This procedure has been used to determine the tissue distribution of such varied compounds as tritium-labeled synthetic corticosteroid Sa-fluoro-1 lp, 16a,17a,21 tetrahydroxy - pregna - 1,4 - diene3.20-dione, tetracycline, terephthalic acid, and 5- [(aryloxy)methyl] 2-oxa-
Table I.
Tritium Recovery from Standard Samples
Disintegrations per Minute Addeda Recovered
Samples
1.953 X I ,953 x 1.953 x 1.953 X 1.953 X 1.953 X 1.953 X 1.953 x 1.953 X 1.953 X 1,953 x 1.953 X
1 2 3 4 5 6 7 8 9
10 11 12
lo5
yo Recovery
1.883 1.931 1.850 1.935 1.874 1,802 1.876 1.815 i ,835 1.757 1.930 1.788
105 105 IO5 lo5 lo5 lo5
~
105
lo5
lo5 105 lo5
96.41 98.87 94.72 99.07 95.95 92.26 96.05 92.93 93.95 89.96 98.82 91.55 95.05
~~
3.03
std. dev.
13 14 15
2 . 5 3 x 104 1 . 3 4 x 104 2 . 6 2 x 103
2 . 7 2 X lo4 1 . 4 1 x 104 2 . 7 6 x 103
93.0 950 94.9
Standard samples are sucrose or filter paper to n.liich were added aliquots of a solution of 7-tritio-tetracycline of knonn radioactivity (specific activity 13 pc./mg. as determined using a Yational Bureau of Standards tritium source).
zolidinone, with excellent recoveries and reproducibility. Although this apparatus was designed for samples containing tritium, it would appear t o be equally useful for the determination of carbon-14. The trapping of the effluent radioactive carbon dioxide is accomplished by bubbling into an aqueous base (’7, 11). Retention of reversibly bound tritium by the glass surface of the combustion tube is known t o occur (6,12, 15) and is thought to be due to formation of a surface layer of the isotope and/or the exchange of tritium Kith the hydroxyl hydrogens of the silicates. While the results of consecutive combustions of standard samples presented in Table I demonstrate t h a t this “memory effect” does not lower significantly the recovery of tritium even Then very small quantities of the isotopes are being determined, tritium retention can result in high blank values and cross contamination of samples when a large series of samples or highly labeled materials are combusted. This contamination should be monitored daily by the routine combustion of non-
Table II.
Compound Sucrose ( 1 ) (2) (3)
(4) (5) (6)
(7) (8)
(9) (10) (11) (12)
radioactive samples and measurement of radioactivity. After the combustion of large series of samples (30 to 40) this contamination is in the neighborhood of IO0 c.p.m., which is three times the normal background count rate. An effective means of removing this “bound” tritium is to pass oxygen saturated with water through the tube overnight with the temperature maintained a t 600’ C., or purging the tubes for 20 minutes with steam. ACKNOWLEDGMENT
The authors gratefully acknowledge the assistance of the Lederle hiechanical Research and Development Shop and the helpful suggestions of David R. Christman of the Brookhaven Kational Laboratory. LITERATURE CITED
(1) Buchanan, D. L., Corcoran, B. J., AN.4L. CHEM. 31, 1635 (1959). (2) Christman, D. R., Day, N . E.,
Hansell, P. R., Anderson, R. C., Ibid.,
27, 1935 (1955). (3) Davidson, J.
D.,
Feigelson,
P.,
Hydrogen Recovery from Standard Samples
Weight of Water of Combustion, 11g. Sample Theory Found ’ 474.5 1054.2 724.1 513.3 811.5 592.6 660.9 643.6 647.4 430.9 420.7 431.8
277.9 618.1 424.1 300.6 475.2 347.0 387.0 376.9 379.2 252.4 246.3 252.9
% Recovery
262.4 574 4 397.2 280.2 443.4 334.4 372.0 319,7 351.3 238.9 231.2 264.5
Av.
94.4 92.9 93.7 93.2 93.3 96.4 96.1 92.8 92.6 94.7 93.9 104.6 94.9 f 3 . 3
std. dev.
VOL. 32, NO. 11, OCTOBER 1960
0
1467
Interiz. J . A p p l . Radiation Isotopes 2, l(1957). ( 4 ) Eidinofl’, M. L., Knoll, J. E., Science 112, 250 (1950). (5) Funt, B. L., Hetherington, A,, Ibzd., 125, 986 (1967). (6) Graff, J., Rittenberg, D., ANAL.CHEW 24,878 (1952). ( 7 ) Kamen, 11.D., “Isotopic Tracers in Biology,” p. 308, Academic Press, New York, 1967. (8) Kerr, V. S., Hayes, F. N.,Ort, G.,
Intern. J . A p p i . IZnrlzniion Isotopes 1, 284 (1957). (9) Kirsten, W.,ANAL. CHEV. 25, 74 (1953). (10) Ott, D. G., Richmond, C. R.,
Trujillo, T. T., Foreman, H., .Vucleonics
17, (9), 106 (1959). (11) Passman, J. hl., Radin, S . S., Cooper, J. A. s., A A i L . CHEM. 28, 484 (1956). (12) Payne, P. R., Campbell, I. G., White, D. F., Bzochem. J . 50,500 (1952). (13) Radin, N. W.,Fried, R , .%YAL.
CHEJI.30, 1926 (1958). (14) White, C. G., Helf, S., iVucleonics 14, ( l o ) , 46 (1966). (16) Williams, D. L., Ronzio, A. It., J . A m . Cheni. SOC.72, 5787 (1950). (16) Wilzbach, K. E., Kaplan, L., Brown, \V. C., Science 118, 522 (1953). (17) Wilzbach, K. E., Van Dyken, -4.R., Kaplan, L., ASAL. CHEX 26, 880 (1964).
RECEIVED for review December 28, 1959. Accepted July 19, 1960.
Backgrounds for Liquid Scintillation Counting of Colored Solutions R. J. HERBERG [illy Research laboratories, Indianapolis, Ind.
b It is often difficult or impractical to obtain background solutions of the same color intensity as those of sample solutions. The change of quenching with color is different for isotope and background solutions so that the usual internal standard techniques are not directly applicable. Since the reciprocal absorbance at 400 mfi of colored solutions is a linear function of their relative counting efficiencies, reference of measurements at this wave length to standard curves permits easy correction of counting rates of colored solutions of different color intensities to the same color intensity.
I
often desirable to count tissue solutions, solutions of dyes, urine samples, and other colored solutions nith a liquid scintillation counter. For low activity samples subtraction of an appropriate background is of utmost importance. Ideally the background sample differs from the unknown sample only in that it contains no added radioactivity; solution composition and color are identical. This identity of color is difficult to achieve for tissue solutions. Urine samples from different individuals, and even the same individual a t different times, xi11 differ in color. The blood color of experimental animals will depend on the oxygenation of the animal’s blood. The uncertainty as to how to prepare adequate background samples, makes i t necessary to know how counting efficiency of backgrounds and samples changes with color. One also needs to know if the change of counting efficiency with color is the same for different levels of the same isotope. Little has been published concerning changes in counting efficiency with color for various kinds of solutions. Domer and Hayes have indicated that for suspensions, sample and background T IS
1468
ANALYTICAL CHEMISTRY
are quenched differently ( 3 ) . Several methods are usable for determination of color quenching in isotope solutions including the usual internal standard technique. Davidson (2) has suggested the ratio of a sample’s count in two channels as a method for quenching determination. The selection of channels for minimum error with this method has been discussed by Baillie ( 1 ) . Guinn (6) has proposed a method nhereby a sample is counted in single channels and again in coincidence. All of these methods implicitly assume that isotope and background solutions are quenched alike by color. That this assumption is not alwaxs true will be shown by the following experiments involving different coloring agents and solvent systems. EXPERIMENTAL
Equipment and Chemicals. Counting was done with a liquid scintillation spectrometer (Packard Tri-Carb Model 314X). Crystalite screw cap 20-ml. vials were used. Spectral measurements were made with a Bausch & Lomb Spectronic 20 colorimeter with test tubes of 15.5-mm. internal diameter. The phosphors used, 2,5-diphenyloxazole (PPO) and 1,4bis - (5 - phenyl - 2 - oxazolyl) - benzene (POPOP), were of scintillation grade (Arapahoe Chemicals, Inc.). Hyamine hydroxide was prepared as a 1.0X solution in methanol by the method of Passmann et al. (8) as modified by Eisenberg
(4)*
The coloring agents used were a n alcoholic potassium hydroxide human blood digest solution, a saturated toluene solution of methyl red (W. A. Taylor and Co., Baltimore; p H 4.4 to 6.0), and an aqueous solution of bromothymol blue (Matheson; HzO soluble). All other chemicals were of reagent grade. The carbon-14 (CI4) and hydrogen-3 (H3) isotopes that were added to solutions as a source of radioactivity
consisted of toluene solutions of C14 palmitic acid and H3 cholesterol, respectively. The volume of isotope solution added to a vial did not exceed 50 pl. The total volume of liquid in a vial did not exceed 15.0 ml. All samples containing C14 activity were counted a t a photomultiplier voltage of 1170; samples a i t h H3 activity were counted a t a voltage of 1480. These are the voltages which gave the masimum count rate for C14 and H3, respectively, in the colorless solvent systems. A 10- to 100-volt channel was used normally. For two-channel counting, the middle discriminator was set to bisect the 10 to 100 count; the setting is given for the particular experiments. I n all experiments samples were counted repeatedly with interspersed standards. C O U N T I N G CHARACTERISTICS SYSTEMS
OF VARIOUS
Blood Digest Solutions. The solvent consisted of a mixture of 3.0 ml. of l.OM methanolic Hyamine hydroxide and 12.0 ml. of toluene. The following sets of samples (13 per set) were prepared: a, empty vials; b, vials with solvent; c , vials with solvent and CI4 activity; d, vials with solvent and scintillator; and e, vials with solvent, scintillator, and C14 activity. For sets d and e 12.0 ml. of toluene containing 0.5% PPO and 0.01% POPOP was mixed with 3.0 ml. of Hyamine hydroxide solution. To the corresponding vials of sets 2 through 5 were added varying volumes of an alcoholic potassium hydroxide blood digest so that each set ranged in color from a clear solution to a pronounced red-brown. The digest was sufficiently concentrated so that no more than 100 pl. was added. A similar group of 5 sets was prepared with H3 activity. All samples were countrd for 30 minutes. At a given tap setting, the empty vials count very uniformly; the count rates, 20.70 0.97 and 66.56 + 0.62 c.p.m. a t taps 7 and 11, respectively, con-
*
