Liquid Scintillation Counting of Tritiated Organic Compounds MARVIN 1. WHISMAN, BARTON H. ECCLESTON, and F. E. ARMSTRONG Bartlesville Petroleum Research Cenfer, Bureau o f Mines, U. S. Department o f Interior, Bartlesville, Okla.
b The use of tritium-tagged hydrocarbons in analytical research has made it necessary to evaluate some of the parameters involved in liquid scintillation-counting techniques. From these evaluations, a routine procedure for measuring the radioactivity of tritiated organic compounds and the confidence limits of results were established. This paper evaluates the more significant variables and sets forth the conditions and procedure adopted by this laboratory.
L
IQUIDSCINTILLATIOS COUKTING was
developed less than 10 years ago 1954 have complete internal-sample. liquid scintillation spectrometers been commercially available. Many publications have presented the techniques and practical applications of these instruments, but only a few (1, 3, 9) have touched on the factors which are of primary concern to the analytical chemist wishing to apply tracer techniques when studying reaction mechanisms. evaluating separation techniques, and identifying combustion products. These factors are the reproducibility of rcsults obtained in counting tritiated materials by a liquid scintillation spectrometer, and the parameters of the analysis, which control thc precision and accuracy of results. The Kilzbach (8)exchange labeling method is used in this laboratory to tag selected hydrocarbon components of gasoline. The tagged hydrocarbons are used in tracer studies of gasoline storage stability. Reaction products resulting from storage are considered important if they are found in the form of a gum. Because the amount of gum formed during a normal storage period is small and the number of compounds contributing to the gum is large, it is reasonable to expect the reacted portion of any tagged compound to be small. For this reason it has been necessary to select a liquid scintillation-counting technique which is not only precise but also rapid and economical. This paper presents data obtained while evaluating some of the variables in liquid scintillation counting and outlines the procedure adopted a. the result of this evaluation.
(4, 6) and only since
484
ANALYTICAL CHEMISTRY
APPARATUS AND REAGENTS
Apparatus. A Packard Tri-Carb (Model 314) liquid scintillation spectrometer was used. Counting containers were 5-dramJ Crystalite screwneck vials, with black Bakelite, polyethylene-lined caps (Wheaton Glass Co., BIillville, N. J.). Liquid measurements were made with microliter pipets in a variety of sizes, calibrated to contain from 25 through 500 pl. and volumetric pipets with liquid capacities of 15 and 20 ml. Reagents. Toluene, Phillips pure grade (dried by passing through a column nacked with 4 8 Molecular Sieves). Phosphor, 2,5-diphenyloxazole (Pilot Chemicals. Inc.. Watertown. Mass.). Wave-length shifter, 1,4-di [2-(5-phenyloxazolyl) ]benzene (Pilot Chemicals, Inc., Watertown, hlass.). Scintillator solution, prepared by dissolving 5 grams of the phosphor and 0.1 gram of wave-length shifter 1,4di [2-(5-phenyloxazolyl)]benzene in 1 liter of dry toluene. Internal standard, tritiated toluene with a radioactivity of 2.2 X lo6 d.p.m./ml. (disintegrations per minute per milliliter) (New England Nuclear Corp., Boston, Mass.), was used to standardize tritiated toluene solutions of higher and lon-er radioactivities prepared by this laboratory for use as internal standards.
n-here ?z is the total number of counts and F.S.D. is fractional standard deviation. This deviation is a result of the random nature of radioactivity decay, and a high total number of counts is desirable to reduce this error t o a minimum. The following voltage settings were maintained: multiplier phototube, HVT5, 960 volts; red-channel discriminator gate, 10 to 50 volts; greenchannel discriminator gate, 50 to 100 volts. -4 background count is obtained on 13 ml. of the scintillator solution. This count, obtained once each day and applied t o all data obtained during that day, should be a t least a 10minute count. The radioactivity of the samples is calculated as follow: Radioactivity (d.p.m. per nil.)
=
n here C, = c.p.m. of vial containing sample
only Ca = c.p.m. of vial containing blank scintillator solution Cr2 = theoretical d.p,m. per ml. of standard C, = c.p.m. of vial containing sample plus internal standard K 1 = volume of internal standard used, microliters Kl = volume of saniple used, microliters
PROCEDURE
Measure 15 ml. of scintillator solution into each of two vials. Carefully pipet into each vial identical quantities of sample to give approximately 5000 to 10,000 c.1l.m. (counts per minute). Rinse the microliter pipet into the vial three times R ith the scintillator solution-sample mixture. Into one of the vials carefully pipet a quantity of intcrnal standard (tritiated toluene), which nil1 give a t least 10 times the total counts expected from the sample. The total counts from the sample plus the internal standard should not exceed 300,000 c.p.m. Cap both vials, and mix their contents by shaking briefly. Place in the sample rack in the freezer chest of the spectrometer a t -10' C. for 1 to 2 hours. Count each sample for 3 to 10 minutes, dqirnding upon the total radioactivity rrcsent. Make enough counts to reduce the fractional statistical counting error to an acceptable minimum. F.S.D. = 1/.\/51
(1)
EXPERIMENTAL DATA
To obtain the optimum volume of scintillator solution for the vials selected for this work and the phosphor system employed, 10 vials were filled with volumes of scintillator solution, ranging from 2.5 to 22 ml. Then 50 ,ul. of a tritiated-toluene standard were pipetted into each vial, and the counting efficiency of each prepared sample was determined. E, =
c,/c, x
100
(3)
\There E , = counting efficiency, per cent C , = c.p.m. per ml. obtained experimentally C t = theoretical activity in d.p.m. per ml. The top curve ( A ) in Figure 1 shows the result of this series of determinations. Maximum counting efficiencies
were obtained between 7.5 and 15.0 ml. of scintillator solution. Curve B in Figure 1 shows the same relationship between scintillator solution volume and counting efficiency when the sample is light yellow. Some quenching occurs as counting efficiencies are generally lower for the colored samples throughout the range of volumes used. Curves C, D , and E of this figure indicate the large drop in efficiency, resulting from counting a highly colored gum. Quenching is believed to be a function of both color and chemical composition. Based on the data from Figure 1, 15 ml. of scintillator solution were arbitrarily selected for counting gaso l i n e ~ and light-colored hydrocarbon samples. For highly colored samples. such as gasoline gums, 20 ml. of solution are used. A commercial sealed standard of tritiated toluene m-as counted 10 times for 50 minutes over a period of several days in order to determine the deviation imposed by instrumental stability and counting statistics The standard deviation ( 2 ) of this series was +0.24% (15,000 i 37 c.p.m.). As a further test for deviation caused by instrument stability and counting statistics. 12 laboratory prepared samples were counted twice for 10 minutes and the standard deviation for the pairs of data was calculated. This value was 1 0 87, (15,000 =t117 c.p.m.). To evaluate the variables created by optical differences in counting vials and pipetting errors, a third group of 10 samples was prepared. The standard deviation calculated, after single 10-minute counts on each sample were obtained, was 10.8% (14,800 & 126 c.p.m.). This value compares closely with the deviations attributed to instrument instability and counting statistics which leads to the conclusion that neither pipetting nor optical differences in vials contribute a significant error. Table I lists the data and standard deviations obtained from single 10minute counts of 20 laboratory prepared samples. The standarcd deviation of columns A , B, and C are 1 1 . 4 % ) i1.7%, and *1.5%, respectively. of the average count for each column. The sample radioactivities calculated from the data in columns A , B. and C are presentrd in column D . The standard deviation is k0.14 X 1P d.p.m. ’ml., about =t2.096of the average value tabulated. To obtain a confidence limit for any single determination a t 95% probability, the standard deviation would be multiplied by Student’s t (to.0: 9 = 2.26); the resulting precision figure is =t0.32 X lo5 d.p.m./ml. or about 14.57, of the average value. Using the standard deviations calculated for columns A and B of this table, one can arrive a t an ideal ratio of internal standard radioactivity
I
6r
Table I. Single Determinations on 20 laboratory-Prepared Samples
D Calculated Sample Radioactivity,
SCINTILLATOR SOLUTION VOLUME, r
Figure 1. Effect upon counting efficiency of scintillator solution volume A. 6. C. D. E.
Tritiated toluene Straw-colored, tagged gasoline 2.5 mg. of brown, tagged gasoline gum 5.0 mg. of brown, tagged gasoline gum 10.0 mg. of brown, tagged gasoline gum
lc.p.m.) to sample radioactivity (c.p.m.). The count rate of the internal standard is determined by comparing the count rate of the sample with that of an identical quantity of sample plus added internal standard. The following equation is used: CIS
= CIS
.I.
s
- cs
(4)
where
ClS
= count rate of the internal
Cis+ s c‘s
standard as affected by the sample in c.p.m. = combined count rate for the sample plus internal standard in c.p.m. = count rate of the sample in c.p.m.
The standard deviations of these count rates as established in Table I are *1.4% for CIS+ 9 (column -4) and +1.7% for CS (column B ) . The ratio of radioactivity of the internal standard to that of the sample is important in determining the precision of CIS (Equation 4). The standard deviation of Cls approaches the standard deviation of CIS+ s (the difference between i1.4 and + 1.7y0is not considered significant) when the counting rate of CIS becomes large compared with that of CS. As CIS becomes very small when compared m-ith C S , the percentage of error in C I S becomes very large. The standard deviation of the combined term, CIS+ s - Cs, may be determined (Y) on the basis of the total count rate as follows: S.D.(cIs,
d(0.014 Cs + rs)’
+ ( 0 . 0 1 7 C ~ ) ~(5)
To obtain the fractional standard deviation the right-hand side of Equation 5 is divided by CIS. The following equation represents the variation in Equation 5 with the change in ratio of c s to C I S . CS
--
CIS
48.65X - 0.586
(6)
A Sample Plus Internal B C Standard, Sample, A - B, D.P.M/ C.P.M. C.P.M. C.P.M. RI1.(X1O6) m49 6.94 69045 63996 6.68 68943 64079 6.94 69316 64250 69116 7.02 64010 6.92 68729 63719 7.16 68126 5124 63002 5086 69491 6.94 64405 7.08 69008 63864 6.88 63714 68702 7.12 66151 61199 Std. dev. &958 f87 zk933 1 0 . 1 4 Each sample contained 15 ml. of scintillator solution plus: column A , 50 d. of tagged gasoline and 50 pl. of tritiated toluene standard; column B , 50 pl. of tagged gasoline.
where
x = d(0.014 Cs + 15)’ + (0.017 Cs)* CIS
From an inspection of the curve plotted from Equation 6, a ratio of 10 to 1 for count rates in c.p.m. for internal standard and sample was chosen as one that would give an acceptable deviation for C I S . The gain in precision in changing the ratio from 10: 1 to 20: 1 n-ould be only l/lo that obtained by increasing the ratio from 1:1 to 1 O : l . Another parameter investigated was the effect upon counting efficiencies of relatively large sample volumes because of dilution of the phosphor. Sample volume was varied from 50 p1. to 1 ml. with no significant effect upon counting efficiency. Undoubtedly, 1 ml. is not the upper limit to sample volume but, because of high specific activities involved in these studies, it has not been necessary to investigate larger samples. Coincidence losses for this instrument were determined by comparing counting efficiency with sample count rate. At approximately 300,000 c.p.m.. rcsolution time becomes an important factor in counting. For this reason an upper limit of 300,000 c.p.m. was established for total apparent radioactivity of any sample vial. I t is possible to have a sample so “hot” that the pulse amplifiers are partly blocked, and an incorrect count of less than 300,000 can be observed. This possible error \% as avoided by keeping a record of the ratio between counts in the red and green channels. If complete or partial blocking of the scalers occurs from coincidence loss, the green count is very 101%or zero. When the tritium content of a sample is very high, it is wise to make a 1000 to 1 dilution for verifying the original count. VOL. 32, NO. 4, APRIL 1960
485
CONCLUSIONS
For the instrument, sample containers, and the phosphor system used, the optimum amount of scintillator solution is 10 to 15 ml. Slight deviations from any established volume within this range will cause negligible error in final calculated radioactivities. It should be possible to measure the selected volume with a graduated cylinder rather than a calibrated pipet. For the internal standard technique of analysis, the ideal ratio of internal standard radioactivity to sample radioactivity would be infinite. Practically, a ratio of 10 to 1 will produce counting efficiencies with about the same expected error as that attributed to the measurement of sample plus internal standard. Experimentally this value was found to be *1.4%. Coincidence losses are not significant below a count rate of 300,000 c.p.m. Careful use of microliter pipets will not introduce a significant error The effect of optical differences in the counting vials described in this paper is insignificant.
Quantities of nonquenching sample of at least 1 ml. can be used (in 15 ml. of scintillator solution) without causing significant loss in counting efficiency. The standard deviation of any single determination of a tritiated hydrocarbon is =k2.0’%’,. Some variables that might affect counting efficiency were not evaluated in this paper. Among these are the effect of dissolved oxygen in the scintillator solution and sample (6) and the effect of voltage-gate settings on the Packard liquid scintillation spectrometer. Preliminary investigations of discriminator settings indicate that counting efficiencies might be increased from 15 or 16% to about 2570 by changing the gate setting from 10 and 50 volts to 5 and 60 volts. The increase in efficiency comes principally from the larger number of pulses included by reducing the lower gate setting from 10 to 5. For reasons of reliable instrument stability, i t was felt that the 10 setting on the lower discriminator would tend t o minimize the error caused by occasional
increases in line noise or by more gradual increases in amplifier tube noise. LITERATURE CITED
(1) Bel!, C.. G., Jr., Haye:! F. S . , “Liquid Scintillation Counting, p. 166, Pergamon Press, London, 1958. (2) Bennett, C. A., Franklin, N. L., “Statistical Analysis in Chemistry and the Chemical Industry,” p. 21, Wiley,
New York, 1954.
(3) Davidson, Jack D., Feigelson, Philip, Intern. J. Appl. Radiation and Isotopes 2, NO. 1, 1-18 (1957). (4) Kallman, H., Furst, hI., Phys. Rev.
79,857 (1950). (5) Pringle, R. W., Black, L. D., Funt, B. L., Sobering, S., Ibid., 92, I582 (1953). (6) Reynolds, G. T., Harrison, F. B., Salvini, G., Ibid., 78, 488 (1950). ( 7 ) Wilson, E. B., Jr., “An Introduction to Scientific Research,” p. 273, McGraw-Hill, New York, 1952. (8) Wilzbach, K. E., J . Am. Chem. SOC. 79, 1013 (1957). (9) Zeigler, C. A., Chleck, D. J., Brinkerhoff, J., ANAL.CHEM.29, 1774 (1957). RECEIVEDfor review July 10, 1959. Accepted January 18, 1960. Work supported in part by the Department of the Army, Ordnance Project TB5-0010C.
Separation of Rhodium from Iridium by Copper Powder G. G. TERTlPlS and
F. E. BEAMISH
University o f Toronto, Toronto, Ontario, Canada
b A method for the separation of rhodium from iridium uses copper as a selective precipitant for rhodium in 1 .ON hydrochloric acid. Both milligram and microgram quantities, 10 mg. to 50 y, of the two metals can b e separated and subsequently determined. Losses of either rhodium or iridium to iridium or rhodium precipitate are negligible and in accordance with the filtrate losses of rhodium and iridium.
T
HIS RESEARCH arises from the lack of convenient precipitants for the determination of rhodium and iridium in a solution of both metals. Until recently, no satisfactory quantitative method of separation was available. Three acceptable methods for the separation of rhodium from iridium and the subsequent determination of these metals have been recorded (4, 6, 14). The titanium(II1) chloride (6) procedure is suitable for macro amounts, although the subsequent removal of excess reagent by cupferron is difficult. The Berman-McBryde method (4) is considered more effective than the antimony (14) method for separating
486
ANALYTICAL CHEMISTRY
microgram amounts of these metals, although there is some difficulty in recovering iridium from the anionic exchanger. The present report deals with the application of copper powder as a selective precipitant for rhodium in 1.ON hydrochloric acid solution of both rhodium and iridium metals in a range from 10 mg. to 50 y and the dissolution of the metals and their separation from copper by cation exchange procedure in forms convenient for gravimetric or colorimetric determination. The optimum conditions for a n acceptable separation of rhodium from iridium in a solution of both metals were studied. These included the volume of the solution, the acidity in the range from 0.1 to 2.ON, the heating time from 15 to 150 minutes, and the temperature from 60” to 9 5 O C. The amount of copper was kept in excess. APPARATUS,
REAGENTS, AND SOLUTIONS
STANDARD
Beckman Model A2 glass electrode p H meter and Beckman Model B spectrophotometer were used for all p H readings and absorption measurements, respectively.
Ion Exchange Columns. Large Column. A borosilicate glass tube 23 cm. in inside diameter was joined t o a draining tube about 5 mm. in inside diameter. The resin bed was 16 t o 17 em. in depth. Small Column. The borosilicate glass tube n.as 1 em. in inside diameter. The draining tube was 4 mni. in inside diameter. The resin bed iTas 5 to 6 cm. in depth. Exchanger. The exchanger m-as Dowex 50X cationic resin in sodium form, of 20 to 50 mesh, supported in the column on a small plug of glass ~vool. The exchanger was regenerated just before use with 3N hydrochloric acid until the eluents n-ere colorless and free of iron and copper, as shown by a spot test of potassium thiocyanate and rubeanic acid, respectively. The excess acid was removed from the exchanger by washing with water until the eluent was neutral to litmus paper. Chlorination Apparatus. A Vycor tube 19 mm. in inside diameter and 50 em. in total length tvith a n elongated end (outlet) t o enable joining to a rubber tube, Jvas heated by a n electric furnace of 9.53 em. in length. Liquid chlorine (Canadian Industries, Ltd.), Thiobarbituric acid (660-Eastman Organic Chemicals) , highest purity.
