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 OF VARIOUS SYSTEMS
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. T h e 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-
*
stitute 62.4 and 85.7% of the count of a mixture of 3.0 ml. of methanolic 1.OM Hyamine hydroxide plus 12.0 ml. of toluene containing 0.5% PPO and O.Olyo POPOP. Vials with solvent counted at a rate greater than the empty vials. For colorless samples, instrument, vial, and liquid together constituted 91.5y0 of the t a p 7 background count rate and essentially l O O ~ , of the t a p 11 background count rate. With increasing depth of color of solutions, the instrument, vial, and solvent contributions to background become a smaller and smaller percentage; for C14 this contribution for the deepest colored solutions observed was 49.3%; for H3it was 80.5%. ’CT’hen activity is present but no scintillator, the lighter samples count considerably above background. The blood digest appears to contain pigments which serve as phosphors. A plot of count rate us. volume of added color solutions shows that the two isotopes are quenched a t rates somewhat different from one another and considerably different from their respective backgrounds. It is obviously improper to adjust the count rate of a background solution for a sample solution of a different color intensity b y a factor obtained b y the addition to the background solution of isotope internal s t a n h d . OPTICAL CHARACTERISTICS
Since internal standard addition was not directly useful for adjustment of background when its color intensity differed from that of t h r sample solution, the counting efficiency of solutions was studied as a function of color intensity. The optical transmission of a blood digest solution containing C14 and H3 isotopcs and their background solutions was measured a t 25-mp intervals (with H20 as reference solvent). K h r n the reciprocal of the ab3orbance a t 400 m p was plotted US. relative efficiency (the count of a color-free sample was taken as 1.000), a linear relationship resultcd for C14, H3, and their respective backgrounds. The data are shown in Figure 1. The pronounced difference in relative quenching of the isotopes and their background is evident. All data fit a straight line well (least squares fit). The wave length ueed for measurement of transmission greatly influences the shape of the 1/A us. relative rate curve. A plot a t wave lengths other than 400 mp gave distinctly curved lines. The sensitivity curve of a Dumont 6292 multiplier phototube is approximately bell shaped in the 300- to 650-mp region with a broad peak a t 435 mp ( 9 ) . An average absorbance was calculated for both isotopes and .their backgrounds from the formula A = ZwiAi/L1wi where the Ai’s are absorbances a t 360, 400, 450, and 500
mp and the w,’s are the relative photomultiplier sensitivities at these wave lengths (0.45, 0.90, 0.97, and 0.76). The plot of 1/A vs. relative efficiency for each isotope gives straight lines more nearly coincident for C14 and H3 than in the original 1/A plot. The two backgrounds give straight lines that do not coincide exactly with each other nor a t all with the isotope curves. Even with this more comprehensive treatment background and isotope behaved differently. These blood digest solutions covered a considerable range of color. The l,/A US. relative efficiency plots appear linear over the entire l/A range dorrn to values of 1.8 to 2.0. These correspond t o solutions with transmission of 27.8 to 31.67,. SAMPLE
AND
BACKGROUND
COCO
I3
20
;3
30
IVABSORBANCE AT 400
mu
Figure 1 . Vaiiation for blood digest solutions of relative count rate with color intensity
CONSTANTS
Since instrument, vial, and solvent contribute so largely to background, subtraction of some constant from the observed sample count rate and subtraction of some other constant from the observed background rate, might leave a residual rate for background and sample that was quenched alike. This would permit the use of the usual internal standard technique for adjustment of sample and background solutions to the same color intensity. Background and isotope constants necessary for this condition were determined as follows. Algebraic constants were subtracted from each observed C14, H3, C14 background, and H3 background rate. Relative rates, in terms of the first (colorless) member of the series were then expressed by the C14,H3, C14 background, and H3background series. The equation of the straight line (least
squares line) fitting the 1/A us. relative rate data was obtained for each series. (The intercepts and slopes were expressed in terms of the algebraic constants already used.) The intercepts of the C14 and C14 background curves were equated; the slopes of the CI4 and C14 background curves were also equated. Solutions of these two simultaneous equations gave values of the constants to be subtracted from observed sample and background rates t o make the residual quenching rate equal. An exactly analogous procedure !vas carried out for H3 isotope and background. The constants obtained are given in Table I, columns 2 and 3, lines 1 and 10. The background constants are equal within experimental error, to the clear background rate, for both isotopes. The isotope constant. for both isotopes, is of nearly the same value
Isotope and Background Constants for-C14 and H3 in Various SolventColor Systems Clear Clear System Isotope Background Isotope Background
Table I.
C’4 Blood digest Methyl red 1
2 3 4 Bromothymol blue 1 2 3 4
33734
42.1
33041
42 . 0
29692 11622 1948 414.3
47.6 47.6 48.3 47.9
29674 11635 1917 411.5
4i.6 47.6 47.6 47.6
45727 8957
33.7 33.7 32.0
47998 9573 9732 263.4
34.4 34.4 34.4 34.4
63813
91.3
64173
91.5
48169 11350
82.3 82.1
48187 11398 888.5 279.1
82.3 82.3 82.3 82.4
23666 5815 483,6 82.2
82.5 82.5 82.5 82.5
214,6
H3
Blood digest Methyl red 1 2 3 4 Bromothymol 1 2 3 4
279.4 23974 477.1 176.8
82.4 82.8 82.1 82.2
VOL. 32, NO. 11, OCTOBER 1960
1469
I
lor
I
r
l o r
r
V
050-
?
d
040-
03023700 C/M 9570 C I M A 5820 G'M 970 C/M 7 48oG'M 263 C/M 83C/M BACKGROUND BKKGROUND 48000 C/M
OM0 100 ;
0
IO
20
30
. 10 J 20 30
0
40
VABSORBANCE
AT 400
mu
Figure 2. Variation for brornothyrnol blue solutions of relative count with color intensity
as the isotope count in the colorless solution, METHYL RED SOLUTIONS
A set of methyl red-toluene solutions representing four different levels of added C14 isotope and H3 isotope, as well as background, was prepared by the addition of various amounts of a saturated toluene solution of methyl red to five sets of solutions composed of 10.0 nil. of toluene containing 0.5% PPO and 0.01% POPOP. Carbon-I4 activity a t four different levels was then added to four of the sets. A similar group of solutions was prepared with added H3 activity. The solutions were counted repeatedly for IO-minute periods at taps 7 and 11, respectively. When the relative rates for the 10.0 to 100.0-channel are plotted us. 1/A (at 400 mp), straight lines are obtained for 1/A>2.00. The background and isotope constants are shown in Table I, lines 2 through 5 and 11 through 15) columns 2 and 3 . The background constants are close to the clear background for this solvent-color system. The isotope constants are nearly equal to the count of the isotope in a clear solution. BROMOTHYMOL BLUE SOLUTIONS
The solvent consisted of a mixture of methanol, dioxane, toluene, naphthalene, PPO, and POPOP, hereafter called diotol (6). It is a variation of a counting solution proposed by Kinard ( 7 ) . It was made alkaline with aqueous sodium hydroxide and to the corresponding samples of five sets were added various amounts of concentrated aqueous bromothymol blue solution. Carbon-14 isotope a t four levels was added to four of the sets. A similar group was prepared and received H3 activity. Samples were counted repeatedly. The 1/A us. relative count rate curves for the 10.0- to 100.0-channel are shown in Figure 2. The curves fit a straight line well down to 1/A's of a t least 2.00. 1470
ANALYTICAL CHEMISTRY
The background constant, is nearly equal to the clear background for this system. The isotope constants decrease with the isotope level for both isotopes and agree ~vellwith the clear solution count except for the lon-est level of tritium. The calculations for this sample involved very small differences. These constants are shown in Table I, lines 6 through 7 and 15 through 18, columns 2 and 3 . COUNT RATIO METHOD FOR QUENCH DETERMINATION
The ratio of a sample's count in two channels has been suggested by Davidson ( 2 ) and Baillie ( 1 ) as a method for determination of quenching. This method was applied to the methyl red solutions and to the bromothymol blue solutions- previbusly eiamined. For the methyl red set. channels were 10.0 to 35.5 and 35.5 to 100.0. At high voltage taps 7 and 11 each channel contained half of the count observed in the 10.0- to 100.0-channel for the highest isotope level color-free solution for CI4 and H3, respectirely. The bromothymol blue set was counted with channels 10.0 to 28.0 and 28.0 to 100.0. These settings split the colorless solution of highest 1Pvel isotope count equally. The data for bromothymol blue are given in Figure 3. The plot shows the relative rate for the 10.0- to 100.0channel (relative to that of the clear solvent as 1.000) us. the ratio of counts in the 10.0-to 28.0-channel to that in the 28.0- to 100.0-channel. Different levels of CI4 isotope are quenched almost identically. Isotope and background quenching are very different. The different H3 levels and background are quenched similarly but not identically. For the methyl red-toluene set, different C14 levels are quenched similarly but not identically. Isotope and background differ slightly. For all colorsolvent systems studied, the relative
LOWER CHANNEL R A T E I U P P E R CHANNEL RATE (IO 0-23 5)/(235-1000) (100-280)/ (280-1000)
Figure 3. Variation for brornothyrnol blue of relative count rate with channel ratio
count rate us. ratio curves are straight lines to relative rates of 0.5 or lower. DISCUSSION
If sample activities are much greater than background, or if the change in counting efficiency of the background with color is small, differences in color between sample and background solutions can be neglected Ivith a resultant small error. If sample activities are low and the rate of change of background efficiency with color is great, compensation must be made for variation in color of background and sample solutions. By both the optical method and channel ratio method the rate of change of background efficiency varies greatly from one solvent-coloL system to another. I n some cases it changes in the same way as isotope rate; in others it is considerably different. The change of isotope efficiency with color varies from o>e solvent-color system to another and to some extent for different isotope levels within the same system. Both the optical method and the channel ratio method permit adjustment of background rates for color differences between sample and background solutions. Both require a previously determined standard curve showing for a given solvent system the change of background rate with absorbance or channel ratio. This is done as follows. Prepare a solution of the clear solvent and of solutions of several color intensities. Determine the count rate of these and their absorbance a t 400 my. Construct an efficiency us. 1 A curve. This permits selection of the count rate of a background solution of the same color intensity as the sample. Both methods can also be applied to determination of counting efficiencies of isotopes without contamination of the samples by internal standard addition. This requires standard curves for several levels of the isotope in the
givc.11 solvent system. Since for both inetliods relative efficiency is a linear function of the observed variable (absorbance or channel ratio), only a few observations are necded once the appropriate conditions (wave length or discriminator sett,ings and high voltage) :ire determined for this linearity. It is necessary that solutions be counted a t that high voltage which gives thc m:tsimum count rate for the particuhr isotope in the uncolored solvent +,in. ;Is the voltage departs from t h k peak value, t’lie 1/A us. relative c>tficieric,ycurve departs from linearity m ( 1 exhibits a niasimuni. -4 serious dimdvantage, for lower .level counting. of the channel ratio met’hod, is t h a t since a low count rate is split into two rhnnncls thc count ratio of which is needed) a slight drift in the voltage of the middle discriminator will change the ratio grcatly. A combination of the usual iritmial standard technique wit’h the optical (or channel ratio) niethodoptical nietliod for background and intcmal standard method for isot’opeis quicker than eit,lier separately since fewer standard curves are required. ‘rlle data of Table I show that there is no single constant t’hat can be substracted from all count’s, sample and background. to make their quenching curves c>oincident. The isotope constant. varies with the isotope level; it is approximabcly numerically equal t o thc sample count in t’he same solvent systcm without added color, a number Ivhich is itsplf a n unknown quantity. S o practically useful application of this portion of the experiment is evident. ACCURACY AND PRECISION
The accuracy and precision of background adjustment by the optical mcthod depend on the counting time of thcl various solutions and on instru-
Table II.
Net Count Rate of Colored Solutions Containing C’* with Various Adjusted Backgrounds
Cnknown
Background 1 Background 2 Background 3 Recovery, %
260 270: 271 271 i 1 ITc 97.5
ment stability between time of determination of standard curves and time of sample counting. Inforniation about these factors is given by an experiment vAth bromocresol green in solutions containing toluene, ethyl alcohol, concentrated ammonium hydrolidc. PPO, and POPOP. Standard curves were determined for CI4 isotope and the corresponding background. Three unknowns all containing the bame amount of radioactivity but different amounts of color, Lvere prrpared and counted; three background., all of different color intensity and all different in color intensity from the unknowns, were prcpared and counted. All solutions were counted Fith channels 10.0 to 40.5 and 40.5 to 100. Unknowns and their backgrounds ere counted once for 10 minutes. Both isotope and background relative efficiency os. 1/A plots lvere linear over the whole range of color, and differed greatly in slope from each other. For H 3and its background, the standard curve plot of relative efficiency us. channel ratio was nearly a vertical line (slope 542 for H3) so that this procedure was not useful. d b sorbanees of all solutions were measured a t 400 mp. F e t count rates were calculated with each of the three backgrounds adjusted by the optical method to the color intensity of each of the three bamples. The results are shonn
-
2
3
288 280 294 287 ==! 1 4 7 103 2
283 276 292 284 =!= 1 6 c c 102 1
1
in Tablc 11. The precision for earh sample is good n-hatever background was used. The over-all accuracy is also good for the counting time employed. ACKNOWLEDGMENT
The author thanks C. N. Rice of this laboratory for valuable discussion and advice during this investigation. LITERATURE CITED
(1) Baillic, I,. -A,,
“Determination of Liquid Scintillation Counting Efficiency by Pulse Height Shift,” submitted to
Intern. J . I l p p l . Radiation and Isotopes. (2) Davidson, J. D., “Liquid Scintillation Counting,’’ p. 95, Pergamon Press, S e w York, 1958. (3) Donicr. F. R., Hayes, F. S.,Nzcdeonics 18, 100 (1960). ( 3 ) Eisenherg, F., Jr., “Liquid Scintillation Counting.” p. 123, Pwgarnon Press, New l-ork, 1958. (5) Guinn, T’. P., Ibid., p. 176. ( 0 ) Herberg, R. J., ASAL. C H E X 32, 42 (1960). ( i )Kinard, F. E., Rev. Sci. I n c f r . 2 8 , 293 (1957). (8) Passmann, J. M., Radin, S . S , Coouer. J. A. D.. AKAL. CHE3f. 28, 484 jl950:). (9) Swank, R. K., “Liquid Scintillation Counting,” p. 29, Pergamon Press, Sew York, 1958.
RECEIVEDfor review April 4, 1900. Accepted August 8, 1960.
Elimination of Anion Interferences in Flame Spectroscopy Use of (Ethylenedinitri1o)tetraacetic Acid A. C. WEST’ and W. D. COOKE Baker Laboratory, Cornel1 University, Ithoco, N. Y.
A method has been devised for eliminating anion interference in flame spectroscopy. (Ethylenedinitri1o)tetraacetic acid, when added to the solutions being analyzed, enhances emissivity and maintains a constant level of intensity which is independent of the anion present. Elimination of interferences of a variety of anions includ-
F -
L A M E SPECTROPHOTOMETRIC METHODS have been used for the deter-
ing phosphate and sulfate was found for calcium, magnesium, cobalt, copper, chromium, and manganese, some of the exDerimental variables in flame
mination of a \vide variety of metals a t low concentrations. Their general
spectroscopy have been examined and an instrument is described which has an absolute stability of 2% over a period of 2 months.
Present address, Department of Chemistry, iyilliams college, ~ ~ i l l i a m s t o , ~ m , Mass.
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1
VOL. 32, NO. 1 1 , OCTOBER 1960
1471
