Loss of 32P-Phosphate and 14C-Carbonate Activity During Liquid Scintillation Counting of Aqueous Samples Walter C. Weimer,’.’ Max G. Rodel,* and David E. Armstrong Water Chemistry Program, University of Wisconsin, Madison, Wis.
Loss of radioactivity in excess of natural decay was observed in aqueous samples of 32P-P04-3 and 14c-co3-2 counted by liquid scintillation in a dioxane-base counting solution. Loss of 32P-P04-3 activity was increased by the presence of the HC03- ion and ranged up to 23% in 8 hr and 46% in 4 days. Common solubilizing agents were ineffective in preventing 32P-P04-3 loss and only partially effective in preventing 14c-co3-2 loss. The 32P-Po4-3activity was recovered by shaking and recounting the sample, but 14c-co3-2 activity was incompletely recovered. Settling of insoluble materials within the counting solution apparently was responsible for 32P-Po4-3 activity loss. A dual mechanism of precipitation of 14c-co3-2 and volatilization of 14C-C02 was responsible for 14C loss. Acidification of the counting solution prevented 32P-P04-3 activity loss; addition of phenethylamine to the counting solution prevented volatilization of 14C-C02, but no treatment was precipitation. found to prohibit 14c-co3-2
During a recent investigation (1) involving the liquid scintillation counting of aqueous samples containing radiophosphorus, it was observed that samples recounted within several hours to a few days after the initial counting exhibited a greater decrease in measured activity than predicted by radioactive decay. Inorganic 32P-Po4-3in filtered lake water, distilled water, synthetic lake waters, and 0.375N and 0.750N HCOONH4 solutions and 32P-and 33P-labeled inositol hexaphosphate in 1.50N HCOONH4 solutions exhibited this accelerated decrease in measured activity. Since these samples were prepared in a commonly used dioxane-base liquid scintillation solution [a modification of the solution described by Bruno and Christian (2)], this phenomenon could have serious implications in laboratories where samples are stored prior to counting. Such an activity loss, which apparently varies markedly with samples of different composition, might invalidate much of the data obtained when utilizing 32P-Po4-3 in aqueous environmental samples. Although a similar phenomenon was reported previously ( 3 ) , the problem is not generally recognized by those performing environmental research. Mueller ( 3 ) postulated that the loss of activity in aqueous samples was caused by phase separation and precipitation of the radioisotope within the counting vial, but no preventive measures were suggested. In this investigation the loss of 32P-Po4-3 and I4CC03-2 activity was evaluated. These radioisotopes are widely used in aqueous environmental chemistry investigations. The effects of several chemical parameters on 32Pactivity loss, and the effects of several amending reagents on the rates of loss of 32P-P04-3 and 14c-co3-2 activity were investigated. Determination of the factors controlling activity loss and evaluation of the effectiveness of potential preventive measures were emphasized. Present address, Battelle-Northwest, P.O. Box 999, Richland, Wash. 99352. Present address. Bechtel Cora. P.O. Box 3965. San Francisco. Calif. 94119. 966
Environmental Science & Technology
Materials and Methods The liquid scintillation solution (cocktail) used for counting (Table I) was a modification of that described by Bruno and Christian (2) for use with aqueous samples. Dimethyl POPOP was obtained from the Packard Instrument Co., Inc., and ethylene glycol monoethyl ether (ethyl cellosolve) was a purified grade obtained from the Fisher Chemical Co. All other cocktail components were Fisher “Scintanalyzed” reagents purified specifically for liquid scintillation use. Organic reagents used for cocktail modification studies (Table 111) were obtained from Packard. A sample prepared for counting (hereafter designated as the counting solution) consisted of 10 ml of cocktail, 3 ml of 1,4-dioxane, and 2 ml of aqueous sample. All samples were counted in a Packard Tri-Carb Model 3320 Liquid Scintillation Spectrometer. Initial activity of the sample generally was adjusted to be IO5 to IO6 counts per minute (CPM) to minimize both the time required for counting and statistical variations in estimations of the disintegration rate. Since the efficiency of the counting solution varied between samples, each sample was corrected to a standard counting efficiency through the use of the automatic external standard (AES) procedure ( 4 ) . A quench curve of AES CPM vs. counting efficiency was prepared using nitromethane as the quenching agent. Both lake water samples and distilled water samples were evaluated. The lake water samples were obtained from Lake Wingra, a hard-water lake in Madison, Wis. All lake water samples were filtered through 0.45 pm membrane filters prior to use to remove particulate matter and biological activity. The pH of the lake water samples was approximately 8.5. Experimental Results 32P-P04-3 Counting in Aqueous Systems. The accelerated decrease in measured 32Pactivity was initially observed during the liquid scintillation counting of 32P-and 33P-labeled inositol hexaphosphate in 1.5N HCOONH4. All subsequent experiments involved the use of inorganic 32P-P04-3 in various aqueous samples. The rates of the decrease in measured 32Pacitivity (in excess of that due to radioisotope decay) for two filtered lake water samples and a distilled water sample are shown in Figure 1.Over an 8-hr period, lake water Sample 1 exhibited a decrease in measured activity of approximately 23% in excess of that due to decay. The rate of loss of activity differed considerably for the two lake water samples and was slower for the distilled water samples. This variation in the rate of activity loss between comparable samples prohibits the use of an “activity loss vs. time” standard curve to correct the activity measurements. Accompanying the decrease in measured activity in the 32P energy channel of the scintillation counter was a corresponding increase in the activity measured in a channel set to observe a lower energy isotope, 14C. This type of shift suggests the occurrence of quenching within the sample ( 4 ) . However, the efficiency of the counting solution, as evaluated by the AES count rate, remained unchanged for both channels, verifying that the fluors in the cocktail were not being degraded and that quenching was not occurring.
I
The lost activity was generally recovered in the 32Pchannel by a vigorous shaking and recounting of the sample vial. This behavior suggested that the radioisotope was concentrated in the lower portion of the counting vial. Examination of several sets of samples verified that the loss of activity was common to all solutions containing radiophosphorus. Several different batches of cocktail were evaluated; all exhibited the same characteristics. Several batches of 32P-P04-3stock solution were also investigated, and all exhibited similar patterns of radioactivity loss. These results confirm that the observed radiophosphorus behavior was not due to a single batch of faulty reagents. Therefore, several sets of experiments were performed to evaluate the mechanism(s) by which the 32Pactivity was apparently lost. Effect of Cocktail Components on 32P-P04-3 Counting. Several types of cocktails were used to count 32PPo4-3in both filtered lake water and distilled water samples to evaluate the effect of specific cocktail constituents on the measured rate of activity loss. These formulations included solutions in which either dimethyl POPOP, PPO, napthalene, or ethyl cellosolve was omitted. These modified cocktails were mixed with the proper ratios of dioxane and aqueous sample containing 32P and then placed into folin tubes and allowed to remain undisturbed a t room temperature. At selected time intervals, 2-ml aliquots were withdrawn from the surface of the folin tube solutions, added to a counting vial containing 10 ml of regular cocktail and 3 ml of dioxane, and counted immediately. After 10 days, the folin tubes were sampled both before and after a thorough mixing of the contents.
Table I. Composition of Liquid Scintillation Solution (Cocktail)a Component
Concentration
10.5 g/I
PPO
(2,5-Diphenyloxazole) Di-methyl POPOP [ 2,2-p-P h en y I ene b is (4-met h y I 5-phenyl)oxazole] Naphthalene Ethyl Cellosolve (Ethylene glycol monoethyl ether) So1vent:dioxane. a M o d i f i c a t i o n of B r u n o a n d C h r i s t i a n sol u t i o n .
0.45 g/I
75 911 200 ml/l
( 2 ) liquid scintillation
221
.B 6'O: 0
..-
a t 12
I
24
I
36
Elapsed
48
60
,
72
I
1
84
96
Time ( h r )
Figure 1. Loss of 32P-P04-3 activity (corrected for radioactive decay) during liquid scintillation counting
Similar patterns were observed for the measurements of the activity in all of the lake water samples (data not presented). At least 80% of the measured 32P activity was lost from the lake water samples during the first 22 hr. The greatest loss, 89%, was seen for the scintillation solution in which the ethyl cellosolve had been omitted. All samples continued to lose activity at comparable rates until approximately 6% of the initial activity remained in solution after 10 days. After thorough mixing of the folin tubes on day ten, virtually all of the 32Pactivity added initially was recovered. The distilled water samples exhibited considerably less loss of 32Pactivity than did the lake water samples (data not presented). During the first 22 hr there was 5.5 to 6% activity loss (approximately 2% more than loss due t o isotopic decay) for these samples. After 10 days, 42 to 46% of the initial activity had disappeared from solution (isotopic decay accounted for 39% of this activity decrease), with the greatest decrease shown by the distilled water sample prepared with the unmodified cocktail. Samples examined after a thorough mixing on day ten exhibited the theoretical counting rate. The behavior of the lake water samples during the first 24 hr of this cocktail modification study suggested that the major component of the cocktail, the dioxane solvent, enhanced the 32Pactivity decrease. T o evaluate the effect of dioxane, folin tube samples of lake water and distilled water containing 32P-P04-3were prepared and mixed with dioxane. The ratio of aqueous sample to dioxane was the same as that used in a total cocktail sample. Samples were withdrawn after various time intervals and counted. The distilled water samples exhibited a decrease in activity equivalent to isotopic decay only. However, the lake water samples showed a greatly enhanced decrease in measured activity. After 27 hr, only about 5% (corrected for decay) of the initial activity remained. At the end of 1week, less than 1% of the added activity remained. All of the added activity was recovered, however, when a thoroughly mixed sample was counted. Samples containing distilled water and 32P only or filtered lake water and 32Ponly were also prepared as controls. The controls showed no activity decrease in excess of that due to isotope decay. Apparently, the dioxane in the counting solution enhanced the decrease in measured 32Pactivity for the aqueous samples containing lake water. Effect of Electrolytes on 32P-P04-3 Counting. The observation that filtered lake water samples showed a more rapid loss of activity than distilled water samples suggested that the activity loss was related to ionic strength. To examine this possibility, several synthetic waters encompassing a range of ionic compositions and concentrations were prepared. The loss of activity observed for most of the waters was slight and showed only minor relation to ionic strength (Table 11). The NaCl, CaC12, CaS04, and HCOONH4 solutions exhibited small losses. In the Na2S04 system, some loss of activity was observed, but duplicate runs reproduced poorly. Because of Na2S04 sample of intermediate ionic strength showed the greatest activity loss, no clear correlation with ionic strength was demonstrated. In contrast to the C1- and so4-2systems, the NaHC03, KHC03, and Ca(HC03)z systems exhibited a definite relationship between salt concentration and both the rate and the amount of activity loss (Table 11). The more concentrated solutions displayed the most rapid and complete activity loss. Although there was a difference between the effects of the NaHC03 as compared to the Ca(HC03)z and KHC03 samples, no other data suggested Na+ to be a cation of special significance. The salt concentration relationVolume 9, Number 10, October 1975
967
~~~
Table II. Effect of Ionic Strength on szPActivity Loss Activity remaining, "/.,a after time intervals of ~
Sample
Filtered lake water, 540 pmholcmb Acidified filtered lake water Distilled water 0.034M NaCI, 4000 pmho/cmb 0.0034M NaCl 0.00034M NaCl Acidified 0.034M NaCl 0.004M CaSO 0.0004M C a s 6 Acidified 0.004% CaSO, 0.04M CaCl 0.004M C a d 0.0004M C a d Acidified 0 . 0 4 h CaCI, 0.75M HCOONH, 0.3M HCOONH Acid if i ed 0 . 7 5 d HCOON H, 0.4M N a ,SO 0.04M Na,S6 0.004M Na,S6 0.0004M N a S 6 Acidified 0.dM da,SO, 0.04M NaHCO,, 4000 pmho/cmb 0.004M NaHCO 0.0004M N a H C d , Acidified 0.04M NaHCO, 0.04M KMCO 0.004M K H C d 0.0004M K H C 6 , Acidified 0.04M KHCO, 0.004M C a ( H C 0 ) 0.0004M C a ( H C 6 3 , Acidified 0.004M Ca(HCO,),
~
4 days
4 daysshakenc
70
54
98
99 99 99
99 99 99
99 88
98 97 98
99 98 100 100 98 99 99 99 99 99 99 99 101 93 97 97 100 98 72
98 97
6 hr
24 hr
92
99 99
100
98 100 100 100 99 99 100
91
97 99 95 96 100 90 98 89 100 101
93 94 100 97 95 100 93 93 87 101 87 71 101 91 98 81 99 98
65
58
99 101 99 96 98 97
79 91
100
98 98
100
99
100
100
97 97 100
100
81 90 89 100 86 90 99
99 98
100
98 99 100 97 98 99 99 94 93
100
99 100 99 100 99 98 97 99 100 93 95 97 100 95 98 100
a Percent o f t h e o r e t i c a l value at each elapsed t i m e . Specific conductance. F o u r - d a y samples shaken p r i o r t o r e c o u n t i n g .
ships observed indicate that the effects of specific anions were more significant than those of specific cations. Filtered Lake Wingra water, which exhibited the greatest activity loss after four days of all the samples evaluated, is normally saturated with CaC03. Because of the behavior of this lake water sample and of the other HC03- salt solutions, apparently the potential loss of 32P activity was related to the presence of HC03- in a water sample. 14c-co3-2 Counting in Aqueous Systems. The behavior of the aqueous radiophosphorus samples in the dioxanebase cocktail posed questions concerning the behavior of other radioisotopes that are used in similar samples. Beform is a widely used isotope in cause 14C in the 14c-co3-2 water chemistry research, an investigation of the behavior of 14c-co3-2in the previously described liquid scintillation solution was conducted. Unstoppered folin tube samples containing only 14CC03+, filtered lake water, and dioxane, were prepared and sampled as described in the radiophosphorus experiments. Within 24 hr, 73% of the added activity was lost; 97% was lost within 3 days. After 14 days only 1%of the original activity remained in solution. A sample taken after thorough mixing of the tubes indicated that 30% of the original activity was recovered. This low recovery was in sharp contrast to the complete recovery of 32P activity by thorough mixing. 968
Environmental Science & Technology
The rates of 14C activity loss from four replicates of lake water and distilled water samples prepared as total counting solution samples are shown in Figure 2. The error bars represent the range in values for the four replicates. Vigorous shaking of the vials a t the conclusion of the experiment had no effect on the activity of the distilled water samples. For the lake water samples, however, shaking increased the observed counts by 2 to 20%. These results suggest two mechanisms of activity loss for lake water samples and a single mechanism of loss for the distilled water samples. Effect of Additives on 32P-P04-3 and 14c-co3-2 Counting. Several amending agents for the liquid scintillation cocktail were evaluated to determine their effectiveness in retarding or prohibiting the accelerated decrease in measured 32P activity. Table I11 is a listing of the various reagents evaluated and a general interpretation of their effects. The addition of acid to the cocktail (Table 111) or directly to the counting solution (Table 11) in the amount of 3 meq H+/1 of solution prevented the loss of activity from filtered lake water and synthetic water samples. The addition of acid had the slightly negative effect of reducing the counting efficiency by approximately 5%. Long-term storage for periods of a month allowed further degradation of the counting solution and further decreased the counting efficiency. However, the loss of counting efficiency was less than 10% even after extended storage. The addition of carrier inorganic phosphate (40 wg of Po4-3-P in 15 ml of total solution) did not affect the loss of activity (Table 111). This addition represented a large quantity of stable P (2.67 mg/l) in comparison to the amounts of radiophosphorus present. Consequently, the added P04-3-P would have altered considerably the sorption of 32P-P04-3 if sorption were the factor causing the loss of 32P.Because the addition of carrier inorganic phosphate did not affect the loss of activity, sorption was apparently not the controlling process. Several other additives commonly used in cocktail preparations were also evaluated for preventing loss of 32P activity. The majority of these additives had no beneficial effect; some produced distinctly deleterious effects. The selection of most of these specific agents was based upon information from previous investigations. The use of methyl alcohol in place of ethyl cellosolve was suggested by the composition of a commonly used cocktail (5). Triton X-100 and Hyamine Hydroxide (both registered trademarks of Rohm and Haas, Inc.) are often used to solubilize materials in the counting solution (6). These agents were generally
a
l To
, I
,
, 3
2
,
4 US
r
0
1
2
3
4
,
6
,
8
ad
wt tor
7 8 k h mhr
Elapsed
Time
s
Flgure 2. Loss of ''C-C03-'
ing
,
5 6 7 b &Him -tar
,
,
10
S
#
11
8
12
t
1
8
,
I3
14
I
vlmprs
9
nwia
10
I1
12
13
14
Is
(Days)
activity during liquid scintillation count-
Table 1 1 1 . Evaluation of Effect of Additives to Liquid Scintillation Solution on 32P Loss Type of
aqueous Reagent
Sarnplea
Effect observed
Methyl Cellosolve replaced by methyl alcohol Triton X-100.added to counting vial in place of a n equivalent volume of cocktail solution; 1 drop, 10 drops, 1 ml, 3 ml, or 5 ml per vial Hyamine Hydroxide added to counting vial in place of a n equivalent volume of cocktail solution; 1 mi or 3 ml per vial Hyamine Hydroxide added as above Carrier inorganic P; 2 pg or 40 pg per vial 1 meq H+ per liter of cocktai I solution 3 meq H+ (or more) per liter of cocktail solution
D.W.
Accelerated decrease in 32Pactivityb Accelerated decrease in 32Pactivityb
L.W.
L.W.
Accelerated decrease in 32Pactivityb
D.W.
No accelerated decrease in 32Pactivity
L.W.
Accelerated decrease in 3 2 Pactivity Accelerated decrease in "P activity Accelerated decrease in 3zPactivity prohibited
L.W. L.W.
a L.w.., f i l t e r e d , caicareous lake w a t e r ; D.w., distilled w a t e r . ~ " A c celerated decrease" indicates t h a t c o u n t e d a c t i v i t y decreased a t a m o r e rapid rate t h a n predicted b y radioactive decay.
ineffective. Although the Hyamine Hydroxide prevented the loss of additional activity from the distilled water samples, the fluorescent characteristics of the counting solution were slowly destroyed, occasionally within 4 days after preparation. Two reagents were evaluated for effectiveness in prohibiting or retarding the loss of 14c-co3-2activity. The reagents, phenethylamine and Hyamine Hydroxide, are used to trap gaseous 14C02 for liquid scintillation counting (7, 8). Two milliliters of either phenethylamine or Hyamine Hydroxide (replacing 2 ml of the cocktail) were added to four replicates of 14c-co3-2 in distilled water or lake water in counting vials. These samples were counted a t designated time intervals for 10 days. The phenethylamine was an effective additive; no activity loss was observed for the distilled water samples during the 10 days. However, some loss of activity was observed for the lake water samples (Figure 2). The Hyamine Hydroxide was partially effective, but decreased the counting efficiency of the cocktail. No activity loss within 10 days was seen for the distilled water samples containing Hyamine Hydroxide. Some loss of activity, however, was observed from the lake water sample containing Hyamine Hydroxide (Figure 2). After day five, the sample could not be counted because of low counting efficiency.
Discussion Mechanism of 32P-P04-3 Loss. Generally when 32PPo4-3 loss occurred, vigorous shaking of the container recovered the lost activity. This behavior was observed initially in the counting vials and confirmed in the folin tube experiments. The loss of activity was apparently not due to phosphate sorption by the glass vial since the extent of loss was not affected by addition of significant quantities of carrier inorganic phosphate. Added inorganic phosphate would decrease the proportion of 32P-phosphate sorbed. Further, shaking of the vials would not cause phosphate desorption from the glass to account for the recovery of activity upon shaking. This evidence, shifts in the energy spectrum, and counting geometry considerations indicated
that the loss of 32P-Po4-3activity was due to physical settling of solid radiophosphorus to the bottom of the counting vial. Apparently, the mixture of the aqueous sample with dioxane-base cocktail stimulated gradual precipitation of nonvisible particulate matter within the counting vial. Phosphate salts may precipitate directly, but the evidence obtained using specific salt solutions indicated that other less soluble ions, such as carbonate species, accelerated the formation of a solid phase. The radiophosphorus loss apparently was enhanced by the tendency of phosphate to be sorbed by fine particles. Several samples that had exhibited the typical loss of 32P activity were vigorously shaken and then filtered through nylon filters having a 1-w pore size. From 18-95% of the theoretical activity was retained by the filters; approximately 1%of the activity remained in the unrinsed scintillation vials. The remainder of the activity was recovered in the filtered effluent. In comparison, when samples of lake water containing 32P-P04-3 were filtered through these nylon filters, only 2-3% of the total activity was retained by the filter, confirming that the activity removed from the complete samples was particulate in nature and was not due to 32P-P04-3 sorption onto the filters. Additional information concerning the rate of formation of the precipitate was obtained by filtering complete samples within 15 min after the 32P-P04-3-containing aqueous samples were mixed with the cocktail solution. Variable proportions of the total activity (65-95%) in the samples were removed by filtration. Apparently precipitate formation begins immediately upon mixing of the aqueous sample with the cocktail solution. Lost phosphorus activity was recovered by resuspension of particulate matter and the associated 32P-Po4-3. This resuspended precipitate resettled more rapidly than during the initial settling because a time lag for particle formation and agglomeration was not required. Figure 3 shows loss of' activity following the shaking of a sample that had been previously incubated and exhibited a loss of activity (approximately 40-45% beyond that due to decay). A comparison of this loss of activity curve (Figure 3) with those presented in Figure 1shows that the loss of activity after incubation and shaking was considerably more rapid than the initial loss. The shape of this curve (Figure 3) suggests that the loss of the 32Pwas due to the settling of particulate material. Counting geometry suggests that the maximum activity loss through settling within the counting vial should be 45-50%, as observed.
:I40t rn
Data ia for a rinqk 32P-PQ
To
2
4
atandard Iolutian in dlatllid
6
8
10
e.
I
I2
Elapsed Time ( h r ) Figure 3. Loss of 32P-P04-3 activity during liquid
scintillation counting after allowing the counting solution to equilibrate in a quiescent vial for one month and then shaking the sample prior to counting Volume 9, Number IO,October 1975
969
The sbrption-settling hypothesis was supported by the activity was lost in an acidiobservation that no 32P-Po4-3 fied counting solution (Table 11). Possibly, the postulated precipitate does not form under acidic conditions; this would be the case if carbonates were involved. Furthermore, phosphate is less readily adsorbed a t low pH because of the decreased charge density on the phosphate ion. Apparently, in the acidified solution, sorption of 32P-Po4-3 was prevented, and the radiophosphorus remained in solution. Mechanisms of 14C-C03-2 Loss. Observations of 14C loss indicated that two different mechanisms may have removed 14c-co3-2activity from the total sample preparation. The dominant mechanisms of loss (the sole mechanism observed in distilled water systems) was prevented by the addition of a COz-trapping agent, whereas the other mechanism was not affected. These results suggest that the major loss of activity was due to the volatilization of l4CO2 from the total sample preparation into the air space in the sample vial, apparently caused by a low solubility of COS in the counting solution. Investigations employing vials completely filled with a total sample of lake water, cocktail, and 14c-co3-2(maintaining a normal ratio of aqueous sample to cocktail) confirmed that this filling procedure reduced the ultimate extent of 14C loss and prohibited the loss entirely for eight days. The delay in activity loss for these samples was probably due to complete inhibition of COz volatilization and a slow development of the second mechanism of removal. This process apparently involved precipitation as observed in the radiophosphorus experiments. In this case, precipitation likely involved the 14c-co3-2 species directly. The proposed mechanisms of 14C removal from the counting solution involve 14C in the carbonate form (or l4CO2). Measurements of photosynthetic 14c-co3-2 fixation, a common environmental application involving 14C counting, generally do not involve the counting of solutions Instead, the amount of 14C incorpocontaining 14c-co3-2. rated into the algal cells during the photosynthetic process is measured by disrupting the cells in the liquid scintillation solution and counting. Data collected in this laboratory indicate that under experimental conditions utilizing dense algal cultures, some of the cellular 14C may not dissolve in the scintillation solution. When this occurs, a timewise decrease in the 14C activity occurs due to settling of the algal cell fragments. Under normal culture conditions, all of tha algal cell components are apparently dissolved in the scintillation solution, and the radioactive products of photosynthesis do not settle from the sample or escape through volatilization. The loss of 14c-co3-2 may be significant during the storage of aqueous 14c-co3-2. Therefore, caution should be exercised during the preparation and counting of all solutions of 14c-co3-2.
970
EnvironmentalScience fi Technology
Summary and Conclusions This investigation involved the evaluation of some problems that may arise during the liquid scintillation counting of radiophosphorus or radiocarbon activity in environmental aqueous samples prepared in dioxane-base counting solutions. Apparently, settling of nonvisible material removed s2P activity from solution in samples containing 32P-Po4-3.The cocktail solvent, 1,4-dioxane, was the cocktail component causing the formation and settling of particles. This phenomenon was observed in both distilled water and lake water, but the presence of carbonates and an increased ionic strength of the aqueous sample generally accentuated the loss of activity. The “lost” 32Pactivity was recovered by a vigorous shaking of the sample followed by immediate recounting. The loss of activity was completely prevented by the addition of 3 meq of H+/l to the cocktail. A similar loss of radioactivity was observed for counting Samples of lake solutions containing aqueous 14c-co3-2. water exhibited a small 14C activity loss apparently due to settling. The major amount of 14C loss in both lake water and distilled water samples appeared to involve an irreversible volatilization of 14C02. Addition of phenethylamine prevented the volatilization of 14C but was ineffective in preventing the loss of activity attributed to settling. No technique for long-term preservation of 14c-co3-2 samples was found. Acknowledgment The authors appreciated the cooperation of the University of Wisconsin Engineering Experiment Station. Literature Cited (1) Weimer, W. C., PhD Thesis, University of Wisconsin, Madi-
son, Wis., 1973. (2) Bruno, G. A,, Christian, J. E., Anal. Chem., 33, 1216-18 (1961). (3) Mueller, E. B., in “The Current Status of Liquid Scintillation Countine.” E. D. Bransome. Jr... Ed.. DD 181-8. Grume Stratton, New YoLk, N.Y., 1970. (4) Wang, C. H., Willis, D. L., “Radiotracer Methodology in Biological Science,” pp 134-5, Prentice-Hall, Englewood Cliffs, N.J., 1965. (5) Bray, G. A., Anal. Biochem., 1,279-85 (1960). (6) Rapkin, E., Packard Instrument Co., Inc., Tech. Bull., 3, Downers Grove, Ill. (1961). (7) Passman, J. M., Radin, N. S., Cooper, J. A. D., Anal. Chem., 28,484-6 (1956). (8) Woeller, F. H., Anal. Biochem., 2,508-11 (1961). ,
Received for review J u l y 25, 1973. Resubmitted March 3, 1975. Accepted J u n e 26, 1975. This investigation was supported i n part by Environmental Protection Agency Project No. 16010 EGR administered through the University of Wisconsin Water Resources Center, and by Eastern Deciduous Forest Biome, U S - I B P , funded by the National Science Foundation and Interagency Agreement AG 199, 40-193-69 with the Atomic Energy Commission-Oak Ridge National Laboratory.
