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Liquid scintillation counting at ambient temperature
ANALYTICAL
liquid
17, 294-%9
BIOCHEMISTFzY
Scintillation
Counting
T. c. HALL2 Department
(1966)
AND
at Ambient
Temperature1
C. J. WEISER
of Horticultural Science, University St. Paul, Minnesota 66101
of Minnesota,
Received June 15, 1966
It has long been recognized that scintillation solutions based on dioxane have great merit in the determination of low-energy isotopes in aqueous solution, and that the addition of naphthalene to such scintillators considerably reduces the quenching effect of water (1). However, the use of refrigerated (0-4O) cabinets has greatly limited the practical application of these systems. The introduction of scintillation counters capable of high efficiency resolution of p emission together with a very constant low background count rate at temperatures between 0’ and 25’ has demanded the reevaluation of dioxane-naphthalene scintillators for aqueous sample counting. APPARATUS
AND
MATERIALS
The Beckman (model CPM-200) liquid scintillation spectrometer was normally operated at room temperature (21’ -C 3”). The use of refrigeration to bring the temperature of the counter below 6” did not significantly alter the background count rates of 23.7 cpm for the 3H channel and 39.5 cpm for the 14C channel. For sealed reference samples (Beckman Set No. 192) counted at a gain value of 460 the following efficiencies were noted: 3H in the 3H channel, 49.6% with an automatic external standardization (AES) figure of 1.53; 14C in the 14C channel, 92% with an AES of 1.35; and for 14C in the 14C less 3H channel, 69.0%. The AES figure for the reference background sample was noted to be 1.26, but very comparable efficiencies were obtained for standard solutions of 14C-toulene (Hazleton Nuclear Science Corporation) and 3H-n-hexadecane (Nuclear Chicago) added to a toluene scintillator which gave similar background counts to the reference background. All counts were taken to a 2 u error of 5% or less. ‘Scientific Journal Series paper number 5996 of the Minnesota Agricultural Experiment Station. *Present address: Department of Horticulture, The University of Wisconsin, Madison, Wisconsin 53706. 294
AMBIENT
SCINTILLATION
COUiiTING
295
Low-potassium foil-lined screw-cap vials were obtained from Packard, as were the scintillation-grade phosphors used in preparing the scintillators. Other chemicals were of reagent grade; p-dioxane WRS from Eastman Kodak and toluene from Allied Chemical. Toluene scintillators were prepared as described previously (2). The dioxane scintillator composition was: 150 gm naphthalene (recrystallized from alcohol), 10 gm PPO, 0.3 gm POPOP in 1 liter p-dioxanc. No attempt was made to purge the scintillators of oxygen since it was noted that nitrogen-treated scintillator became increasingly quenched over a period of 4-6 hr in the counting vials at room temperature. The quenching was easily detected by a decreasing AES figure over this time. The AES value for nonpurged scintillator did not alter markedly over a period of several days. RESULTS
AND
DISCUSSlOX
While small volumes (0.1-0.25 ml) of aqueous solution can be counted at high efficiency with toluene-ethanol scintillators (2), and larger volumes at lower efficiency with other systems (3, 4)) the necessity for solubilizing agents such as 2-ethoxyethanol, Hyamine, and ethanol in relatively large volumes causes severe quenching. Since solubility problems with dioxane-naphthalene scintillators are largely avoided at room temperature, the use of these systems permits high efficiency counting with water concentrations up to 20%. Formic acid may be used to acidify alkaline solutions prior to counting. As shown in Table 1, aqueous samples of 0.05-0.50 ml have little effect on the efficiency or quenching of 14C in 5 ml dioxane scintillator, while severe effects are evident with the toluene scintillator. The actual counting efficiency for any individual sample can be readily obtained from a graph of the AES value vs. efficiency (Fig. 1), always provided that it3 AES value is known. Sample volume does not have to be known. Such ease of recording efficiency is a great asset when converting counts per minute to disintegrations per minute and to molar equivalents. The graph in Figure 1 does not show the volume relationship between efficiency and quenching, which is demonstrated for aqueous sample volumes up to 2.00 ml in Table 1. Thus increasing the volume of dioxane scintillator to 10 ml to maintain a single phase with 2 ml aqueous sample gave AES figures which correspond (Fig. 1) with efficiencies in good agreement with experimental efficiencies (Table I), despite the fact that the graph was based on results from 5 ml scintillator. At the discriminator settings employed, there were virtually no counts recorded in the 14Cless 3H channel from samples containing 3H, an ideal situation when involved in experiments requiring double labeling with
296
HALL
Efficiency
and AEP
Ratio
AND
TABLE 1 for Aqueous
nH channel Sample
vol
and seintiUstor
0.05 ml aq. Toluene Dioxane 0.10 ml aq. Toluene Dioxane 0.15 ml aq. Toluene Dioxane 0.20 ml aq. Toluene Dioxane 0.25 ml aq. Toluene Dioxane 0.50 ml aq. Toluene Dioxane 1.00 ml aq. Toluene Dioxane 2.00 ml aq. Toluene Dioxaned
EffiC., y.
WEISER
Samples
“C less rH channel
WI! channel
AES
Effiie.,
AES
of W
Effic.,
%
AES
and 3H
Ethanol added to to1uene sointillator to maintain phase, nIla
%
samplec 16.9 30.4
0.432 1.202
81.2 84.3
0.481 1.194
27.6 57.3
0.488 1.181
0
sample
-
1.189
81.7 84.3
0.467 1.168
27.0 57.1
0.483 1.180
0
29.8 16.5 29.1
0.410 1.169
-
-
-
-
0
-
-
81.6 84.4
0.467 1.167
27.0 56.7
0.455 1.172
0
15.4 26.9
0.381 1.150
80.9 85.2
0.429 1.150
24.0 56.3
0.427 1.153
0.2
10.6 24.0
0.181 1.082
76.2 83.5
0.247 1.117
13.7 52.7
0.248 1.086
1.4
6.3 17.9
0.035 0.933
68.7 81.0
0.065 0.947
6.2 44.3
0.035 0.968
3.2
3.0 20.3
0.001 1.032
54.7 82.3
0.004 1.065
1.0 47.7
0.005 1.035
6.2
sample
sample
sample
sample
sample
sample
a AES: Automatic External Standardization ratio. b Additional volumes of water were added, and also ethanol to the toluene scintillator in the counting vials, as indicated. “0.05 ml aqueous samples of 3H-phenylalanine (102,605 dpm) or W-phenylttlanine (54,949 dpm) was added to 5 ml toluene or dioxane scintillator. d An additional 5 ml dioxane scintillator was added to maintain phase. Composition of the scintillators is given in the text.
these isotopes. Approximately 26% 14C was counted in the 3H channel. For highly colored, or strongly acid or alkaline solutions, the glass filter paper method of Davies and Cocking (5) is particularly useful, and even 3H could be counted at efficiencies of over 20% by this procedure with our equipment. It was noted that certain supplies of dioxane gave high background count rates, and some attention has to be paid to the source of this solvent. Occasionally spurious high background count rates were observed following the addition of the sample to the scintillator. Brief chill-
Dioxane
Toluene-ethanol
scintillator
Co
2. -.I
--
1.200
-
-L---.I-1.000
External
wntlllator
0 ‘-\.‘: LI
--.-J.--
0.000
standardization
0.600
0.400
0.200
9 0.000
ratio
1. Comparison of quenching effect of water on two scintillators. From an experimentally obtained AES figure, the efficiency of counting for a sample may be directly obtained from the gmph. The rpsulk from Table 1 arp incorporated in this figure (see testJ. FIG.
ing (20-30 set) in a refrigerator after preparation of the sample resolved this complication. The necessity for a silicone oil light path connection between the sample vial and the single photomultiplier tube of earlier equipment often gave rise to high residual background counts if the vials were reused (2). While silicone oil is not required in modern automatic scintillation counters, low levels of activity were sometimes noted after normal cleaning procedures. Washing in a 2% solution of Decon 7,”
298
HALL
AND
WEISER
concentrate (lMedical-Pharmaceutical Developments Ltd., Shoreham by Sea, Sussex, England) at 50” for 10 min was effective in removing these trace levels of contamination. Although 32P can readily be counted in the systems described above, a peak of activity was sometimes observed approximately coincident with
Lower
discriminator
setting.
FIG. 2. Spectra of 3H, 93, and ?l?. This figure was obtained by taking the count rate in a 2% window (e.g., with lower discriminator set at 200 units and the upper at 220 units) for each of the isotopes presented: (a) q , ‘H; (b) A, “C; (c) 0, 3zP; (d) 0, ‘*P. Curves a, b, and c were taken at a gain of 440 for aqueous samples in toluene-ethanol scintillator, curve d at a gain of 460 for an aqueous sample in a dioxane scintillator. System d was therefore less quenched, which accounts for the =P peak response being farther to the right than in curve c.
the peak response of the counter to 3H (Fig. 2, curve c). Cab-0-Sil may be used to reduce this complication in experiments involving double labeling with 3H and 32P, but addition of the sample to scintillator with rapid mixing to reduce direct contact of the 32P sample and the glass vial was often effective in mitigating the effect (Fig. 2, curved). The dioxane scintillators should find a particular value in the assay
AMBIENT
SCINTILLATION
COUNTIKG
of low levels of activity in eluates from column chromatography. cially where the use of flow cells is not suitable or convenient.
299
espe-
SUMMARY
Ambient-temperature liquid scintillation counting of aqueous tianlple:: of up t.o 2 ml volume realized efficiencies of over 807~ for ‘*C and 20% for 3H in a dioxane-naphthalene scintillator. The use of ext,ernal standardization for rapid determination of counting efficiency is present.eci together with some characteristics of a newly available commercial scintillation count.er normally operated nt room temperature. ACKNOWLEDGMENTS The Louis W. and Maud Hill Family Foundation is sincerely thanked for an apparatus grant to C. J. W. and support for a Post-dortoral Frllowshir, to T. C. H. REFERENCES 1. FURST, 2. HALL, 3. BRUNO, 4. BROWN, 5. Davr~s,
M., T.
BROWN, F. H., Nucleorkx 13, So. 4, 58 (1955). C., AND COCKING, E. C., Riochenl. J. 96, 626 (1965). G. A., AND CHRISTIAN, J. E., Anal. Chem. 33, 1216 (1961). W. O., AND BADMAN, H. G., &o&em. J. 78, 571 (1961). J. W., .4ND COCKING, E. C., Biochim. Riop,hy~. Acfn 115> 511 (19661. KALLMANN,
H.,
AND
liquid
17, 294-%9
BIOCHEMISTFzY
Scintillation
Counting
T. c. HALL2 Department
(1966)
AND
at Ambient
Temperature1
C. J. WEISER
of Horticultural Science, University St. Paul, Minnesota 66101
of Minnesota,
Received June 15, 1966
It has long been recognized that scintillation solutions based on dioxane have great merit in the determination of low-energy isotopes in aqueous solution, and that the addition of naphthalene to such scintillators considerably reduces the quenching effect of water (1). However, the use of refrigerated (0-4O) cabinets has greatly limited the practical application of these systems. The introduction of scintillation counters capable of high efficiency resolution of p emission together with a very constant low background count rate at temperatures between 0’ and 25’ has demanded the reevaluation of dioxane-naphthalene scintillators for aqueous sample counting. APPARATUS
AND
MATERIALS
The Beckman (model CPM-200) liquid scintillation spectrometer was normally operated at room temperature (21’ -C 3”). The use of refrigeration to bring the temperature of the counter below 6” did not significantly alter the background count rates of 23.7 cpm for the 3H channel and 39.5 cpm for the 14C channel. For sealed reference samples (Beckman Set No. 192) counted at a gain value of 460 the following efficiencies were noted: 3H in the 3H channel, 49.6% with an automatic external standardization (AES) figure of 1.53; 14C in the 14C channel, 92% with an AES of 1.35; and for 14C in the 14C less 3H channel, 69.0%. The AES figure for the reference background sample was noted to be 1.26, but very comparable efficiencies were obtained for standard solutions of 14C-toulene (Hazleton Nuclear Science Corporation) and 3H-n-hexadecane (Nuclear Chicago) added to a toluene scintillator which gave similar background counts to the reference background. All counts were taken to a 2 u error of 5% or less. ‘Scientific Journal Series paper number 5996 of the Minnesota Agricultural Experiment Station. *Present address: Department of Horticulture, The University of Wisconsin, Madison, Wisconsin 53706. 294
AMBIENT
SCINTILLATION
COUiiTING
295
Low-potassium foil-lined screw-cap vials were obtained from Packard, as were the scintillation-grade phosphors used in preparing the scintillators. Other chemicals were of reagent grade; p-dioxane WRS from Eastman Kodak and toluene from Allied Chemical. Toluene scintillators were prepared as described previously (2). The dioxane scintillator composition was: 150 gm naphthalene (recrystallized from alcohol), 10 gm PPO, 0.3 gm POPOP in 1 liter p-dioxanc. No attempt was made to purge the scintillators of oxygen since it was noted that nitrogen-treated scintillator became increasingly quenched over a period of 4-6 hr in the counting vials at room temperature. The quenching was easily detected by a decreasing AES figure over this time. The AES value for nonpurged scintillator did not alter markedly over a period of several days. RESULTS
AND
DISCUSSlOX
While small volumes (0.1-0.25 ml) of aqueous solution can be counted at high efficiency with toluene-ethanol scintillators (2), and larger volumes at lower efficiency with other systems (3, 4)) the necessity for solubilizing agents such as 2-ethoxyethanol, Hyamine, and ethanol in relatively large volumes causes severe quenching. Since solubility problems with dioxane-naphthalene scintillators are largely avoided at room temperature, the use of these systems permits high efficiency counting with water concentrations up to 20%. Formic acid may be used to acidify alkaline solutions prior to counting. As shown in Table 1, aqueous samples of 0.05-0.50 ml have little effect on the efficiency or quenching of 14C in 5 ml dioxane scintillator, while severe effects are evident with the toluene scintillator. The actual counting efficiency for any individual sample can be readily obtained from a graph of the AES value vs. efficiency (Fig. 1), always provided that it3 AES value is known. Sample volume does not have to be known. Such ease of recording efficiency is a great asset when converting counts per minute to disintegrations per minute and to molar equivalents. The graph in Figure 1 does not show the volume relationship between efficiency and quenching, which is demonstrated for aqueous sample volumes up to 2.00 ml in Table 1. Thus increasing the volume of dioxane scintillator to 10 ml to maintain a single phase with 2 ml aqueous sample gave AES figures which correspond (Fig. 1) with efficiencies in good agreement with experimental efficiencies (Table I), despite the fact that the graph was based on results from 5 ml scintillator. At the discriminator settings employed, there were virtually no counts recorded in the 14Cless 3H channel from samples containing 3H, an ideal situation when involved in experiments requiring double labeling with
296
HALL
Efficiency
and AEP
Ratio
AND
TABLE 1 for Aqueous
nH channel Sample
vol
and seintiUstor
0.05 ml aq. Toluene Dioxane 0.10 ml aq. Toluene Dioxane 0.15 ml aq. Toluene Dioxane 0.20 ml aq. Toluene Dioxane 0.25 ml aq. Toluene Dioxane 0.50 ml aq. Toluene Dioxane 1.00 ml aq. Toluene Dioxane 2.00 ml aq. Toluene Dioxaned
EffiC., y.
WEISER
Samples
“C less rH channel
WI! channel
AES
Effiie.,
AES
of W
Effic.,
%
AES
and 3H
Ethanol added to to1uene sointillator to maintain phase, nIla
%
samplec 16.9 30.4
0.432 1.202
81.2 84.3
0.481 1.194
27.6 57.3
0.488 1.181
0
sample
-
1.189
81.7 84.3
0.467 1.168
27.0 57.1
0.483 1.180
0
29.8 16.5 29.1
0.410 1.169
-
-
-
-
0
-
-
81.6 84.4
0.467 1.167
27.0 56.7
0.455 1.172
0
15.4 26.9
0.381 1.150
80.9 85.2
0.429 1.150
24.0 56.3
0.427 1.153
0.2
10.6 24.0
0.181 1.082
76.2 83.5
0.247 1.117
13.7 52.7
0.248 1.086
1.4
6.3 17.9
0.035 0.933
68.7 81.0
0.065 0.947
6.2 44.3
0.035 0.968
3.2
3.0 20.3
0.001 1.032
54.7 82.3
0.004 1.065
1.0 47.7
0.005 1.035
6.2
sample
sample
sample
sample
sample
sample
a AES: Automatic External Standardization ratio. b Additional volumes of water were added, and also ethanol to the toluene scintillator in the counting vials, as indicated. “0.05 ml aqueous samples of 3H-phenylalanine (102,605 dpm) or W-phenylttlanine (54,949 dpm) was added to 5 ml toluene or dioxane scintillator. d An additional 5 ml dioxane scintillator was added to maintain phase. Composition of the scintillators is given in the text.
these isotopes. Approximately 26% 14C was counted in the 3H channel. For highly colored, or strongly acid or alkaline solutions, the glass filter paper method of Davies and Cocking (5) is particularly useful, and even 3H could be counted at efficiencies of over 20% by this procedure with our equipment. It was noted that certain supplies of dioxane gave high background count rates, and some attention has to be paid to the source of this solvent. Occasionally spurious high background count rates were observed following the addition of the sample to the scintillator. Brief chill-
Dioxane
Toluene-ethanol
scintillator
Co
2. -.I
--
1.200
-
-L---.I-1.000
External
wntlllator
0 ‘-\.‘: LI
--.-J.--
0.000
standardization
0.600
0.400
0.200
9 0.000
ratio
1. Comparison of quenching effect of water on two scintillators. From an experimentally obtained AES figure, the efficiency of counting for a sample may be directly obtained from the gmph. The rpsulk from Table 1 arp incorporated in this figure (see testJ. FIG.
ing (20-30 set) in a refrigerator after preparation of the sample resolved this complication. The necessity for a silicone oil light path connection between the sample vial and the single photomultiplier tube of earlier equipment often gave rise to high residual background counts if the vials were reused (2). While silicone oil is not required in modern automatic scintillation counters, low levels of activity were sometimes noted after normal cleaning procedures. Washing in a 2% solution of Decon 7,”
298
HALL
AND
WEISER
concentrate (lMedical-Pharmaceutical Developments Ltd., Shoreham by Sea, Sussex, England) at 50” for 10 min was effective in removing these trace levels of contamination. Although 32P can readily be counted in the systems described above, a peak of activity was sometimes observed approximately coincident with
Lower
discriminator
setting.
FIG. 2. Spectra of 3H, 93, and ?l?. This figure was obtained by taking the count rate in a 2% window (e.g., with lower discriminator set at 200 units and the upper at 220 units) for each of the isotopes presented: (a) q , ‘H; (b) A, “C; (c) 0, 3zP; (d) 0, ‘*P. Curves a, b, and c were taken at a gain of 440 for aqueous samples in toluene-ethanol scintillator, curve d at a gain of 460 for an aqueous sample in a dioxane scintillator. System d was therefore less quenched, which accounts for the =P peak response being farther to the right than in curve c.
the peak response of the counter to 3H (Fig. 2, curve c). Cab-0-Sil may be used to reduce this complication in experiments involving double labeling with 3H and 32P, but addition of the sample to scintillator with rapid mixing to reduce direct contact of the 32P sample and the glass vial was often effective in mitigating the effect (Fig. 2, curved). The dioxane scintillators should find a particular value in the assay
AMBIENT
SCINTILLATION
COUNTIKG
of low levels of activity in eluates from column chromatography. cially where the use of flow cells is not suitable or convenient.
299
espe-
SUMMARY
Ambient-temperature liquid scintillation counting of aqueous tianlple:: of up t.o 2 ml volume realized efficiencies of over 807~ for ‘*C and 20% for 3H in a dioxane-naphthalene scintillator. The use of ext,ernal standardization for rapid determination of counting efficiency is present.eci together with some characteristics of a newly available commercial scintillation count.er normally operated nt room temperature. ACKNOWLEDGMENTS The Louis W. and Maud Hill Family Foundation is sincerely thanked for an apparatus grant to C. J. W. and support for a Post-dortoral Frllowshir, to T. C. H. REFERENCES 1. FURST, 2. HALL, 3. BRUNO, 4. BROWN, 5. Davr~s,
M., T.
BROWN, F. H., Nucleorkx 13, So. 4, 58 (1955). C., AND COCKING, E. C., Riochenl. J. 96, 626 (1965). G. A., AND CHRISTIAN, J. E., Anal. Chem. 33, 1216 (1961). W. O., AND BADMAN, H. G., &o&em. J. 78, 571 (1961). J. W., .4ND COCKING, E. C., Biochim. Riop,hy~. Acfn 115> 511 (19661. KALLMANN,
H.,
AND