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Tritosol: A new scintillation cocktail based on Triton X-100
ANALYTICAL
BIOCHEMISTRY
Tritosol:
63, 555-558 (1975)
A New Based
Cocktail
X-l 00
on Triton UWE
D-3000
Scintillation
FRICKE
Department of Pharmacology, Medical Hannover-Kleefeld, Karl-Wiechert-Allee
School of Hannover, 9, German Federal
Republic
Received March I, 1974; accepted September 19, 1974 A scintillation cocktail consisting of 3.0 g PPO, 257 ml Triton X-100, 106 ml ethanol, 37 ml ethylene glycol, and 600 ml xylene is described. A linear relationship between counting efficiency and the external standard ratio could be demonstrated over a wide range of quenching. The counting efficiencies (unquenched) for 3H are about 47%, for r4C about 87%. and for Wa about 80%.
Scintillation cocktails based on Triton X-100, a nonionic surfactant, seem to become more and more usual in liquid scintillation counting (l-4). These scintillation cocktails mostly can accept a large volume of aqueous sample, but a great disadvantage is the poor linearity between efficiency and the external standard ratio over a wide range of quenching (5). Therefore, the purpose of the present study was to develop a scintillation fluid based on Triton X- 100, which should fulfill this requirement, and to investigate its applicability with respect to some of the commonly used radioisotopes: 14C, 3H, and 45Ca. MATERIALS
AND
METHODS
Standards of 14C-labeled toluene, 3H-labeled toluene, and of a hydrochloric acid solution of 45Ca, and the premixed scintillation cocktail Aquasol were supplied by New England Nuclear Corp., Boston, MA. Triton X-l 00 in technical quality was purchased from Serva, Heidelberg (Germany), ethylene glycol was obtained from Riedel de Haen, Berlin (Germany), xylene (mixture of isomers), and the other chemicals were of reagent grade and were supplied by Merck Comp., Darmstadt (Germany). The recommended newly developed scintillation cocktail (Tritosol) was of the following composition: 3.0 g PPO (2,5diphenyloxazole), 257 ml Triton X-100, 37 ml ethylene glycol, 106 ml ethanol, and xylene to make 1000 ml. In all experiments 10 ml of the respective scintillation cocktail (see Results and Discussion) and -as quenching agent increasing amounts of water (up to 3.0 ml) were used. All counting was performed on a 555 Copyright 0 1975 by Academic Press, Inc. Printed All rights of reproduction in any form reserved.
in the United
States.
556
UWE
FRICKE
Nuclear Chicago Mark II liquid scintillation spectrometer using 20-ml polyethylene counting vials (Hormuth, Heidelberg, Germany). RESULTS
AND
DISCUSSION
Besides the newly developed scintillation cocktail Tritosol for comparison purposes one of the Triton X- 100 containing scintillation cocktails tested by Anderson and McClure (2), consisting of 3.0 g PPO, 250 ml Triton X-100, and 750 ml xylene, and the commercial preparation Aquasol were used. Figure 1 illustrates the relationship between the counting efficiency for [3H]-toluene and the external standard ratio or the added amounts of water and the external standard ratio, respectively, using the different scintillation fluids. Whereas with Aquasol and with the recommended new scintillant a linear relationship between the respective two parameters could be obtained over a wide range of quenching, the above scintillation mixture according to Anderson and McClure (2) did not give satisfactory results. The discontinuance in the latter curves which consistently was observed in all respective experiments is obviously attributed to the instability of the emulsion. A separation of the aqueous from the organic phase was observed already by Patterson and Greene (1) and Dobrota and Hinton (3) under certain conditions. The counting efficiencies (unquenched) obtained with the three tested scintillation fluids were: about 52% with the scintillant according to Anderson and McClure (2), about 52% with Aquasol, and about 47% with the new scintillation mixture. After addition of 3.0 ml water to the respective scintillants the counting efficiency decreased below 20% using the scintillation cocktail of Anderson and McClure (2), to about 35% with the commercial cocktail Aquasol, and to about 32% using Tritosol. Corresponding investigations were performed for [ 14C] -toluene or [““Cl -chloride with the recommended new scintillation cocktail only. The unquenched counting efficiency was about 87% with a corresponding external standard ratio of 1.87 (‘“C) and about 80% with a corresponding external standard ratio of 1.75 (““Ca), respectively. Addition of 3.0 ml water resulted in a decrease both of the efficiency and external standard ratio to about 82%/1.60 (‘*C) and to about 76%/l .46 (45Ca), respectively. In the experiments with [45Ca]-chloride a slight increase of the efficiency from about 78 to 82% has been observed by adding water up to 1.0 ml. Therefore, when using more polar solutes addition of water is recommended. Furthermore, the influence of alkaline or acidic solutions, urea, sucrose, and high salt concentrations (NaCl) on the stability of the new scintillant was tested. No deviations from the above results could be detected when (a) alkaline solutions up to 3.0 ml of 0.5 N NaOH and acidic solutions up to 3.0 ml of 1.O N HCl or (b) aqueous solutions
TRITOSOL-NEW
SCINTILLATION
557
COCKTAIL
: TRITOSOL
z
@
AQUASOL
15
20
25 ESR
.-c
0 Cocktail
50
ANDERSON
affer
r
_ -----.._.
-_
2 .(---
e ii
--. --.-.
.. '*-.--,* --
and MCCLURE
.* L
0
'1. 15
20
25 ES!?
30
10 I 1.5
I 2.0
I 2.5
EXT.
STAND.
RATIO
1. The relationship between counting efficiency and external standard ratio for [3H] toluene using Tritosol (panel A), Aquasol (panel B), and one of the Triton X-100 containing scintillation cocktails tested by Anderson and McClure (2) (panel C). The results of two different experiments are given. The inserts represent the respective dependence of external standard ratio (ESR) on sample water content. FIG.
(sample volume: 1.0 ml) of urea up to 5.0 M, sucrose up to 0.3 M, and of sodium chloride up to 1.O M were added. Other variables such as cooling (4°C) and long-term storage (about 10 days) for repeated counting of the samples did not influence the measurements. Chemoluminescence has not been seen in any case, not even at short time (5 min) after mixing the samples.
UWE
558
FRICKE
Thus, it can be stated: (I) The new scintillation mixture Tritosol has proved to accept a rather large amount of aqueous solutions (up to 3.0 ml/lo.0 ml scintillant) without a great loss in counting efficiency, (2) Acidic (1.0 N HCI), alkaline (0.5 N NaOH) solutions, and other biological solutes (e.g., sucrose: 0.3 M, urea: 5.0 M, NaCl: 1.O M) did not influence the stability of Tritosol. (3) A linear relationship between the counting efficiency and the external standard ratio over a wide range of quenching could be demonstrated with the new scintillation mixture. Besides, the new scintillant can be formulated at very low costs. ACKNOWLEDGMENT The skilful technical assistance of Miss Christa Busche is greatfully appreciated.
REFERENCES 1. PATTERSON,
M. S., AND GREENE, R. C. (1965) Anal. Chem. 37, 854-857. L. E., AND MCCLURE, W. 0. (1973) Anal. Biochem. 51, 173-179. 3. DOBROTA, M., AND HINTON, R. H. (1973) Anal. Biochem. 56, 270-274. 4. CARTER, G. W., AND VAN DYKE, K. (1973) Anal. Biochem. 54, 624-627. 5. TURNER, J. C. (1967) in Sample Preparation for Liquid Scintillation Counting, 2. ANDERSON,
Radiochemical Centre, Amersham, England.
The
BIOCHEMISTRY
Tritosol:
63, 555-558 (1975)
A New Based
Cocktail
X-l 00
on Triton UWE
D-3000
Scintillation
FRICKE
Department of Pharmacology, Medical Hannover-Kleefeld, Karl-Wiechert-Allee
School of Hannover, 9, German Federal
Republic
Received March I, 1974; accepted September 19, 1974 A scintillation cocktail consisting of 3.0 g PPO, 257 ml Triton X-100, 106 ml ethanol, 37 ml ethylene glycol, and 600 ml xylene is described. A linear relationship between counting efficiency and the external standard ratio could be demonstrated over a wide range of quenching. The counting efficiencies (unquenched) for 3H are about 47%, for r4C about 87%. and for Wa about 80%.
Scintillation cocktails based on Triton X-100, a nonionic surfactant, seem to become more and more usual in liquid scintillation counting (l-4). These scintillation cocktails mostly can accept a large volume of aqueous sample, but a great disadvantage is the poor linearity between efficiency and the external standard ratio over a wide range of quenching (5). Therefore, the purpose of the present study was to develop a scintillation fluid based on Triton X- 100, which should fulfill this requirement, and to investigate its applicability with respect to some of the commonly used radioisotopes: 14C, 3H, and 45Ca. MATERIALS
AND
METHODS
Standards of 14C-labeled toluene, 3H-labeled toluene, and of a hydrochloric acid solution of 45Ca, and the premixed scintillation cocktail Aquasol were supplied by New England Nuclear Corp., Boston, MA. Triton X-l 00 in technical quality was purchased from Serva, Heidelberg (Germany), ethylene glycol was obtained from Riedel de Haen, Berlin (Germany), xylene (mixture of isomers), and the other chemicals were of reagent grade and were supplied by Merck Comp., Darmstadt (Germany). The recommended newly developed scintillation cocktail (Tritosol) was of the following composition: 3.0 g PPO (2,5diphenyloxazole), 257 ml Triton X-100, 37 ml ethylene glycol, 106 ml ethanol, and xylene to make 1000 ml. In all experiments 10 ml of the respective scintillation cocktail (see Results and Discussion) and -as quenching agent increasing amounts of water (up to 3.0 ml) were used. All counting was performed on a 555 Copyright 0 1975 by Academic Press, Inc. Printed All rights of reproduction in any form reserved.
in the United
States.
556
UWE
FRICKE
Nuclear Chicago Mark II liquid scintillation spectrometer using 20-ml polyethylene counting vials (Hormuth, Heidelberg, Germany). RESULTS
AND
DISCUSSION
Besides the newly developed scintillation cocktail Tritosol for comparison purposes one of the Triton X- 100 containing scintillation cocktails tested by Anderson and McClure (2), consisting of 3.0 g PPO, 250 ml Triton X-100, and 750 ml xylene, and the commercial preparation Aquasol were used. Figure 1 illustrates the relationship between the counting efficiency for [3H]-toluene and the external standard ratio or the added amounts of water and the external standard ratio, respectively, using the different scintillation fluids. Whereas with Aquasol and with the recommended new scintillant a linear relationship between the respective two parameters could be obtained over a wide range of quenching, the above scintillation mixture according to Anderson and McClure (2) did not give satisfactory results. The discontinuance in the latter curves which consistently was observed in all respective experiments is obviously attributed to the instability of the emulsion. A separation of the aqueous from the organic phase was observed already by Patterson and Greene (1) and Dobrota and Hinton (3) under certain conditions. The counting efficiencies (unquenched) obtained with the three tested scintillation fluids were: about 52% with the scintillant according to Anderson and McClure (2), about 52% with Aquasol, and about 47% with the new scintillation mixture. After addition of 3.0 ml water to the respective scintillants the counting efficiency decreased below 20% using the scintillation cocktail of Anderson and McClure (2), to about 35% with the commercial cocktail Aquasol, and to about 32% using Tritosol. Corresponding investigations were performed for [ 14C] -toluene or [““Cl -chloride with the recommended new scintillation cocktail only. The unquenched counting efficiency was about 87% with a corresponding external standard ratio of 1.87 (‘“C) and about 80% with a corresponding external standard ratio of 1.75 (““Ca), respectively. Addition of 3.0 ml water resulted in a decrease both of the efficiency and external standard ratio to about 82%/1.60 (‘*C) and to about 76%/l .46 (45Ca), respectively. In the experiments with [45Ca]-chloride a slight increase of the efficiency from about 78 to 82% has been observed by adding water up to 1.0 ml. Therefore, when using more polar solutes addition of water is recommended. Furthermore, the influence of alkaline or acidic solutions, urea, sucrose, and high salt concentrations (NaCl) on the stability of the new scintillant was tested. No deviations from the above results could be detected when (a) alkaline solutions up to 3.0 ml of 0.5 N NaOH and acidic solutions up to 3.0 ml of 1.O N HCl or (b) aqueous solutions
TRITOSOL-NEW
SCINTILLATION
557
COCKTAIL
: TRITOSOL
z
@
AQUASOL
15
20
25 ESR
.-c
0 Cocktail
50
ANDERSON
affer
r
_ -----.._.
-_
2 .(---
e ii
--. --.-.
.. '*-.--,* --
and MCCLURE
.* L
0
'1. 15
20
25 ES!?
30
10 I 1.5
I 2.0
I 2.5
EXT.
STAND.
RATIO
1. The relationship between counting efficiency and external standard ratio for [3H] toluene using Tritosol (panel A), Aquasol (panel B), and one of the Triton X-100 containing scintillation cocktails tested by Anderson and McClure (2) (panel C). The results of two different experiments are given. The inserts represent the respective dependence of external standard ratio (ESR) on sample water content. FIG.
(sample volume: 1.0 ml) of urea up to 5.0 M, sucrose up to 0.3 M, and of sodium chloride up to 1.O M were added. Other variables such as cooling (4°C) and long-term storage (about 10 days) for repeated counting of the samples did not influence the measurements. Chemoluminescence has not been seen in any case, not even at short time (5 min) after mixing the samples.
UWE
558
FRICKE
Thus, it can be stated: (I) The new scintillation mixture Tritosol has proved to accept a rather large amount of aqueous solutions (up to 3.0 ml/lo.0 ml scintillant) without a great loss in counting efficiency, (2) Acidic (1.0 N HCI), alkaline (0.5 N NaOH) solutions, and other biological solutes (e.g., sucrose: 0.3 M, urea: 5.0 M, NaCl: 1.O M) did not influence the stability of Tritosol. (3) A linear relationship between the counting efficiency and the external standard ratio over a wide range of quenching could be demonstrated with the new scintillation mixture. Besides, the new scintillant can be formulated at very low costs. ACKNOWLEDGMENT The skilful technical assistance of Miss Christa Busche is greatfully appreciated.
REFERENCES 1. PATTERSON,
M. S., AND GREENE, R. C. (1965) Anal. Chem. 37, 854-857. L. E., AND MCCLURE, W. 0. (1973) Anal. Biochem. 51, 173-179. 3. DOBROTA, M., AND HINTON, R. H. (1973) Anal. Biochem. 56, 270-274. 4. CARTER, G. W., AND VAN DYKE, K. (1973) Anal. Biochem. 54, 624-627. 5. TURNER, J. C. (1967) in Sample Preparation for Liquid Scintillation Counting, 2. ANDERSON,
Radiochemical Centre, Amersham, England.
The