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Liquid scintillation counting of C14O2 with phenethylamine
508
SHORT COMMUNICATIONS REFERENCES
1. FLEISCHER, S., LIETZE, A., WALTER, A., AND HAUROWITZ, F., Proc. Sot. Exptl. Biol. Med. 101, 860 (1959). 2. WIGGANS, D. S., RUMSFELD, H. W., JR., AND BURR, W. W., JR., Federation Proc. 15, 384 (1957). 3. GOLDSWORTHY, P. D., AND VOLWILER, W., J. Biol. Chem. 230, 817 (1958). 4. JEFFAY, H., OLURAJO, F. O., AND JEWELL, W. R., Anal. Chem. 32, 306 (1960). 5. JEFFAY, H., AND ALVAREZ, J., Anal. Chem. 33, 612 (1961).
HENRY JULIAN
JEFFAY ALVAREZ
Department of Biological Chemistry University of Illinois College of Medicine Chicago, Illinois Received June 12, 1961
Liquid
Scintillation
Counting
of Cl402
with
Phenethylamine’
Carbonic-C” has been determined by scintillation counting as gas (1) or as aqueous carbonate (2). Another technique is the incorporation into an organic scintillator solution by means of a compound that can “bind” carbon dioxide. Compounds suitable for this purpose should, in both the reacted and unreacted form, be soluble in a mixture in which toluene is the principal component and, at the same time, show little pulse quenching. The partial vapor pressure of the reactant should be low, and the reaction with carbon dioxide must be rapid and quantitative. Thus far, this task has been solved with some success (3,4) by the use of ‘LHyamine” hydroxide [p- (diisobutylcresoxyethoxyethyl)dimethylbenzylammonium hydroxide], a compound that can be prepared from the commercially available quaternary chloride, Hyamine 10-X (reg. trademark Rohm & Haas, Inc.). Upon reaction with COZ, this base forms a toluene-soluble bicarbonate. The shortcomings in the use of this reagent are an appreciable quenching and high cost. A recent communication (5) reports the use of a tertiary alkyl primary amine (Primene 81-R; Rohm & Haas, Inc.). However, a still later paper (6) states that this compound “did not quantitatively absorb carbon diox1 The work Commission
described
in
this
report
~11s supported
by
the
U. S. Atomic
Energy
SHORT
COMMUNICATIONS
509
ide.” Consequently, we have turned our attention to other compounds. We have observed that carbamates formed by the reaction of CO, with amines quench pulses only slightly. Of the unreacted amines, primary aliphatic compounds quench far less than secondary ones.’ A series of available primary amines was then tested to determine which would give t,he best compromise with respect to the requirements stated in the first paragraph.3 The carbamate from phenethylamine (2-phenylethylamine) is very soluble in alcohols but much less so in toluene. With a sufficient stoichiometric excess (about 3 moles amine/mole CO,), the reaction is quite rapid. The product formed in the presence of alcohol was found (by elemental analysis for C, H, and Nj to have the empirical formula C,,H,,N,O,, which corresponds t.o phenethylammonium phenethylcarbamate. This compound decomposes near 75’. Currently, the following preparat,ion is employed: 27 ml of redistilled phenethylamine,* 27 ml of absolute methanol, 500 mg 2,5-diphenyloxazole (PPO), and 10 mg 1,4-bis- [2- (5-phenyloxazolyl) ] benzene (POPOP). This mixture is diluted to 100 ml with toluene. The alcohoi serves to increase the solubility of the product. At, the same time, it lowers the partial vapor pressure of the amine, thus minimizing gasphase reaction that leads to deposition of the carbamate at pointa removed from the amine solut,ion. By the addition of some toluene at this point, the volume of solution is deliberately increased for more convenient handling, while the two phosphors are at a concentration equal to t.hat of the stock toluene scintillator added later during sample preparation. The apparatus used here consists of u diffusion system, with wide cross sections, in which the gas (from a cold trap or a generator) is released directly into a counting vial through an adapter. The vial contains the necessary amount of CO, reagent which is kept, near 0” and agitated by a magnetic stirrer, and about 20 min is allowed for t.he gas uptake with an initial and final pressure of 100-150 mm. We define a “millimole equivalent.” of reagent. as the quantity capable of reacting quantit.atively with 1 mmole CO? under the conditions just described. This corresponds to 1.1 ml of the Hyamine base (1 &I in methanol) or 1.5 ml of the phenethylamine-alcohol-toluene mixt,ure. ‘For example, for equal molar concentrations, quenching increases in the ordel tc-butylamine < di-n-butylamine < tri-n-butylamine. ’ Abandoned (mostly for reasons of insolubility of the carbarnates) were: bellxylamine, t-octylamine (kindly supplied by Rohm & Haas, Inc.), n-dodecylamine, ;md 4-(aminomethpl)cyclohexanemethanol. ’ Matheson, Coleman & Bell Division, Cat, No. 6066; price $11.25 per kilogram
510
SHORT
COMMUNICATIONS
Counting efficiencies were compared for preparations in which either 1 M Hyamine or the phenethylamine mixture were employed. All samples throughout were brought to 10 ml with toluene scintillator solution, and to final concentrations of 0.5% PPO and 0.01% POPOP. In a Packard Tri-Carb spectrometer at -10” (background 25 counts/min), 2 mmoles CO, could be counted with 60% efficiency when the phenethylamine mixture was used.5 The ‘Lmillimole equivalent” Hyamine sample gave less than 30% efficiency. In a custom-built spectrometer operating in coincidence at room temperature, 5 mmoles CO, could be introduced into 10 ml (containing 7.5 ml of phenethylamine mixture) and counted at 50% efficiency. At t,his temperature, part of the toluene can be replaced by naphthalene; this replacement results in even better efficiency.g The corresponding Hyamine sample (employing 5.5 ml of the 1 M solution) proved to have an efficiency close to zero. It is to be concluded that the phenethylamine mixture is superior to the “millimole equivalent” Hyamine preparations in regard to counting efficiency. From other experiments it appears reasonable to assume that the slight reduction in relative methanol content causes only part of t.his difference. It should be pointed out that with both reagents the efficiency shows little dependence on the amount of gas reacted. For the most accurate work the internal standard method (addition of a known amount of W-labeled toluene) is recommended. That the C?“O, is quantitatively absorbed into the phenethylamine solution was demonstrated by separately generating different known amounts of the gas (from accurately weighed BaWO, of known specific activity) into a fixed quantity of the amine. A straight-line relationship was established on plotting the liquid scintillation counting rate against the quantity of C’“0, used. SUMMARY
Phenethylamine has been found to be superior to the widely used “Hydroxide of Hyamine 10-X” as a compound to convert C’“O, into a soluble form for liquid scintillation counting. REFERENCES 1. KRAKAU, G., AND SCHNEIDER, H., Atomkernenergie 3, 515 (1958). 2. FUNT, B. L., AND HETHERINGTON, A., Science 129, 1429 (1958). 3. PASSMAN, J. M., RADIN, N. S., AND COOPER, J. A. D., Anal. Chem.
28, 484
(1956).
‘Samples containing CO2 in excess of 2 mmoles/lO ml of total solution showed incipient crystallization when kept at -10” for prolonged periods. ‘Concerning this phenomenon, see Kallmann, H., and Furst, M., ~TL “Liquid Scintillation Counting,” pp. 14-17. Pergamon Press, New York, 1958.
SHORT
511
COMhIUNICATIONS
F., in “Liquid Scintillation Counting,” (C. G. Bell, Jr., and F. N. Hayes, eds.), p. 123. Pergamon Press, New York, 1958. OPPERMANN, R. A., NYSTROM, R. F., NELSON, W. O., AND BROWN, R. E., Intern.
2. EISENBERG, 5.
J. Appl. Radiation and Isotopes ‘7, 38 (1959). R. G., PEETS, E. A., GORDON, S., AND BUYSKE,
6. KELLY,
D. A.,
Anal. Biochem.
FRITZ
H. WOELLER
267 (1961).
Lawrence
Radiation Laboratory University of California Berkeley, California Received March 14, 1961
2,
SHORT COMMUNICATIONS REFERENCES
1. FLEISCHER, S., LIETZE, A., WALTER, A., AND HAUROWITZ, F., Proc. Sot. Exptl. Biol. Med. 101, 860 (1959). 2. WIGGANS, D. S., RUMSFELD, H. W., JR., AND BURR, W. W., JR., Federation Proc. 15, 384 (1957). 3. GOLDSWORTHY, P. D., AND VOLWILER, W., J. Biol. Chem. 230, 817 (1958). 4. JEFFAY, H., OLURAJO, F. O., AND JEWELL, W. R., Anal. Chem. 32, 306 (1960). 5. JEFFAY, H., AND ALVAREZ, J., Anal. Chem. 33, 612 (1961).
HENRY JULIAN
JEFFAY ALVAREZ
Department of Biological Chemistry University of Illinois College of Medicine Chicago, Illinois Received June 12, 1961
Liquid
Scintillation
Counting
of Cl402
with
Phenethylamine’
Carbonic-C” has been determined by scintillation counting as gas (1) or as aqueous carbonate (2). Another technique is the incorporation into an organic scintillator solution by means of a compound that can “bind” carbon dioxide. Compounds suitable for this purpose should, in both the reacted and unreacted form, be soluble in a mixture in which toluene is the principal component and, at the same time, show little pulse quenching. The partial vapor pressure of the reactant should be low, and the reaction with carbon dioxide must be rapid and quantitative. Thus far, this task has been solved with some success (3,4) by the use of ‘LHyamine” hydroxide [p- (diisobutylcresoxyethoxyethyl)dimethylbenzylammonium hydroxide], a compound that can be prepared from the commercially available quaternary chloride, Hyamine 10-X (reg. trademark Rohm & Haas, Inc.). Upon reaction with COZ, this base forms a toluene-soluble bicarbonate. The shortcomings in the use of this reagent are an appreciable quenching and high cost. A recent communication (5) reports the use of a tertiary alkyl primary amine (Primene 81-R; Rohm & Haas, Inc.). However, a still later paper (6) states that this compound “did not quantitatively absorb carbon diox1 The work Commission
described
in
this
report
~11s supported
by
the
U. S. Atomic
Energy
SHORT
COMMUNICATIONS
509
ide.” Consequently, we have turned our attention to other compounds. We have observed that carbamates formed by the reaction of CO, with amines quench pulses only slightly. Of the unreacted amines, primary aliphatic compounds quench far less than secondary ones.’ A series of available primary amines was then tested to determine which would give t,he best compromise with respect to the requirements stated in the first paragraph.3 The carbamate from phenethylamine (2-phenylethylamine) is very soluble in alcohols but much less so in toluene. With a sufficient stoichiometric excess (about 3 moles amine/mole CO,), the reaction is quite rapid. The product formed in the presence of alcohol was found (by elemental analysis for C, H, and Nj to have the empirical formula C,,H,,N,O,, which corresponds t.o phenethylammonium phenethylcarbamate. This compound decomposes near 75’. Currently, the following preparat,ion is employed: 27 ml of redistilled phenethylamine,* 27 ml of absolute methanol, 500 mg 2,5-diphenyloxazole (PPO), and 10 mg 1,4-bis- [2- (5-phenyloxazolyl) ] benzene (POPOP). This mixture is diluted to 100 ml with toluene. The alcohoi serves to increase the solubility of the product. At, the same time, it lowers the partial vapor pressure of the amine, thus minimizing gasphase reaction that leads to deposition of the carbamate at pointa removed from the amine solut,ion. By the addition of some toluene at this point, the volume of solution is deliberately increased for more convenient handling, while the two phosphors are at a concentration equal to t.hat of the stock toluene scintillator added later during sample preparation. The apparatus used here consists of u diffusion system, with wide cross sections, in which the gas (from a cold trap or a generator) is released directly into a counting vial through an adapter. The vial contains the necessary amount of CO, reagent which is kept, near 0” and agitated by a magnetic stirrer, and about 20 min is allowed for t.he gas uptake with an initial and final pressure of 100-150 mm. We define a “millimole equivalent.” of reagent. as the quantity capable of reacting quantit.atively with 1 mmole CO? under the conditions just described. This corresponds to 1.1 ml of the Hyamine base (1 &I in methanol) or 1.5 ml of the phenethylamine-alcohol-toluene mixt,ure. ‘For example, for equal molar concentrations, quenching increases in the ordel tc-butylamine < di-n-butylamine < tri-n-butylamine. ’ Abandoned (mostly for reasons of insolubility of the carbarnates) were: bellxylamine, t-octylamine (kindly supplied by Rohm & Haas, Inc.), n-dodecylamine, ;md 4-(aminomethpl)cyclohexanemethanol. ’ Matheson, Coleman & Bell Division, Cat, No. 6066; price $11.25 per kilogram
510
SHORT
COMMUNICATIONS
Counting efficiencies were compared for preparations in which either 1 M Hyamine or the phenethylamine mixture were employed. All samples throughout were brought to 10 ml with toluene scintillator solution, and to final concentrations of 0.5% PPO and 0.01% POPOP. In a Packard Tri-Carb spectrometer at -10” (background 25 counts/min), 2 mmoles CO, could be counted with 60% efficiency when the phenethylamine mixture was used.5 The ‘Lmillimole equivalent” Hyamine sample gave less than 30% efficiency. In a custom-built spectrometer operating in coincidence at room temperature, 5 mmoles CO, could be introduced into 10 ml (containing 7.5 ml of phenethylamine mixture) and counted at 50% efficiency. At t,his temperature, part of the toluene can be replaced by naphthalene; this replacement results in even better efficiency.g The corresponding Hyamine sample (employing 5.5 ml of the 1 M solution) proved to have an efficiency close to zero. It is to be concluded that the phenethylamine mixture is superior to the “millimole equivalent” Hyamine preparations in regard to counting efficiency. From other experiments it appears reasonable to assume that the slight reduction in relative methanol content causes only part of t.his difference. It should be pointed out that with both reagents the efficiency shows little dependence on the amount of gas reacted. For the most accurate work the internal standard method (addition of a known amount of W-labeled toluene) is recommended. That the C?“O, is quantitatively absorbed into the phenethylamine solution was demonstrated by separately generating different known amounts of the gas (from accurately weighed BaWO, of known specific activity) into a fixed quantity of the amine. A straight-line relationship was established on plotting the liquid scintillation counting rate against the quantity of C’“0, used. SUMMARY
Phenethylamine has been found to be superior to the widely used “Hydroxide of Hyamine 10-X” as a compound to convert C’“O, into a soluble form for liquid scintillation counting. REFERENCES 1. KRAKAU, G., AND SCHNEIDER, H., Atomkernenergie 3, 515 (1958). 2. FUNT, B. L., AND HETHERINGTON, A., Science 129, 1429 (1958). 3. PASSMAN, J. M., RADIN, N. S., AND COOPER, J. A. D., Anal. Chem.
28, 484
(1956).
‘Samples containing CO2 in excess of 2 mmoles/lO ml of total solution showed incipient crystallization when kept at -10” for prolonged periods. ‘Concerning this phenomenon, see Kallmann, H., and Furst, M., ~TL “Liquid Scintillation Counting,” pp. 14-17. Pergamon Press, New York, 1958.
SHORT
511
COMhIUNICATIONS
F., in “Liquid Scintillation Counting,” (C. G. Bell, Jr., and F. N. Hayes, eds.), p. 123. Pergamon Press, New York, 1958. OPPERMANN, R. A., NYSTROM, R. F., NELSON, W. O., AND BROWN, R. E., Intern.
2. EISENBERG, 5.
J. Appl. Radiation and Isotopes ‘7, 38 (1959). R. G., PEETS, E. A., GORDON, S., AND BUYSKE,
6. KELLY,
D. A.,
Anal. Biochem.
FRITZ
H. WOELLER
267 (1961).
Lawrence
Radiation Laboratory University of California Berkeley, California Received March 14, 1961
2,