Lisuid Scintillation Counting of Radioactive Sulfuric Acid and Other Substances NORMAN S. RADIN and RAINER FRIED Northwesfern University Medical School and Veferans Administration Research Hospit a/, Chicago, 111.
b A method i s described for the determination of sulfur-35 in large amounts of sulfuric acid using a liquid scintillation counter. The acid i s converted to a neutral salt of a high molecular weight aliphatic primary amine and i s dissolved in toluene-ethyl alcohol. The efficiency of counting i s almost independent of the amount of sulfate, but does depend on the amount of amine. Other acids, such as radioactive citric, phosphoric, and hydrochloric acids can be counted b y this technique. Preliminary findings on the counting of monosaccharides are presented also.
T
OLUENE is the preferred solvent for liquid scintillation counting. Many toluene-insoluble substances which form salts can be dissolved in toluene by the use of an acid or base of high molecular weight and high hydrocarbon content. A quaternary ammonium hydroxide has been used in this way for counting radio:xtive carbon dioxide ( 7 ) , hydroxy acids (Q), purines (z?), amino acids and proteins (8, IO), acidic polysaccharides ( G ) , and even whole tissues (I). High molecular n-eight quaternary amines are not commercially available as the free bases, and it is necessary to convert the salt form to the base form. It seemed likely that the more strongly acidic radioactive substances could be dissolved b y a veaker base, such as a primary amine, and the need to form the free base n-odd then be obviated. Primene 81-R (Rohni & Haas Go., Philadelphia, Pa.) is suitable for several such applications. This amine is a mixture of similar amines with average molecular weights of 191 and contains a primary amine group attached to a tertiary carbon atom. This point of attachment improves the stability and tends to prevent chemical reactions n-ith the sample. The carbon chain contains many branched methyl groups, Ivhich improve the solubility. This paper describes details of the use of Primene for sulfate counting, as well as prelininary trials n i t h other substances.
APPARATUS
Samples are counted in 4-dram screw 1926
ANALYTICAL CHEMISTRY
cap vials Ivith a Tri-Carb liquid scintillation counter (Model 314, Packard Instrument Co., LaGrange, Ill,), A small square of tin foil, 0.001 inch thick, is placed over the mouth of the vial before screwing down the cap. The excess foil is then pushed up to avoid blocking the light. The temperature of the freezer is set so that the photomultiplier tubes and lead shield are kept at about 1-13' C. Precipitation of the terphenyl, the primary scintillator, may occur if the sample is stored in the lower part of the freezer, and samples are stored a t a level close to that of the counting chamber. The solvent in the radioactive samples is removed with a rotary vacuum evaporator (Rinco Instrument Co., Greenville, Ill.) which is fitted with a water-cooled condenser leading directly downward to a water aspirator. The evaporations are performed in a 300-ml. Florence flask, which is attached to the evaporator by a rubber stopper. MATERIALS
A slightly acidic solution of Priniene acetate is made b y making u p a solution 0.5M in Primene and 0.55M in acetic acid in toluene (J. T. Baker Chemical Co., ACS grade). The Primene should be redistilled in vacuo if it contains more than a slight color. The scintillation solution is 0.4% terphenyl and 0.01% 1,4-di [2-(5-phenyloxazolyl) ] benzene (4) in toluene containing 5% absolute alcohol. For the sulfate experiments, a nearly carrier-free solution of Fulfuric acid was prepared by passing radioactive sodium sulfate through a small column of Dowex 50-H+. Additional nonradioactive sulfuric acid was added in varying amounts. PROCEDURE
The radioactive acid, in a few milliliters of water, is placed in a 300-ml. Florence flask, with 25 ml. each of toluene and methanol, and a suitable amount of Primene acetate solution. The mixture is evaporated under vacuum to a sirup in a 40' C. water bath. The methanol and toluene help t o remove the water and excess acetic acid. The methanol also helps to prevent splashing. To ensure that all the water has been removed, 25 ml. of toluene are added, the flask is srrirled to dissolve the sirup, and the evaporation is continued.
The sirup is dissolved in the scintillation solution by shaking or sn-irling a t least 5 minutes, and an aliquot is transferred to the counting vial. The vial is cooled for a t least 1 hour before counting. Virtually the entire sample can be counted, if desired, by pouring the mixture into the vial. The loss n i t h 10 ml., on careful drainage, is only about 1.5% and is rather reproducible. RESULTS A N D DISCUSSION
Effect of Free Amine. Tests with free Primene and sulfuric acid, processed as described, indicated t h a t the presence of free excess amine has very little effect on t h e counting efficiency, although there seems to be more variability in t h e counting efficiency. The salt of a volatile acid (acetic) n as used because t h e free base smells had and because t h e amine is slightly volatile and distills into the evaporator with the water. At the higher levels of amine, the resultant possible variation in the amount distilled would cause variations in the counting efficiency. Noreover, the free amine might react n ith organic compounds accompanying the sulfuric acid (as from a biological extract) to produce quenching substances. A neutral counting solution n as considercd to be more stable. Amount of Amine. Tablr I s h o w the results 11-hen various amounts of Primene acetate were used with 0.05 mmole of S3j-sulfuiic acid. The Primene acetate-sulfate sirup TI as transferred t o 25-nil. rolunietric flasks and made u p t o volume with t h e scintillation solution. Twenty-niilliliter aliquots Tvere used for counting and all samples contained the same amount of radioactivity. The counting efficiency is independent of amount of amine a t low levels (the first three values are identical within the precision of the method) and decreases slovi.ly with increasing levels. Evidently larger amounts of sulfate could be counted with good efficiency. If variable amounts of sulfate are to be counted, the simplest procedure is to use a constant, large amount of Primene. Counting is begun immediately after inserting the sample into the counting
Table 1.
Counting Rate a s a Function of Primene Content" Amount of Observed Primene, Activity, Mmoles C.P.M. 0.12
19,750 19, i 3 0 19,910 19;200 18; 910 18,180
0.20
0.40 2.00 4.00 8.00
Amounts of Primene acetate actually used were 25% greater than amounts listed here, as only 20 out of 25 ml. were counted. Table II. Counting Rate as a Function of Sulfate Content" Amount of Observed Sulfate, Activity, Mmoles C.P.M. 0.5
Av. 1.0
Av. 2.0
-4v. 4.0
Av.
33,390 34,020 33,700 33,760 33,320 33,540 33,340 33,510 33,420 32,550 33,330 32,940
Amount in each vial was actually about 57% of the amount listed (explanation in text).
chamber, as there is no phosphorescence effect under ordinary lighting conditions. A test with a blank (containing nonradioactive sulfate and Primene) showed no phosphorescence even a t the higher voltage needed for tritium counting. Several S36 samples m r e counted immediately after preparing and cooling, and a t intervals over a period of 1 week. No change in counting rate other than decay was observed. The slight variations in the machine were corrected b y a C14 standard, which matched the S35 scintillation spectrum moderately well. The absolute efficiency of counting S3bulfate with this method could not be determined directly, because no standard seemed to be available. The addition of 0.05 ml. of a secondary C14 standard solution to a sample made from 0.25 mmole of aniine acetatesulfate gave a counting rate very nearly equal to that obtained with the standard and scintillation solution alone. Because the efficiency of counting C*4 under these conditions is about 70% (Y), and because the scintillation spectrum of S35 is similar to that of (314, the efficiency of counting S35 in this sample is probably about 65%. Only the scintillation pulses of 10 volts and over are presented. Amount of Sulfate. Table I1 shows t h e effect of varying amounts of
sulfuric acid, using 10 mmoles of Primene acetate. All samples contained t h e same amount of S35. T h e sirup was dissolved in 15 ml. of scintillation solution, and a 10-ml. aliquot was counted. T h e amount of sulfuric acid, within t h e limits of t h e range studied, is almost without effect on the counting efficiency. This corresponds to a lack of self-absorption correction, for a considerable range of amounts, and only a rough knowledge of the amount of sulfate being counted is needed. The ralues of duplicate samples are included to show the typical variability encountered. A 0.5% variation can be observed with any sample, merely by changing the position of the vial in the counting chamber. Much of the remaining variability is probably due t o variations in the diniensions of the vials. Samples containing only trace amounts of sulfate gave counts that diminished with time. It is possible that adsorption onto the vial or tin foil takes place. Benzidine Sulfate. T h e usual method of counting $335 involves oxidation to sulfuric acid, precipitation as t h e benzidine salt, and plating of t h e salt for Geiger counting. Because of self-absorption, this method is soniewhat less efficient, particularly a t high levels of sulfate, but has t h e advantage t h a t the weight of sulfate can be determined simultaneously. Khere desirable to determine the m i g h t of sulfate b y this method, the benzidine salt can be counted by liquid scintillation, using free Priniene t o ninke it dissolve. A test with 50 mg. of benzidine sulfate, 0.5 ml. of Primene, and 10 ml. of S3%ontaining scintillation solution revealed that benzidine quenched the counting rate b y about 65y0. The counting rate did not become stable until several hours had passed. For optimum efficiency, it would be better to dissociate the benzidine salt with alkali and ether, remove the alkali b y cation exchange, and convert the sulfuric acid to the Priniene salt. Interfering Compounds. I n studies of sulfate metabolism in biological systems, it is convenient t o avoid combustion of t h e organic material, Cations and amino acids can be removed readily b y ion exchange, b u t nonionic and anionic material will still remain. T o investigate t h e effect of typical contaminants, these were added t o 10 mmoles of Primene acetate and 2 mnioles of S35-sulfuric acid, and the samples were processed as before, S o effect was observed with 10 mg. of glucose, 1 mmole of hydrochloric acid, or 0.5 mmole of phosphoric acid. The counting rate n-as 1oLvered 2.5y0 by 1 mmole of citric acid. It is safer to check the efficiency of unfamiliar
systems with a small amount of standard toluene-soluble S35. Counting Other Isotopes. I n these tests, glucose, chloride, phosphate, and citrate dissolved in the scintillation mixture. Therefore, these substances could be counted n-ith high efficiency if they themselves n ere radioactive. Presumably other caiboxylic acids, labeled n i t h tritium or (214, could be counted, as well a s phosphate and sulfate esters. 1Iucic acid, the highly polar hydroxy acid, and alanine, a n amino acid, did not dissolve appreciably in this system. nor did t h e use of Priniene base or the addition of methanol help. Results with glucose suggest that this inethod can be adapted to the counting of carbohydrates. It is likely that a n 5-glycosyl derivative forms with the amine. Some experimentation n ith larger amounts of glucose suggest that the following approach is satisfactory. T o a counting vial vere added 50 mg. of glucose, 0.5 ml. of free Primene, and 4 nil. of methanol. A piece of tin foil and a cap viere placed looselj- over the vial, the vial was lon-ered part way into n 65" C. n-ater bath, and, after the air in the vial \vas displaced b y methanol vapors, the cap was screwed don n tightly. Heating was continued for 2 hours, with occasional shaking until the glucose dissolved. the T-ial was cooled, and 15 nil. of toluene containing 78 nig. of diphenyloxazole (3) wcre added. The addition of 0.05 nil. of C1' qtandard solution revealed a counting efficiency of roughly 45%. The loner efficiency 11as due primarily to the methanol, \\ hich was needed to dissolve the larger amount of glucose derivative. Probably more efficient solvents could be found ( 2 ) . After several days of storage in the cold, a few crystals precipitated. d n other trial, with 3 hours of heating gave a slightly yellowish,solution. which did not yield any crystals for a xveck. Glucuronolactone rapidly gave a bronn color with free Priniene, even at room temperature. The use of Primene acetate as in the sulfate method, but with a cooler water bath during the evaporations, gave a slightly colored sirup which dissolved readily in the toluene-ethyl alcohol scintillation solution. The efficiency of counting, with 50 mg. of lactone, 2.5 mmoles of acetate, and 10 ml. of scintillation solution, was roughly 55%. The solution gradually turned brown. Possibly a secondary or tertiary amine m-ould be more suitable for such sensitive substances. An earlier paper on the liquid scintillation counting of sulfate describes a complex mixture, which apparently holds very small amounts of sulfuric acid in solution ( 5 ) . Sodium sulfate, even in small amounts, has not in our experience given stable counts with simple scintillaVOL. 30, NO. 12, DECEMBER 1958
* 1927
tion media, presumably because of gradual precipitation.
Institute of Neurological Diseases and Blindness, Public Health Service. LITERATURE CITED
ACKNOWLEDGMENT
(1) Agranoff, B., Northwestern Univer-
The authors wish to express their appreciation to Beverly Brown and Barbara Roecker for carrying out preliminary experiments on this problem, and to the Rohm & Haas Co. for gifts of their high molecular m-eight amines. The work was supported in part by grant No. B-1179, from the National
sity Liquid Scintillation Counting Symosium, August 22, 1957, Evanston, Ill. (2p Davidson, J. D., Feigelson, P., Intern. J . A p p l . Radiation and Isotopes 2, l(1957). (3) Hayes, F. N., Hiebert, R. D., Schuch, R. L., Science 116,140 (1952). Ott, D. G., Kerr, V. K., (4) Hayes, F. N., Nucleonics 14, No. 1, 42 (1956). (5) Helf, S., Castorina, T. C., White,
c. G., Graybush, R. J., ANAL. Cmar. 28, 1465 (1956). (6) Markovitz, A., University of Chicago,
personal communication. (7) Passmann, J. M., Radin, N. S., CooDer. J. A. D.. ANAL.CHEM.28, 484 (1956).' (8) Radin, N. S., Sorthwestern University Liquid Scintillation Counting Symposium, August 21, 1957, Evanston, Ill. (9) Radin, N. S., Martin, F. B., Brown, J. R., J . Bwl. Chem. 224,499 (1957). (10) Vaughan, M., Steinberg, D., Logan, J., Science 126,446 (1957). RECEIVEDfor review February 7, 1958. Accepted July 28, 1958.
Spectrophotometric Determination of p -tert- Buty Icatec hol a nd 0-Amino p he no1 in 2-Methyl-5-vinylpyridine KURT H. NELSON and M.
D. GRIMES
Phillips Petroleum Co., Bartlesville, Okla. p-tert-Butylcatechol in 2-methyl-5vinylpyridine can be determined b y dissolving the sample in 1.ON hydrochloric acid and extracting the p-fertbutylcatechol with diethyl ether. The p-tert-butylcatechol i s then extracted from the ether with 1.ON sodium hydroxide and i s air oxidized to the colored quinoid form for spectrophotometric measurement a t 485 mp. oAminophenol is determined b y dissolving the 2-methyl-5-vinylpyridine in n-heptane and extracting the oaminophenol with 0.1 N hydrochloric acid, The acid phase i s buffered with ammonium acetate-acetic acid and the o-aminophenol i s oxidized with hydrogen peroxide, after which its absorbance i s measured at 435 mp. About 40 mFutes is required to analyze a sample for both inhibitors.
I
N THE MANUFACTURE of 2-methyl-5-
vinylpyridine, inhibitors are added to prevent polymerization and other undesirable reactions during shipment and storage. Usually, p-tert-butylcatechol is added as the inhibitor, but oaminophenol may also be added for this purpose. Enough of these inhibitors is added to give an initial concentration in the range of 0.0 to O.3y0p-tert-butylcatechol and 0.0 to 0.1% o-aminophenol. Because the concentrations of the inhibitors decrease with time, a method for the determination of the concentration level of either inhibitor is necessary to assure continued effectiveness. The direct application of ultraviolet spectrophotometry to the determination of the inhibitors did not appear feasible because the 2-methyl-5-vinylpgridine 1928
ANALYTICAL CHEMISTRY
is a very strong absorber in the spectral region around 280 to 286 mp and would mask the bands arising from the p-tertbutylcatechol and o-aminophenol. p-tert-Butylcatechol in butadiene has been measured by a ferric chloride colorimetric procedure (6), by a ceric sulfate titration method (?'), and by ultraviolet measurements after removal of the butadiene by evaporation (1). It has also been determined colorimetrically in 2-methyl-5-vinylpyridine after extraction into sodium hydroxide solution, During the extraction, the p-tertbutylcatechol is air oxidized to a colored quinoid form ( 3 ) . No method for determining o-aminophenol in 2-methyl-5-vinylpyridine was found in the literature. o-Aminophenol in other materials has been determined by titration with standard perchloric or hydrochloric acid in glycol-hydrocarbon solvents, using potentiometric or thymol blue end points ( 2 ) . Another procedure describes the oxidation of o-aminophenol with an excess of standard ceric sulfate, followed b y back titration of the remaining ceric sulfate with ferrous sulfate (8). o-Aminophenol has also been determined b y fluorescence measurements after reaction with benzoyl chloride (6). None of these existing methods, except the procedure for p-tert-butylcatechol in 2-niethyl-5-vinylpyridineI appeared applicable to the present case. Preliminary experiments, however, showed that the procedure for p-tertbutylcatechol gave erroneous results when o-aminophenol was also present. Therefore, the procedures described below were developed.
REAGENTS
BUFFER SOLUTIOS. Dissolve 200 grams of reagent grade ammonium acetate in 500 ml. of distilled water, add 10.0 ml. of glacial acetic acid, and dilute with distilled mater to 2000 ml. in a volumetric flask. 2-1\kTHYI&VINSLPYRIDINE. Distill 1000 ml. of 2-methyl-5-vinylpyridine a t 10 nim. pressure and 64' C. Collect approximately 800 ml. of distillate after discarding the first 20 ml. p-teTt-BUTYLCATECHOL. Purify by distillation a t 4 mm. pressure and 140' C. Prepare a stock solution by dissolving 1.000 gram of purified material in 2methyl-5-vinylpyridine and diluting t o 100 ml. with 2-methyl-5-vinylpyridine. To prepare standard solutions, transfer 0.0, 5.0, 10.0, 15.0, and 20.0 ml. of the stock solution to 50-ml. volumetric flasks and dilute to volume with 2methyl-5-vinylpyridine. 0-AMINOPHENOL. Recrystallize from ethyl alcohol. Prepare a stock solution by dissolving 0.2500 gram of purified material in 2-methyl-5-vinylpyridine and diluting to 50 ml. with 2-methyl-5vinylpyridine. To prepare standard solutions, transfer 0.0, 2.0, 4.0, 6.0, 8.0, and 10.0 ml. of the stock solution to 50-ml. volumetric flasks and dilute to volume with 2-methyl-6-vinylpyridine. APPARATUS
SPECTROPHOTOMETER. A spectrophotometer capable of recording the spectrum in the 300- t o 700-mp wave length region is preferable. A Cary Model 11 recording spectrophotometer was used in this investigation. A Beckman Model DLT or B spectrophotometer may be used by measuring absorbances at three wave lengths. Quartz or Corex cells with a 1-em. light path are also used.
