Acetyl Group Determination Using Acetic Anhydride
C14
ROYAL H. BENSON' and RICHARD B. TURNER2 Radioisotope Research Laboratory, Veterans Adrninistrafion Hospital, Houston, rex.
b Acetyl groups are determined by measuring the specific activity of compounds acetylated with C14 acetic anhydride of known specific activity. This method ofFers accuracy and freedom from many common errors and is applicable to milligram quantities of unknown compounds. The acetylated material may b e recovered unchanged after analysis.
T
HE DETERMINATION of acetylatable hydrogen has been important in the investigation of the structure of many compounds. Some commonly used methods include that of Verley and Bolsing (4, in which phenolic groups are titrated with acetic anhydride in pyridine, the method of Pregl and Grant (Z),of hydrolysis of the acetyl derivative, distillation of the liberated acetic acid followed by titration of the distillate, and the method of DeRalt and Glenn ( I ) , by titration of unreacted acetic anhydride-pyridine reagent. These methods and their modifications are proved procedures but, in some cases, do not give valid results. Errors are usually introduced when unexpected decompositions occur, or unexpected acidic components are present. It appeared that the treatment of compounds containing acetylatable hydrogen with C14 acetic anhydride of known specific activity, followed by the determination of the specific activity of the acetylated product, would 1 Present address, Monsanto Chemical Co., Texas City, Tex. * Present address, The Rice Institute, Houston, Tex.
give accurate acetyl group values with a minimum of errors. APPARATUS A N D REAGENTS
Carboxyl-labeled C14 acetic anhydride, specific activity approximately 2 /IC. per mmole, is prepared in 90% yield from fused C14 sodium acetate and acetyl chloride by refluxing 24 hours in dry ether. After fractionation, the product is kept under nitrogen. Acetic anhydride prepared by this procedure, and stored in this manner, assayed better than 98 mole % purity after 1 year. PROCEDURES
Acetylation in Pyridine. T o 1 mmole of compound in 1 ml. of anhydrous pyridine in a 5-ml. flssk add 2 mmoles of Cl4 acetic anhydride for each expected hydroxyl group. Heat the mixture at 125' C. for 4 hours, cool, pour into 50 ml. of cold water, and rinse into a separatory funnel with 50 ml. of ether. Extract the ethereal layer three times with dilute HC1, once with dilute KaOH and wash with water and saturated NaCl solution. Dry by filtration through anhydrous Na2S04. If the product does not crystallize readily following evaporation of the ether, dissolve the residual oil in benzene and chromatograph on 15 grams of activated silicic acid. The acetylated product is usually eluted by a mixture of benzene and chloroform. rlppearance of the product in the eluate is readily determined by evaporating a small aliquot to dryness and counting the radioactivity in the residue. The fractions containing the product are combined, evaporated to dryness, and the product purified by crystallization and recrystallization. Acetylation Catalyzed by Sodium
Table I. Acetyl Group Determinations A4cet,vl Wt. of Samde ~. .
Compound Ethyl lithocholate Methyl deoxycholate Methyl cholate Oubagenin tetraacetate B Oubagenin tetraacetate A Gossypol Acetic anhydride
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0
Groubs Calculated
Sample Counted, Mg.
C.P.M. f 1%
C.P.N./ Mmole, All.
Acetyl Groups Found
4 6 2
19.245 11.201 6,946 5.368 11.437 7.800 7.741
60 1 647 536 386 1060 878 2 140
13,948 28,357 42,338 40,561 56,191 86,673 28 , 199
0.99 2.01 3.00 2.88 3.98 6.15 2.00
ANALYTICAL CHEMISTRY
Kit
T o 0.1 ml. of C14 acetie anhydride in a 5-ml. flask, add slightly less than the theoretical amount of I N NaOH. Evaporate the solution t o dryness, fuse, and grind the residual solid t o a powder. Add 1 mmole of hydroxy compound and 2 mmoles of C14 acetic anhydride for each expected hydroxyl group. After heating a t 125' C. for 4 hours, the product is treated as before, with omission of the HCl extraction. Determination of Specific Activity. The method of Schwebel, Isbell, and Moyer (3) is used. Weigh a sample of acetylated material, 5 to 20 mg., into a 1-ml. recalibrated volumetric flask and dilute to volume with dimethylformamide (redistilled). After mixing, pipet 0.5 ml. of solution into an aluminum planchette 1/g inch deep, 15/18 inch in outside diameter, '/a inch in inside diameter resting in the flow counter (Tracerlab). After preflushing and counting, a duplicate is prepared from the remaining 0.5 ml. and counted. The specific activity of the acetic anhydride is determined in the samc manner. Acetate.
It is advisable also to count a fixed standard C14 source for corrections due to changes in counter efficiency. The acetylated compounds may be recovered after counting by evaporation of the solvent. All specific activities are calculated in counts per minute per millimole of compound. The number of acetyl groups is determined by dividing the specific activity of the acetylated compound by one half the value obtained for the specific activity of acetic anhydride. DISCUSSION
Table I lists the results for acetyl group determinations on six compounds containing from one to six acetyl groups. The accuracy of the method, indicated by the results, ranges from i1 to =k4%',, depending on the substance analyzed. The t\vo sources of error inherent in this method are the determination of specific activity and the purity of the acetylated product. To obtain the results shown in the table, duplicate samples of each product were counted to at least 40,000 counts to obtain =k 1% accuracy a t the 95% confidence level. Our experience has shown that sample prep-
aration and geometry can be duplicated with precision greater than the conntr ing error. The error due t o the degree of purity of the acetylated product was elitninated by purification to constant specific activity. For routine determinations the accuracy obtained by these rather extreme measures may not be necessary. The position of the labeled carbon atom in the acetic anhydride is not of great importance. The possibility of an error due to the isotope effect may be eliminated by the use of acetic anhydride-2-GI4; however, an error from this source has not heen detected. Acetylation in pyridine is probably the most widely applicable acetylation method. In the case of gossypol however, extensive decomposition was encountered. Acetylation, catalyzed
by sodium acetate, proved successful without observable decomposition. The specific activity of the acetic anhydride and the sensitivity of the available counting equipment determine the size of sample required. If acetic anhydride of 20 pc. per mmole is prepared, then only 0.5 mg. or less of acetylated material is necessary for this det.ermination, with the equipment described. If a liquid scintillation counter is available, the determination of specific activity is simplied, sample size requirements are even smaller, and counting error may be reduced because of the greater sensitivity of such instruments. This method offers several advantages over previous acetyl group methods. First, there is no hydrolysis of the derivative and, consequently, no possible
errors from unexpected decomposition or incomplete hydrolysis. Second, there is no distillation involved, eliminating the errors of loss, or errors from unexpected volatile components. Third, there is no titration, eliminating the errors due to unexpected acidic components. Finally, the sample of acetylated material may be completely recovered, unchanged, for further work. LITERATURE CITED
(1) DeWalt, C. W., 1Glenn, R. A,, ANAL. CHEM.24,1789 (1952).
J.,, "Quantitative Mibroanalysis," 4th ed., p. 161, Blakiston, Philadelphia, Pa., 1945. (3) Schwehel, A., Isbell, H. S., Moyer, .I. D.. J . Research Natl. Bur. Standards
(2) . . Preel. F.. Grant.
Org&
53, 2 2 1 4 (1954). (4) Vcriey, A,, Bolsing, F., Ber. 34, 3354 (1901). RECEIVED for review February 15, 1960. Accepted July 12, 1960.
Tritium Radioactivity Determination of Biological Materials by a Rapid Dry Combustion Technique EDWlN A. PEETS, JAMES R. FLORINI, and DONALD A. BUYSKE Pharmacological Research Department, Experimental Therapeutics Research, lederle Laborotories Division, American Cyanamid Co., Pearl River, N. Y.
b
In pharmacological and biological research it i s often necessary to determine the tritium radioactivity of a large number of samples of biological materials having varied specific activities. A p,roctical method of measuring the radioactivity of large numbers of such samples i s dry combustion of the material to water and carbon dioxide and determination of the radioactivity of the products using a liquid scintillation spectrometer. Since the currently available apparatus can accommodate only single samples of relatively small size, a furnace has been designed which makes possible the rapid simultaneous combustion of three samples of up to 2 grams dry weight.
of biological materials into a solubilizing or suspending system (6, 10, 13, 14) for liquid scintillation counting has been suggested, but again sample size is limited. I n addition, most biological materials are too highly colored to allow satisfactory counting by the liquid scintillation technique. It is apparent, therefore, that complete combustion of such samples to carbon dioxide and tritium-enriched water would offer considerable advantages for this type of determination, Several sealed tube combustion techniques have been pub-
lished (1, 16, 17) but they are limited t o 5- to 25-mg. samples. The apparatus t o be described is an enlarged and modified version of the standard carbon-hydrogen apparatus. It can oxidize simultaneously three samples of up to 2 grams dry veight; the entire heating and cooling cycle requires approximately 45 minutes. Thus, 24 t o 30 samples can be prepared for counting in a normal working day. In addition t o preparing samples containing tritium for radiometric determination, this apparatus is applicable to
B .
IOLOGICAL and
pharmacological studies often necessitate the deterniination of radioactivity in both liquid and solid samples of biological origin. In studies involving tissue distribution and residue levels of drugs in plants and animals, it is not infrequent that 100 or more samples may be collected for radioactivity determinations. Due to the estremely weak energy of tritium, infinitely thick samples are less than 1 mg. per sq. em. (4). Therefore, it is not possible to make direct radiometric measurements on any except very small samples of a solid. Direct incorporation
Figure 1. Combustion furnace showing combustion tubes and one trap VOL. 32, NO. 11, OCTOBER 1960
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