confirmed at higher molecular weights by comparing the ~ ~ ~ a l uobtained es using the acetic anhydride-pyridine and 3-nitrophthalic anhydride-triethylamine methods 011 a 20,000 molecular weight sample of polyethylene glycol, where no absolute knowledge of its hydroxyl content was available. A summary of the precision and accuracy studies are shown in Table I. h close correlation was attained between the two independent methods of analysis on polyethylene glycol 20,000, while the 3-nitrophthalic anhydride method gave good agreement in the polyethylene glycol 400 case. The standard deviations in all cases fell within approximately 11%of the average milliequivalents/gram of hydroxyl in the two materials under consideration. Table I1 lists many of the alcohols studied and demonstrates the applicability of the method. From the data it is readily seen that the method is applicable only to primary alcohols. I n the case of the polypropylene glycol, some serondary alcohol groups are present, the assumption being that synthesis with propylene oxide can produce either primary or secondary terminal alcohols. Similarly, glycerol, containing a secondary alcohol group, is not completely esterified. INTERFERENCES
Primary and secondary aliphatic and aromatic amines will react with the reagent and will interfere with the determination.
Table Ill.
Time and Temperature Effects on Meq./gm. Hydroxyl for Secondary Alcohols
Alcohols Isopropanol
.set-Butanol
Glycerol Glycerol Glycerol Glycerol
T(pp., C. 25 25
0 25 40 60
10 min. 5.35 3.74 24.4 26.9 27.8 30.0
Secondary alcohols do not react quantitatively with the reagent. This is best shown in the data obtained from extended time and increased temperature studies performed on this class of alcohols. Results of this study are shown in Table 111. While both extending the reaction time and increasing the reaction temperature increase the degree of esterification, much more rigorous esterification conditions would be required, in order to attain quantitative results in a reasonably short time. Tertiary alcohols do not esterify with the reagent. The presence of water interferes with the determination by hydrolyzing the anhydride. ACKNOWLEDGMENTS
The authors wish to express their appreciation to J. R. Martin for his interest and helpful suggestions, and also to R. bl. Baillie, Jr., who performed all of the experiments connected with this development.
Meq./gm. Hydroxyl 30 min. 60 min. 120 min. 10.7 13.8 l;, . 5 7.66 10.5 12.2 26.9 29.3 ... 29.6 30.3 30.8 30.6 ... .. 30.2 .,. , , ,
Theory 16.6 13.5 32,6 32 6 32 6 32 6
LITERATURE CITED
(1) Budd, 11. S., AXAL. CHEY.34, 1343 i1962).
Bush, D. G., Iiunzelsauer, L. J., LIerrill, S.H., Zbid., 35, 1251 (1963). ( 3 ) Fritz, J . S.,Schenk. G. H.. Zbid.. 31, 1808 i 19j9). (4) Gutnikov, G., Schenk, G. H., Z b i d . , 34, 1316 (1962). ( 5 ) Kyriacou, U., Ibid., 32, 291 (1960). (6) Nehlenbacher, V. C., "Organic Analysis," Vol. I, - pp. 1-38, Interscience, Sew York, 1903. (7) Robinson, W. T., Cundiff, R. H., Uarkunas, P. C., ANAL. CHEY. 33, 1030 (1961). (8) Siggia, S., Hanna, J. G., I b i d . , 33, 896 (1961). (9) Siggia, S , Hanna, J. G., J . Polymer Sci. 56. 5 6 , 297 11962). (1962). (10) Sigiia, Siggia, S.,~ S., Hanna, J. G., Culmo, R., ANAL.CHEM.33, 900 (1961). (11) Stetzler, K. S., Smullin, C. F., Ibid., 34, 194 (1962). ( 1 2 ) Veraguth, A. J., Diehl, H., J . Am. Chem. SOC.62, 233 (1940). (2j
RECEIVEDfor review April 29, 1964. Accepted August 3, 1064. Division of A4nalyticalChemistry, Fisher Award Symosium honoring John llitchell, Jr., 147th !leering, ACS, Philadelphia, Pa., April 1964.
A Low-Level Carbon-1 4 Counting Technique L. T. FREELAND Plastics Department, Mcrnufacturing Division, E.
b Liquid scintillation spectrometry has proved very useful in the dating of carbon- 14 containing artifacts. However, most carbon- 14 dating methods involve a tedious and usually low yield preparation of a suitable solvent for liquid scintillation spectrometry. The possibility of isotope effects is present in some of these procedures. This paper describes a tmechnique which circumvents the before mentioned limitations. The method requires the combustion of the scimple to COz, absorption of the CO:!in sodium hydroxide solution, followed by reaction with phenylmagnesiuni bromide to form benzoic acid. The benzoic acidC I 4 is esterified to methyl benzoate. This methyl benzoate is the solvent used in the assay of the sample by liquid scintillation spectrometry.
I . du Pont de Nernours & Co., Wilrningfon, Del.
SCINTILLATION spectrometry been used previously to determine both contemporary and lower concentrations of carbon-14 ( I , 2 , 8 ) . The syntheses of the solvent compounds for liquid scintillation counting have been time consuming, involved, and apt to produce chemical yields of usually less than 60% (3, 5 , If). This paper describes a method of stabilizing the liquid scintillation spectrometer and a simple method for synthesis of the solvent compound in high chemical yields. The stabilization of the liquid scintillation spectrometer is necessary because o the long counting times (24 to 72 hours) requ'red by the low concentrations of carbon-14. The method depends upon the complete combustion of the sample to CO, and the reaction of the resulting COz with phenyl-
c"h:s
magnesium bromide to yield benzoic acid which is esterified to methyl benzoate. The methyl benzoate is the radioactive sample as well as the solvent for the phosphor for liquid scintillation spectrometry. The overall chemical yield from original sample to purified methyl benzoate is 85 to 87%. EXPERIMENTAL
Liquid Scintillation Spectrometer. Samples were counted in a Tri-Carb Liquid Scintillation Spectrometer, Model 314-DC (Packard Instrument Co., La Grange, Ill.). T h e freezer containing samples, photomultiplier tubes, and preamplifiers is maintained at 0" C. T h e instrument is operated on an isolated electrical circuit equipped with a constant voltage regulator. The VOL. 3 6 , NO. 1 1 , OCTOBER 1 9 6 4
2055
CALCIUM
CHLORIDE
STIRRER
Figure 2. apparatus Figure 1. paratus
Combustion and
high voltage is set at 920 volts (high voltage tap 5) and discriminators adjusted to 10 to 100 volts to obtain maximum counting rate for carbon-14. To ensure instrument stability and low background counting rates, we have found the following electron tubes give the most desirable results in tbe previous instrument components. Preamplifier
Amplifier
CK 5703-WA Raytheon 6CL6 TungSol
C K 5702-WA Raytheon 6AL5 RCA 12BZ7 GE Discriminator Analyzer Scaler 6AH6 Ray- 12ilT7 Tung-Sol 12AX7 RCd theon 6AG7 RCA 6J6 RCA The spectrometer described above operates over a month's period as follows: the counting efficiency of a carbon-14 standard was 60.5 f 0.3% and background counting rate was 24.5 f 0.2 counts per minute. The stability of this spectrometer is quite satisfactory for assay of contemporary carbon- 14. Combustion of Sample a n d Absorption of COz. T h e combustion of the sample is similar to the Pregl method of direct ovidation in a n oxygen stream (IO). The combustion and absorption apparatus is shown in Figure 1. The combustion tube is packed as follows: section enclosed in top furnace, 4 inches of void space, 8 inches of 0.5% platinum on alumina 1/4-inch pellets; section enclosed in middle furnace, 6 inches of 'Ia-inch copper oxide pellets and 5 inches of coarse copper oxide wire; section enclosed in bottom furnace, 12 inches of '//4-inch copper oxide pellets; section enclosed between furnaces packed with l/ls-inch alumina beads. The absorbers are packed to a depth of 24 inches with Cannon protruded metal distillation packing, type 316 SS, size 0.16-inch X 0.16-inch (Scientific Development Co., Box 795, State College, Pa.). The column is filled 2056
ANALYTICAL CHEMISTRY
Grignard
reaction
absorption ap-
with 1 liter of 4.8M sodium hydroxide, prepared from 50% NaOH sdution (low in carbonate) just prior to use. This is to prevent contamination by absorbed atmospheric CO,. During operation the top furnace is maintained a t 850" to 1000' C. and the other furnaces at 850" to 900' C. The oxygen inlet manometer should indicate a AP of 15 to 20 mm. of H g with a flow of 2.5 liters of oxygen/minute. This flow is maintained by a vacuum pump filled with D C 550 silicone oil. This oil is very stable with no evidence of peroxide build-up. Liquid samples are injected through a n 18-gauge syringe needle or can be added as an aqueous slurry from a pressure-equalizing separatory funnel. Adequate sample should be combusted to yield 0.5 moles of COz. The rate of addition of sample is determined by the chemical composition of the sample and care is taken to prevent any positive pressure reading on the oxygen inlet manometer. At the completion of combustion, the apparatus is vented to the atmosphere, and the absorber column is drained and rinsed with two 250-nil. portions of distilled water. These rinses are added to caustic solution. The column is washed by filling to capacity 3 times with water andonce with 3.6X sulfuric acid and finally rinsed 3 times with distilled water and allowed to drain. This washing procedure prevents build-up of contaminants in the absorber columns. Reaction of CO, with Grignard Reagent. The Grignard synthesis equipment is shown in Figure 2. The caustic solution and rinses obtained from the combustion of the sample are added to the 3-neck flask. The separatory funnel is filled with 360 ml. of 85% phosphoric acid. T h e system is then purged with Seaford grade nitrogen for a minimum of 5 minutes. During this purging the Grignard reaction vessel is cooled to - 10" to -20" C. with a slurry of ice and methanol. Phenylmagnesium bromide 3X (Arapahoe Chemical Co., Boulder, Colo.) is diluted to a molarity of 2.3 with anhydrous diethyl ether. This 2.3-11
Grignard reagent gives a solution of good viscosity and no precipitation of the magnesium salt occurs a t -10" to -20' C. during the reaction with C o n . The nitrogen purge is stopped, and 450 ml. of 2.3-TI Grignard agent is added to the synthesis vessel. The phosphoric acid is added to the caustic solution within 45 minutes, since if more time is required secondary condensation products occur resulting in poor yield. .\ slight pressure of COz is maintained on the synthesis vessel by means of the 250-ml. gas collecting bags. Purification of Benzoic Acid from Grignard Reagent. The contents of the reaction vessel are drained by means of a stopcock into a 3-liter stainless steel beaker which contains 100 ml. of concentrated hydrochloric acid, 200 ml. of distiiled water, and 500 grams of crushed ice. During the draining, the contents of this beaker are stirred vigorously with an air-driven magnetic stirrer. The reaction vessel is rinsed twice with 150-m1. portions of 6-V HCI in methyl alcohol. The contents of t,his beaker are transferred to a separatory funnel and separated. The aqueous layer (lower layer) is extracted twice with 150-ml. portions of diethyl ether. The ether layers are combined and extracted three times with 200-ml. portions of 1.3.11 sodium hydroxide solution. The sodium hydroxide layers are combined and 70 ml. of 85% phosphoric acid is added. The acidified aqueous layer is then extracted 3 times with 150-ml. portions of diethyl ether. The combined diethyl ether extracts are placed in a 5OO-ni1. round bottom boiling flask and the diethyl ether is evaporated to dryness in a hood to obtain benzoic acid. Esterification and Distillation of Methyl Benzoate. I n t o the benzoic acid previously obtained is added approximately 350 ml. of refined and dry methanol (derived from fossil carbon a n d dried over sodium sulfate for 48 hours) and 10 ml. of concentrated sulfuric acid. Thiq solution is refluxed for 6 hours, then diqtilled to remove excess methanol until the distilling head temperature reaches 70' C. The methyl benzoate is diluted with a 200-ml. portion of diethyl ether and extracted with a 200-nil. portion of
distilled water. The ether layer is cooled in wet ice bath for a niinimuni of 5 minutes, then extracted with a 200nil. portion of cool 1.332 sodium hydroxide solution. This cold caustic extraction removes an:; excess benzoic acid from the ether-methyl benzoate layer. I3enzoic acid has been found to hublime during distillation of methyl benzoate and to cause a serious variable quenching phenomena in the methyl benzoate scintillation solution. The ether and water is removed by distillation and then the methyl benzoate fraction that boils from 198” to 203” C. is collected. The methyl benzoate thus ohtaincd is 99.957, pure as substantiated by gas chromatographic analysis, and saponification equivalent. Preparation of Scintillation Phosphor in Methyl Benzoate for Assay. T h e methyl benzoate scintillating solution is prepared to contain 0.094 gram of 2,5-di~)heii~loxazoleand 3.112 prams of naphrhalene in 18.0 grams of methyl benzoate in a polyethylene cwunting vial (Packard Inst. Co.. Vials, Polyethylc~ne 25-nil.). A convenient method for preparation of a number of counting vials a t one time is to dissolve the phosphors in benzene, then allow the solvent to evaporate. Alter drying the vials overnight in a desiccator containing Drierite add 18 ?C 0.0001 grams of previously prepared methyl benzoate. \Then solution is complete, the vial is cooled to 0” C., and counted as discussed previously. Counting times are determined to yield a 1% counting accuracy (9). Calculations. Shown below is the method for calculating; D.P.AI./gram carbon.
with previous assay of carbon-14 in contemporary material ( 7 ) . The calculation of the limiting age of a carbon-14 technique is given by the equation Limiting age =
(:E)
2.303 log
So is the net C.P.M. for contemporary carbon-14, B is the background C.P.M. and T is time assuming a counting time of 1440 minutes (24 hours) (6). The limiting age for the methyl benzoate lowlevel carbon-14 counting technique IS 31,250 years. I n the assay of these low levels of carbon-14, the internal standard technique is necessary to determine accurate counting efficiencies (4), since during distillation some compound distills over and quenches the counting rate of the methyl benzoate. I n countng replicate samples of material from the same distillation, the counting efficiency does not vary by more than &lyea t the 507, counting efficiency level. If the
