increased an already large final uncertainty in the low concentration range. The data in Table I clearly show that precision of the method is well within the uncertainty limits quoted above. When problems of reagent blank, diluent blank, and spike form are better resolved, it is predicted that accuracy and precision values will converge on the value reported herein for precision. The source of and means for reducing the reagent blank are under investigation. Although only a technique for the measurement of elemental carbon has been described in detail, the use of a spike of carbide prior to sample dissolution and a spike of BaC1303 prior to acidification, followed in each case by collection of the gas and measurement of isotopic composition, would enable measurement of total carbon in a sample. The method as described demonstrates improved sensitivity and precision over previous chemical techniques. I n the range of critical in-
terest from the carburization of stain10 to 30 less steel standpoint-i.e., p.p.m., 50 to 150 wg. in a 5-gram *2 to 5 p.p.m. accuracy sample-a uncertainty (95% confidence limit) has been obtained. This is considerably better than the 10 to 20 i 10 p.p.m. essentially given as a lower limit for chemical procedures, and is within the initially established goals for the method. The technique has been tested for accuracy over the range of 10 to 150 pg. of elemental carbon which represents 2 to 30 p.p.m. in a 5-gram sodium sample. Extension to higher concentrations facilitates the analysis and should introduce no problems. Precision in the 10-p.p.m. range has been demonstrated to be below the deviations reported for accuracy.
LITERATURE CITED
(1) Anderson, W. J., Sneesby, G. V., “Carburization of Austenitic Stainless Steel in Liquid Sodium,” NAA-SR5282, September 1 1960. (2) Calvin, M., Heidelberger, C., Reid, J. C., Tolbert, B. M., Yankwich, P. F.,
~
“Isotopic Carbon: Techniques in Its Measurements and Chemical Manipulation,” Wiley, New York, 1949. (3) Lockhart, R. W., Sabol, W. W., ‘%esults of the First Round Robin Analysis for Carbon in Sodium,” GE-APED letter document, February
23, 1963. ( 4 ) Mungall, T. G., Mitchen, J. H., Johnson, D. E., ANAL.CHEM.36, 70 (1964). (5) Pepkowitz, L. D., Porter, J. T., 11, “The Determination of Carbon m Sodium,” KAPL-1444, November 1955. (6) Stoffer, K. G., Phillips, J. H., ANAL. CHEM.27, 773 (1955). (7) Volk, W., “Applied Statistics for
Engineers,” McGraw-Hill, New York, 1958.
ACKNOWLEDGMENT
The authors acknowledge the assistance of Rachel Seite Dunn and Robert Wilbourn, who performed the chemical manipulation of the samples prior to mass spectrographic analyses.
RECEIVEDfor review March 12, 1964. Accepted May 18, 1964. Presented at the Winter Meeting of the American Nuclear Society in New York, November 18, 1963. This work was sponsored by the Atomic Energy Commiesion, contract NO. AT-( 11-l)-GEN-8.
Spectrophotometric Determination of p -Chloroaceta nilide in Phenaceti n-Acid Hy d rolysis Method W. B. CRUMMETT, J. SIMEK, and V. A. STENGER Special Services laboratory, The Dow Chemical Co., Midland, Mich.
b A procedure has been developed which is suitable for the determination of as little as 10 p.p.m. p-chloroacetanilide in phenacetin. Upon refluxing the sample with 48% hydrobromic acid, phenacetin is converted to p-hydroxyaniline hydrobromide whereas p-chloroacetanilide yields pchloroaniline hydrobromide. If the mixture is made alkaline, p-chloroaniline can be extracted selectively with cyclohexane and determined by ultraviolet spectrophotometry. No significant interferences have been encountered in the analysis of commercial samples.
A
to Harvald and coworkers (S), undesirable side effects sometimes observed in patients who have taken considerable phenacetin may be ascribed to p-chloroacetanilide which was present as an impurity. Because the amount of p-chloroacetanilide present in commercial phenacetin may vary from 0 to 2500 p.p.m. depending on the process of manuCCORDING
1834
ANALYTICAL CHEMISTRY
facture and the regulations of the country in which it is sold, it becomes important to have an analytical method which will determine precisely and accurately the p-chloroacetanilide actually present. The polarographic method of the Raney-nickel Jones and Page (4, hydrogenolysis method of Hald (a), and the paper chromatography method of Ritter and coworkers (5) have detection limits of about 500 p.p.m. The paper chromatographic method has been modified by a USP committee (9) to determine the presence of 300 p.p.m. A second spot on the paper chromatogram is, sometimes observed and may be mistaken for p-chloroacetanilide since the R, values are very close. This spot has been shown by N. E. Skelly of this laboratory, to be caused by innocuous N,N-diacetyl phenetedine (6). The authors have sought for a method which would be more sensitive while retaining suitable specificity. Upon acid hydrolysis, phenacetin should yield p - aminophenol ( p - hydroxyaniline)
while p-chloroacetanilide should yield p-chloroaniline. Both compounds are soluble in aqueous acid solutions, but from an alkaline solution only the chloroaniline can be extracted by an organic solvent; the other compound remains in the water layer as a phenoxide. Ultraviolet spectrophotometric determination of p-chloroaniline in an organic solvent is sufficiently sensitive for the purpose. Hydriodic acid is usually used for the cleavage of alkoxy compounds, but there are difficulties with formation of free iodine. One of the authors has pointed out (7) that 48% hydrobromic acid is practically as effective and he has utilized it in an unpublished method for the determination of bis(p-chlorophenoxy)methane by hydrolysis to p-chlorophenol. Much earlier, a mixture of 48% hydrobromic acid with acetic acid had been employed by Stoermer (8) for the dealkylation of phenyl ethers; in the case of anisole he reported 85% conversion. More recently, Anderson and coworkers , ( I )
have used hydrobromic acid for the cleavage and determination of tertbutoxy compounds. I t is possible to obtain quantitative cleavage of a 2-gram sample of phenacetin by refluxing for two hours in the presence of 25 ml. of 4870 hydrobromic acid. Cyclohexane is :j suitable extraction solvent and calibration can be carried out either against p-chloroacetanilide carried through the procedure or directly against a standard solution of p-chloroaniline in cyclohexane. The procedure as developed is sensitive down to about 10 p.p.m. pchloroacetanilide in phenacetin. EXPERIMENTAL
Apparatus. ,4 125-ml. flatbottomed reflux flask with a glass joint for connection with a n aircooled condenser, is used as the reactor. The spectrophotometric dat'a reported in this paper were obtained on a Cary Model 14 instrument, but for routine work other instruments may be sat,isfactory. Silica, absorption cells 10.00 cm. long are needed. Reagents. Hydrobromic acid, 48%, reagent grade. Sodium hydroxide, %yo solution. Dissolve 250 grams of reagent grade sodium hydroxide in 750 ml. of water. Sodium chloride, 25y6 solution. Dissolve 25 grams of reagent grade sodium chloride in 75 ml. of water. Cyclohexane, transparent to ultraviolet radiation between 250 and 350 mp. p-Chloroaniline, East,man Kodak No. 505. p-Chloroacetanilide, Eastman Kodak Yo. 662. Phenacetin. Use the purest material available. If necessary, the compound may be recrystallized from absolute ethanol and dried a t 75" C. Calibration. Accurately weigh a b o u t 25 mg. of p-chloroaniline, dissolve it in cyclohexan.e, and transfer the solut'ion to a 100-ml. volumetric flask. Dilute to the mark with cyclohexane, shake thoroughly, and pipet a 10-ml. aliquot into a, second 100-ml. volumetric flask. Dilute to volume with cyclohexane and shake. Dilute a 10-ml. aliquot of the second solution to 50 ml. in the same way. Transfer a portion of the final solution to a 10.00-cm. silica absorption cell and record the absorbance from 350 to 260 mp on the recording spectrophotometer. Use cyclohexane as a reference liquid. Draw a base line through the minimum a t 268 mp and tangent to the curve near 323 mw. Subtract the base line absorbance at, 293 mp from that indicated on the curve to obtain a net absorbance. Divide the weight of p-chloroaniline present in 50 ml. of the final solution by the net, absorbance ti,t the maximum. This value, coefficient (7, is the number of milligrams of p-chloroaniline per 50 ml. per absorbance unit (in a 10.00-
cm. cell). The value of coefficient C should be about 0.388 mg. per unit. Procedure. Weigh 2.0 grams of sample in a 125-m1. reflux flask. A\dd 25.0 ml. of 48y0 hydrobromic acid and a few boiling chips. Attach the air-cooled condenser and reflux the mixture vigorously on a hot plate in a hood for 2 hours. Remove the flask from the condenser and allow it to stand in the hood until fumes cease, then place it in an ice bath and allow it to stand for about 5 minutes. Add 10 ml. of cyclohexane and 40 ml. of the 25% sodium hydroxide solution. Allow the mixture to stand until it is cool again. Transfer the contents of the flask t o a 125-ml. separatory funnel. Rinse the flask thoroughly with 10 ml. of cyclohexane and add this to the funnel. Stopper the funnel and shake it vigorously for 3 minute.;. A\llowthe layers to separate and drain the aqueous layer into a second separatory funnel. Add 20 ml. of cyclohexane to the aqueous layer and shake as before. Discard the aqueous layer and drain the cyclohexane solution from the second funnel into the first funnel. Allow the cyclohexane solution to drain into a clean 125-ml. separatory funnel. Wash the walls of the first funnel with 5 ml. of cyclohexane and add this to the cyclohexane solution. I d d 5 ml. of 2570 sodium chloride solution and shake vigorously for about 30 seconds. (Sodium chloride solution rather than water is used for this extraction to minimize emulsion formation and improve the separation.) Discard the aqueous solution and repeat the saline extraction, again discarding the aqueous layer. Transfer the cyclohexane solution to a 50-ml. volumetric flask, rinsing the funnel with 5 ml. of cyclohexane. Dilute, if necessary, to the mark with cyclohexane. Record the absorbance of this solution as outlined above under calibration. Run a reagent blank by taking the reagents through the procedure. Likewise run a 2-gram sample of pure phenacetin. The absorption contributed by the blank and phenacetin should be negligible. Calculations. mol. wt. of chloroacetanilide mol. wt. of chloroaniline
-
Xet absorbance a t 298 mp X coefficient C X 1.33 = mg. of p-chloroacetanilide mg. found X 1000 grams of sample p.p.m. p-chloroacetanilide RESULTS AND DISCUSSION
X h e n concentrated hydrobromic acid is added to phenacetin a t room temperature according to the procedure, most of the phenacetin remains undissolved.
Table 1.
Rate of Acid Hydrolysis of Phenacetin
Absorbance Hydrolysis maximum, Net A298 time, min. mp (10.00-em. cell) 10 15 30 60 90 120
298 300 305 305 307 310
Table 11.
phenacetin, grams
0 0
2.255 2.021 0 0
2.022 2.195 2.083 2 010 2.099 2 172 1 978 1.971 2.120 2.034 2.024 2.144 2.005
328 254 85.8 0.238 0.022 0.007
Recovery Data
p-C hloroacetanilide,
p-Chloroacetanilide, Pg. p.p.m. Added Found Added Found 10 10 10
10
20
20 20 20 50
51 101 101 151 201 201 252 252 503 1004
9 ... 9 ... 9 4.4 10 4.9 20 ., , 18 , . . 16 9.9 17 9.1 47 24 ~52 26 99 48 93 46 149 77 192 102 192 ~.~ 95 .. 237 124 259 124 488 235 982 502
... ...
4 5
... ...
8
8 -2.1 _
26 47 43 75 97 91 --
117 128 228 491
On heating, there occurs a vigorous dissolution which is complete before the boiling point is reached. Foaming begins a t the boiling point and continues for about 30 minutes. This is probably due to the release of ethyl bromide and acetyl bromide. d brief study was made of the hydrolysis rate of phenacetin, the results of which are summarized in Table I. The ultraviolet absorbance of cyclohexane extracts of the 10- and 15minute runs is due to a n unknown hydrolysis intermediate or mixture. After 30 minutes, however, the ultraviolet curve is identical with that of p-phenetidine and represents 3,6y0 of the theoretical amount of p-phenetidine that could be formed from 2 grams of phenacetin. Later, trace amounts of other hydrolysis products are apparent as the absorption maximum shifts to longer wavelengths. The net absorbance a t 298 mp of 0.007 after 120 minutes would correspond to 2 p.p.m. p-chloroacetanilide. Results on known mixtures are shown in Table 11. The mixtures were prepared by adding various amounts of a solution of p-chloroacetanilide in methanol (1.005 mg. per ml.) to VOL. 36, N O . 9, AUGUST 1 9 6 4
e
1835
m 0:
0
I
WAVELENGTH, mp
Figure 1. p-Chloroaniline recovered from p-chloroacetanilide added to phenacetin Recrystallized phenacetin ( 2 grams) 20 pg. of p-chloroacetanilide added to 2 grams of 50 pg. of p-chlaroacetanilide added to 2 grams of 100 pg. of p-chlaroacetanilide added to 2 grams of 200 pg. of p-chloroacetanilide added to 2 grams of
1,
2. 3. 4. 5.
indicated portions of phenacetin known to be free of p-chloroacetanilide. The results were corrected for the absorbance due to the reagents and the phenacetin used. With the exception of the results a t the lower limit of the method, the recovery is 97 f 5% of the total amount present. Some of the curves obtained in the recovery study are shown in Figure 1. Curve 2 represents 10-p.p.m. p-chloroacetanilide. This is approaching the lowest concentration at which the shoulder at 308 mp is discernible in the
Table 111.
Analysis of Commercial Products
Sample A B C D E F
1430 68 670 700 3 20 56, 57, 56
(7
H I J K L, Lot L, Lot L, Lot L. Lot L; Lot
1836
p-Chloroacetanilide, p.p.m.
phenacetin phenacetin phenacetin phenacetin
WAVELENGTH,mp
Figure 2. Ultraviolet absorbance of the hydrolysis products of acetanilide and p-chloroacetanilide
----
-Aniline 0.220 mg. per 100 ml. of cyclohexane. 10.00-cm. cells -p-Chloroaniline 0.584 mg. per 100 ml. of cyclohexane. 10.00-cm. cells
original spectrum and is, therefore, the lowest concentration a t which p-chloroacetanilide can be identified as being present. Net absorbances lower than this place an upper limit on the amount that can possibly be present, but they do not characterize this absorbance as being due to p-chloroaniline, the hydrolysis product of p-chloroacetanilide. Results on some commercial samples are shown in Table 111. Samples A through K were sent to the laboratory without designation as to source. The results indicate the wide range of p-chloroacetanilide concentrations which may be found in commercial phenacetin. On the other hand, pchloroacetanilide has never been detected in brand L, known to be produced by a process in which p-chloroacetanilide is not an intermediate.
270
