sis. SRM paint was analyzed by polarography and atomic absorption. The reasonably good agreement between 252Cf activation analysis and the other methods demonstrates the reliability and utility of the isotope source technique for this determination. Irradiation for 2 hours with the 600-/.~g 252Cf source followed by 100 minutes of counting with a 60-cm3 Ge(Li) detector yields about 7 counts of the 204mPb isotope per milligram of lead. For a paint sample weighing 1.5 grams, the lower limit of determination would be of the order of 1%. As noted, analysis of the Ge(Li) spectrum of those elements likely to be major or minor constituents of paint indicates that they will not interfere in the quantitation of the gamma ray lines of 204mPb.Self-shielding effects due to the matrix are expected to be negligible since the nuclear reaction involves fast neutrons and a paint sample is
unlikely to contain appreciable amounts of neutron moderating elements. The results presented here indicate that with a 252Cf source of several tens of milligrams and the 60-cm3 Ge(Li) detector, the determination of -1% of lead in a sample of 1-2 grams of paint could be accomplished with a 15-minUte irradiation and 15-minute counting. The potential exists for largely automating the irradiation and counting of a large number of samples. Hence, it would appear possible that although the initial cost of equipment for this determination would be greater than for alternate methods, the speed and adequate reliability of the isotope source method would make it economically competitive. Received for review August 23, 1973. Accepted November 13, 1973.
Colorimetric Determination of N-Arylhydroxylamineswith 9-ChIoroacridine Richard E. Gammans, James T. Stewart, and Larry A. Sternson' The Bioanalytical Laboratory, Department of Medicinal Chemistry, School of Pharmacy. The Univers/ty of Georgia. Athens, Ga. 30602
During the metabolic conversion of certain aromatic amines to excretable conjugates in the endoplasmic reticulum of liver cells, N-arylhydroxylamines are formed ( I ) which have been implicated in the chemical carcinogenesis ( 2 ) displayed by such compounds. Few analytical procedures are available for the rapid detection and assay of such aromatic hydroxylamines. Boyland and Nery (3) detected 1-3 bg/ml of N-phenylhydroxylamine colorimetrically by complex formation with either salicylidenearylamine N-oxides and ferrocyanide or pentacyano-ammine ferroate in aqueous solutions. Qualitative identification of arylhydroxylamines by thin-layer chromatography with the use of various spray reagents has also been described ( 4 ) . In addition, some gas chromatographic determinations of arylhydroxylamines have been reported (5, 6). The interaction of N-arylhydroxylamines with 9-chloroacridine to give highly-colored solutions has been observed in our laboratory. In this paper, we describe a new colorimetric method for determining microgram quantities of arylhydroxylamines with 9-chloroacridine. EXPERIMENTAL A p p a r a t u s . Spectra and absorbance measurements were made with a Perkin-Elmer Spectrophotometer. Model 202, and a Bausch and Lomh Spectronic 20 colorimeter. Reagents. 9-Chloroacridine was obtained from Eastman Organic Chemicals. N-Phenylhydroxylamine, p-chlorophenylhydroxylamine, and p-tolylhydroxylamine were synthesized by the method reported by Smissman and Corhett (7). CyclohexylhyAuthor to whom corespondence should he directed. C. C . Irving, J . Bioi. Chern., 239, 1589 (1964). J. A. Miller, J. W . Cramer, and E. C. Miller, Cancer Res.. 20, 950 (1960).
E. Boyland and R. Nery, Anaiyst ( L o n d o n ) ,89, 95 (1964) J. Booth and E. Boyland, Biochem. J., 91, 362 (1964). H. B. Hucker. Drug Metab. Disposition. 1, 322 (1973). A . H . Beckett and S. AI-Sarra], J . Pharm. Pharmacal.. 24, 916 (1972). E. E. Smissrnan and M. D. Corbett, J. Org. Chern., 37, 1847 (1972).
A N A L Y T I C A L C H E M I S T R Y , V O L . 46, NO. 4 , A P R I L 1974
droxylamine was prepared by reduction of cyclohexanone oxime ( 8 ) by the procedure of Feuer e t al. ( 9 ) . The melting points of the synthesized compounds were in agreement with literature values (10, 1 2 ) . All other chemicals were commercially available and were utilized as received. Fresh solutions of arylhydroxylamines were prepared daily by dissolving weighed amounts in 9570 ethanol. Solutions of 9-chloroacridine were prepared immediately before use hy dissolving weighed amounts in ethanol and were kept refrigerated. Procedure. A quantity of a n ethanolic solution of arylhydroxylamine was placed in a n appropriate volumetric flask. An ethanohc solution containing an approximate 10-fold excess of 9-chloroacridine was added to the flask. The solution was acidified with 10% v/v aqueous hydrochloric acid, shaken briefly, and allowed t o stand a t room temperature for 5 minutes. Ethanol was added t o volume so that the final concentration of arylhydroxylamine in the flask was equal to or greater than 1 X 10--5M.The absorhance was measured at 450 n m and the measurements were corrected for reagent blanks in the procedure.
RESULTS AND DISCUSSION
9-Chloroacridine interacts with an arylhydroxylamine in the analytical procedure to yield a highly-colored yelloworange solution. The absorption curve in the visible spectrum for a n analytical solution of A'-phenylhydroxylamine shows an absorption maximum a t 450 nm. Preliminary data have revealed that the reaction yields a nucleophilic addition product similar to the one reported for primary aromatic amines (12). Further work on the identification of the compound formed between arylhydroxylamines and 9-chloroacridine is under way in our laboratory. In comparing absorption curves of the colored solutions obtained with equimolar concentrations of the various arylhydroxylamines, it was noted that p-chlorophenylhyA . I . Vogel, " A Textbook of Practical Organic Chemistry." 3rd ed.. John Wiley, New Y o r k , N Y.. 1966. p 344 H . Feuer,*B F. Vincent, and R S Bartlett, J Org Chem.. 30. 2877
(1965). A . Lapworth and L Pearson J Chem. Soc.. 119. 765 (1921). R. D. Haworth and A . Lapworth, J. Chem Soc.. 119. 768 (1921) J. T . Stewart. T D. Shaw, and A . B. Ray. A n a / . Chem.. 41, 360
(1969).
Table 11. Analysis of Known Arylhydroxylamine Mixtures for Arylhydroxylamine
Table I. Calibration Data for Various Arylhydroxylamines
Compound
N-Phenylhydroxylamine
Final concn, M X 10-5
1.5 3.0 6.0
p-Chlorophenylhydroxylamine
1.5
p-Tolylhydroxylamine
3.0 6.0 1.5 3.0 6.0
Arylhydroxylamine“ Absorbancea
0.163 i O.OISb 0.320 j= 0.011 0.008 0.605 0.096 ?L 0.014 0.196 =k 0.008 0.403 + 0.008 0.096 + 0.014 0.196 i 0.008 0.396 i O . 0 0 2
a Measured a t 450 nm and based upon 5 replicate determinations of each solution. Confidence limits a t p = 0.05
droxylamine and p-tolylhydroxylamine produce color which absorbs a t the same wavelength maximum as N phenylhydroxylamine but with diminished intensity. The absorbance data are shown in Table I. The reduced absorption for the substituted derivatives of phenylhydroxylamine may be the result of a less-than-quantitative yield of the colored product under the conditions of the analytical determination. It was suggested that heating the hydroxylamine and acridine on a steam bath might ensure a more complete reaction. However, maximum color development could be obtained upon shaking the solution and allowing it to stand at room temperature for 5 minutes. Repeated readings on a series of different samples indicated good color stability for periods u p to 24 hr. Although stability of‘ the 9-chloroacridine reagent solution has been discussed previously (12, 13), our present work has further revealed that refrigeration of the acridine solution after preparation allows use of the solution for approximately 8 hours before decomposition occurs. The analysis method is a micro procedure, and sensitivity is in the 0.8-3 pg/ml range (10-5M) of arylhydroxylamine. Reagent preparation is simple and rapid since 9chloroacridine is commercially available and can be used without further purification or modification procedures. The time involved in the actual analysis procedure is less than 30 minutes compared to the colorimetric method by complex formation which takes from 2 to 14 hours depending upon the reagent used ( 3 ) . Standard curves can be prepared by plotting absorbance readings us. the volumes taken of equimolar concentrations of various arylhydroxylamines. In all cases, Beer’s law holds for this system. Data from several systems shown in Table I1 indicate that this procedure permits the determination of arylhydroxylamines in the presence of primary aromatic amines (whose colorimetric determination with 9-chloroacridine has been previously reported, vide injra), primary aliphatic amines, phenols, aromatic nitro compounds, aliphatic and aromatic amides, and aliphatic hydroxylamines. It has been shown previously t h a t secondary and tertiary aromatic, heterocyclic, and aliphatic amines and carbonylcontaining compounds did not interfere with colorimetric determinations utilizing 9-chloroacridine. Although one cannot exclude the possibility t h a t these other functional (13) J. T. Stewart, A . B. Ray, and W. B. Fackler, J. Pharm. s o , 50. 1261 (1969).
Mixture
Found, Componentsb
1 N-Phenylhydroxylamine
Aniline 2 N-Phenylhydroxylamine Phenol Acetamide n-Butylamine 3 N-Phenylhydroxylamine Nitrobenzene Acetanilid 4 N-Phenylhydroxylamine C yclohexylhydroxylamine
M X 10-&
yo of Theory
1.496
99.75
=k 3 , 62c
1.489
99.27 i 1 . 5 9
1.485
98.97 f 2.50
1.514 100.90 i 1.81
Final concena Based upon 3 replicate determinations of each solution. tration of phenylhydroxylamine and all other components in mixtures was 1.500 X 10-5M;except aniline which was present in a final concentration of 1.500 X 10-3M. Confidencelimitsatp = 0.05.
groups react with 9-chloroacridine in typical nucleophilic displacement reactions, only primary arylamines produce any colored product under the conditions of the analytical reaction. Measurements reveal t h a t arylhydroxylamines can be quantitated in the presence of a hundredfold excess of primary aromatic amine. Specificity permitting the determination of arylhydroxylamines with 9-chloroacridine in the presence of primary arylamines is obtained by measuring absorbance a t 450 n m , where the absorption characteristics of any 9-chloroacridine-arylamine adduct show minimal interference. Received for review September 10, 1973. Accepted November 20, 1973. This investigation was supported in part by NIH Grant CA-14158-01 from the National Cancer Institute.
CORRECT1ON Application of a Piezoelectric Quartz Crystal as a Partition Detector. Development of a Digital Sensor In a paper by Morteza Janghorbani and Harry Freund, Anal. Chem., 45, 325 (1973), piezoelectric crystals for assay of SOz in air were described using carbowax as coating. This paper did not refer to the previously described, successful, studies on piezoelectric crystals for SOz, by G. G . Guilbault “Use of Tetrachlormercurate as a Coating for Son,’’ Enuiron. Lett., 2, 35 (1971), and the “Use of Piezoelectric Crystals as Sensitive and Specific Detectors for Son,’’ Anal. Lett., 5 , 255 (1972). The latter paper describes the use of carbowax as a coating for SOz, the same as discussed by Janghorbani and Freund.
A N A L Y T I C A L CHEMISTRY, V O L . 46, N O . 4, A P R I L 1974
621
