ml of water to the toluene/Triton X-100 scintillation solution, observed radioactivity was increased to 70% of the Aquasol group. The lowered counts in toluene/Triton X-100 scintillation solution were not due to lower efficiency. Inhibition of Protein Synthesis by Dimethylnitrosamine and Cycloheximide. The effects of two compounds known to inhibit protein synthesis, cycloheximide and dimethylnitrosamine, were measured by the technique described here. As can be seen, incorporation of aH-leucine following treatment with either inhibitor was clearly depressed (Table 11). DISCUSSION
Data have been presented here clearly indicating the advantages of Aquasol for determining radioactivity of proteins. Replicates prepared in Aquasol are highly reproducible; additionally, the observed radioactivity with Aquasol is higher than with a toluene/Triton X-100 (2 : 1) scintillation solution (Table I). Samples prepared in Aquasol accurately indicate the actual protein activity, as shown by a linear response to protein concentration (Figure l), and by a decrease in radioactivity of liver protein following cycloheximide and dimethylnitrosamine. Aquasol has the unique property of forming gels when mixed with water. This gel may keep the protein in
Table 11. Effects of Inhibition of Protein Synthesis of Observed Radioactivity Leucine incorporation,a Treatment Animals (No.) (mean k S.E.) Rat (4) 490 k 49 Control Rat (4) 167 + 19 Dimethylnitrosamine, 20 mg/kg Mouse (3) 438 =IC9 Control Cycloheximide, Mouse (3) 59 + 11 64 m d k g a Expressed as cpm per 66 mg wet weight liver.
solution. On the other hand, samples prepared in toluene/ Triton X-100 (2 : 1) scintillation solution settle on the bottom of the vial where self-absorption becomes an important factor. RECEIVED for review December 1, 1970. Accepted February 1, 1971. Work supported by N.I.H. Grants C-6516 and FR05526.
Luminescence Characteristics of n=Alkylcarbazoles A. W. Perry, P. Tidwell, J. J. Cetorelli, and J. D. Winefordner Department of Chemistry, University of Florida, Gainesville, Fla. 32601 IN THIS REPORT, the fluorescence and phosphorescence characteristics of several n-alkylcarbazoles are given; n-alkylcarbazoles have recently been found in cigarette smoke condensate ( I ) . These species have been shown to have high carcinogenic activity, (1-3) but have not been found in the human respiratory environment (4-6). Sensitive, selective methods of measurement of n-alkylcarbazoles are necessary to measure these species in cigarette smoke condensate ; the present study indicates that fluorimetry and phosphorimetry may have some use in these studies. EXPERIMENTAL
Apparatus. All fluorescence measurements were made with a modified Aminco-Bowman spectrophotofluorometer (No. 4-8202, American Instrument Co., Inc., Silver Spring, Md.). The standard source housing was replaced with an elliptical source condensing system (No. 4-8208, American Instrument Co.) to increase sensitivity. The xenon lamp was powered by a Harrison constant current power supply (No. 6280 A, Hewlett Packard, Palo Alto, Calif.) operated at 7.5 ampere to enhance the signal-to-noise ratio. The starter (1) D. Hoffmann, G. Rathkamp, and S. Nesnow, ANAL.CHEM.,
41,1256(1969). (2) D. Hoffmann and E. L. Wynder, Proc. Amer. Ass. Cancer Res., 7,32 (1966). ( 3 ) E. L. Wynder and D. Hoffmann, Science, 162,862 (1968). (4) R. L. Stedman, Cliem. Rec., 68,153 (1968). (5) E. L. Wynder and D. Hoffmann, “Tobacco and Tobacco Smoke, Studies in Experimental Carcinogenesis,” Academic Press, New York, N. Y . ,1967. (6) D. Hoffmann and E. L. Wynder, Chapter 20 in “Air Pollution,” Vol. 11, A. C. Stern, Ed., Academic Press, New York, N. Y . ,1968.
circuit for the lamp was described by Zweidinger and Winefordner (7). Spectra (and phosphorescence lifetimes) were recorded with the Aminco microphotometer and an Aminco x-y recorder (No. 1620-827). Intensity measurements for preparation of analytical curves were made using a low noise nanoammeter with a galvanometric recorder (8). The slit arrangement was adjusted for maximum sensitivity when making detection limit measurements and for maximum allowable resolution when recording spectra. All phosphorescence measurements were made with the modified Aminco-Bowman spectrophotofluorometer, the Aminco phosphoroscope attachment and the spinning sample cell assembly (7, 9). The same slit arrangement as described above was used for phosphorescence studies. Reagents. All carbazoles were purchased (Aldrich Chemical Co., Inc., Milwaukee, Wis.; K and K Labs, Inc., Plainview, N. Y.;Matheson, Coleman and Bell, Norwood, Ohio); the solvents, ethanol (Union Carbide Corp.) and cyclohexane (Matheson, Coleman and Bell), were prepared as follows. Ninety-five per cent ethanol was vacuum-distilled at 113 mm of Hg, resulting in ethanol of greater than 99 purity and no detectable background; cyclohexane was redistilled at atmospheric pressure in a five-foot vacuum-jacketed column, resulting in a background decrease by approximately one order of magnitude from the original starting solution. Stock solutions (0.1 mg/ml) of each carbazole were prepared. Solutions of lower concentrations were prepared by successive dilutions. (7) R. A. Zweidinger and J. D. Winefordner, ANAL.CHEM., 42, 639 (1970). (8) H. C. Hollifield and J. D. Winefordner, ibid.,40, 1759 (1968). (9) T. C. O’Haver and J. D. Winefordner, J. Cliem. Educ., 46, 241 ( 1969). ANALYTICAL CHEMISTRY, VOL. 43, NO. 6, MAY 1971
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Table I. Fluorescence and Phosphorescence Characteristics of Carbazole and Several n-Alkylcarbazoles Range of linearity PhosphoExcitation Emission of analytical Limit of detection rescence maximum, nm5 maximum, nma curve, decade* (a/mP lifetime,d Species Solvent Fluore Phosf Fluore Phosf Fluor Phos Fluor Phos sec Carbazole 0.001 7.80 EtOH 340 341 360 436 4 4 0.0003 Carbazole 0.001 7.2 360 435 Cyclohexane 340 297 4 4 0.0005 n-Methylcarbazole 0.001 8.4 EtOH 346 336 360 437 4 4 0.ooO8 n-Methylcarbazole 7.5 0.001 Cyclohexane 346 298 360 43 1 4 4 0.0005 n-Ethylcarbazole 340 369 437 4 4 0.001 0.001 7.8 EtOH 339 n-Ethylcarbazole 0.001 8.1 Cyclohexane 340 298 364 433 4 4 0.0008 2-Methylcarbazole 442 4 4 0.001 0.001 8.1 333 357 EtOH 346 2-Methylcarbazole 332 356 443 4 4 0.001 0.001 7.5 Cyclohexane 346 a Maxima are uncorrected for instrumental characteristics. Accuracy of wavelength setting is f 2 nm. The range of linearity could be considerably greater because the highest measured concentration was still on the linear portion. Limits of detection were taken as those concentrations producing a signal twice the background. Shortest measureable lifetime is 0.5 sec. e Measurements taken at 298 “K. f Measurements taken at 77 “K. Lifetimes are taken as time for phosphorescence to decay to lie of the original signal. All decays were exponential and precise to k0.2 sec.
Procedure. Measurements of spectra, analytical curves, limits of detection, and phosphorescence lifetimes were made according to procedures previously discussed (IO). The clean-up procedures and precautions described by Zweidinger, Sanders, and Winefordner (11)were also followed. The limit of detection was defined as that concentration resulting in a signal of two times the variation in the luminescence background of the solvent.
(10) J. D. Winefordner, W. J. McCarthy, and P. A. St. John,
Phosphorimetry as an Analytical Approach in Biochemistry, Chapter in D. Glick, “Methods of Biochemical Analysis,” Vol. 15, Interscience, New York, N. Y . ,1967. (11) R. A. Zweidinger, L. B. Sanders, and J. D. Winefordner, Anal. Chirn. Acta, 47,558 (1969).
RESULTS
The fluorescence characteristics at 298 OK and phosphorescence characteristics at 77 OK of carbazole, n-methylcarbazole, n-ethylcarbazole, and 2-methylcarbazole in ethanol and in cyclohexane are listed in Table I. N o actual analyses of carbazoles in cigarette smoke were carried out, but because of the low detection limits for the carbazoles, the use of fluorimetry and phosphorimetry for quantitative measurement of n-alkylcarbazoles in a rather small amount of cigarette smoke seems possible.
RECEIVED for review November 16, 1970. Accepted January 21, 1971. Research was carried out as part of a study on the phosphorimetric analysis of drugs in blood and urine, supported by a U. s. Public Health Service Grant (GM-11373-08).
New Spectrophotometric Method for Determination of FormaIdehyde B. W. Bailey and J. M. Rankin DiGision of Laboratories and Research, New York State Department of Health, New Scotland Avenue, Albany, N . Y . 12201
THEMOST COMMON of the many methods which have been devised for the determination of formaldehyde in air are the chromotropic acid method ( I ) , the 6-amino-l-naphthol-3sulfonic acid or J-acid method (Z), and the 3-methyl-2-benzothiazolone hydrazone (MBTH) method (3). All three exhibit excellent sensitivity and are applicable over a wide range of concentrations. The chromotropic acid and MBTH methods are spectrophotometric procedures with a Beer’s law range of 0.24-4.0 pg HCHO/ml and 0.05-0.92 pg HCHO/ml, respectively, in (1) A. P. Altshuller, D. L. Miller, and S. F. Sleva, ANAL.CHEM., 33, 621 (1961). (2) E. Sawicki, T.W. Stanley, and J. Pfaff, Anal. Cltirn. Acta, 28, 156 (1963). (3) E. Sawicki, T. R. Hauser, T.W. Stanley, and W. Elbert, ANAL.
CHEM., 33,93 (1961). 782
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aqueous solution. The J-acid method is a spectrofluorometric procedure. Its range of applicability is 0.001-0.2 pg HCHO/ ml, making it by far the most sensitive in use for the determination of formaldehyde. All three of these methods, however, lack selectivity (2). The MBTH method, which shows only minimal differentiation between formaldehyde and the higher aliphatic aldehydes, causes the latter to function as a strong positive interference to the determination of formaldehyde. Both the chromotropic acid and the J-acid methods show a negative interference for the higher aliphatic aldehydes and, therefore, produce anomalously low values for the formaldehyde concentration in aldehyde mixtures. The method of formaldehyde determination proposed here is based on the catalytic effect of formaldehyde on the hydrogen peroxide oxidation of p-phenylenediamine. This effect
