Quantitative Determination of 1-Alkylindoles in Cigarette Smoke Dietrich Hoffmann’ and Gunter Rathkampl Division of Enaironmental Cancerigenesis, Sloan-Kettering Institute f o r Cancer Research, New York, N. Y . IO021 This report is part of a study on the identification of tumorigenic agents in cigarette smoke. For the determination of 1-alkylindoles, the nonvolatile particulate matter of the smoke collected in solvent is distributed between two pairs of solvents and the resulting concentrate is chromatographed on alumina and subsequently analyzed by gas chromatography. The isolated components were identified by retention times, ultraviolet spectra, and mass spectra. 1-Methylindole-2-14C was synthesized and served as internal standard. The mainstream smoke of an 85-mm U. S. cigarette without filter tip contained 420 ng of 1-methylindole, 126 ng of 1-ethylindole, 32 ng of 1,2- and 1,7-dimethylindole, 380 ng of 1,3-dimethylindole, and 99 ng of 1,4-, 1,5-, and 1,6-dimethylindole. Pyrolysis studies suggest tryptophan to be an important precursor for the pyrosynthesis of indoles and l-alkylindoles. This communication reports the first identification of 1-alkylindoles in the respiratory environment. The possible biological significance of 1-alkylindoles in experimental tobacco carcinogenesis needs to be explored.
THESUBFRACTION BI of cigarette “tar” shows the highest carcinogenic activity on mouse skin of all tested tobacco smoke condensate fractions and subfractions ( I , 2). BI amounts to about 0 . 6 x of the dry “tar” and contains polynuclear aromatic hydrocarbons (PAH), chlorinated hydrocarbon insecticides, indoles, and carbazoles (2, 3). Recent subfractionations indicated the presence of 1-alkylindoles in BI. Until now these components have not been identified in the human respiratory environment (4-6). N-Alkylated indoles display different adsorption properties than the parent molecule. In the present method these components are enriched by distribution of cigarette “tar” between two pairs of solvents and by chromatography on alumina. The resulting concentrate is separated into individual components by gas chromatography. 1-Methylindole2-14C was synthesized by direct methylation of indole-2- 14C with methyl p-toluenesulfonate and served as an internal standard. Pyrolysis experiments with nicotine and tryptophan suggest the latter to be a significant precursor for l-alkylindoles in tobacco smoke. EXPERIMENTAL
Apparatus. For the quantitative analysis we smoked the cigarettes individually with a CSM-10 (Cigarette Components Ltd.) whereas for the isolation of 1-alkylindoles we employed Present address, Division of Environmental Toxicology, American Health Foundation, 180 East End Avenue, New York, N. Y. 10028. (1) D. Hoffmann and E. L. Wynder, Proc. Amer. Cancer Res. Assoc., 7 , 32 (1966). (2) E. L. Wynder and D. Hoffmann, Science, 162,862 (1968).
( 3 ) D. Hoffmann, G. Rathkamp, and S. Nesnow, ANAL.CHEM., 41, 1256 (1969).
(4) R. L. Stedman, Chem. Reu., 68, 153 (19683. (5) E. L. Wynder and D. Hoffmann, “Tobacco and Tobacco
Smoke, Studies in Experimental Carcinogenesis,” Academic Press, New York, 1967. (6) D. Hoffmann and E. L. Wynder in “Air Pollution, Vol. 11” A. C. Stern, Ed., Academic Press, New York, 1968, Chap. 20. 366
ANALYTICAL CHEMISTRY, VOL. 42, NO. 3, MARCH 1970
a 30-channel automatic smoker with a vibrating liquid trap (5,7). A Perkin-Elmer gas chromatograph Model 800 with a dual flame ionization detector was used for the analysis. The @-radiation was counted with a Nuclear Chicago Scintillation System 720. Ultraviolet absorbance measurements were obtained with a Cary Model 11 recording spectrophotometer. The mass spectra were determined with a HitachiPerkin-Elmer RMU-6D by the Morgan-Schaffer Corporation (Montreal, Canada); the energy of the bombarding electrons was kept at 70 eV. The NMR spectra were determined in deuterochloroform on a Varian A-60 instrument employing tetramethylsilane as internal reference (y 10.0). Evaporations were completed at reduced pressure with water bath temperatures below 50 “C. The melting points were corrected. Refractive indexes were determined with a ZeissAbbe refractometer connected with a Colora thermostat. For the pyrolysis studies we used a Perkin-Elmer Pyrolysis Unit, which was adjusted to 880 “C by a Chromel-alumel thermocouple and differential voltmeter (J. Fluke Co.). Reagents. All solvents were spectrograde, with the exception of xylene which was reagent grade. For the synthesis of 1-alkylindoles we purified the commercially available indoles by column chromatography (8). Alumina Woelm (activity 11, except as indicated) was obtained from Alupharm Chemicals, Sephadex LH-20 from Pharmacia Fine Chemicals, Gas Chrom P and XE-60 from Applied Science Laboratories. Synthesis of l-Methyl-2-14C-Indole. 4.2 Mg of indole-2IC-picrate (4.08 mCi/mM, Tracerlab) was dissolved under ice cooling in 5 ml of 2 N sodium hydroxide and extracted four times with ether and dried (Na2S04). The resulting 1.41 mg of indole-2-I4C was dissolved in 1.0 ml of xylene and mixed with 6.0 mg of anhydrous sodium carbonate and 9.0 mg of methyl p-toluenesulfonate and heated for eight hours in an 150 “C oil bath. The dried ether extract of the reaction resulted in 1.31 mg of residue, which was chromatographed with n-hexane on 5 grams of alumina (activity I). The radioactive fractions were combined and rechromatographed (5 grams of alumina, activity I). The resulting 1.18 mg of 1methylindole-2-14C had a specific activity of 4.01 mCi/mM (yield 7473. The chemical and radiochemical purity of the labelled indole was ascertained by repeated column chromatography and gas chromatography. The counting efficiency for unquenched 1-methylindole-2- 4C in toluene with 0.4 PPO (2,5-diphenyloxazole) and 0.005 % POPOP (p-bis[2(5-phenyloxazolyl)]benzene) was 72.8 %. Reference Compounds. 1-Methylindole (N-methylindole) was synthesized according to Shirley and Roussel (9), with the modification that a 50x excess of methyl p-toluenesulfonate was used. Under magnetic stirring we refluxed for 50 hours 40 ml of xylene with 10 grams (86 mM) of indole, 24 grams of methyl p-toluenesulfonate (129 mM) and 14 grams of anhydrous sodium carbonate. The dry, neutral portion of the reaction product was purified by repeated column chromatography on alumina (activity I). The purity of the resulting 7.0 grams of 1-methylindole ( 6 2 x yield) was determined by refractometric index [n2’D 1.6062; Lit. ( I o ) (7) F. Seehofer, J. E. Miller, and E. Elmenhorst, Be&. Tabakforsch., 3, 75 (1965). (8) D. Hoffrnann and J. Rubin, Bei/r. Tabakforsch., 3, 409 (1966). (9) D. A. Shirley and P. A. Roussel, J . Amer. Chem. Soc., 75, 375 (1953). (10) R. L. Hinrnan and J. Lang, J. Amer. Chem. Soc., 86, 3796 (1964).
1.60621, gas chromatographic parameters and ultraviolet absorption spectrum. X(cyc1ohexane) max. 294 mp (e 6210), 288 (6520), 282 (6850). The six isomeric dimethylindoles were prepared by the above described method from C-methylindoles and methyl p-toluenesulfonate and not by the earlier reported Fischer indole synthesis or Tyson ring closure (11). The yields for the chromatographically pure dimethylindoles varied between 48-64 %. 1,2-Dimethylindole: mp 56 "C. [Lit. (11) 56 "C]. h(cyclohexane) max. 292 mp (e 7510), 281.5 (8240), 276 (7820). 1,3-Dimethylindole: ~ * O D 1.5902 [Lit. (IO) 1.58961. X(cyclohexane) max. 302.5 mp (e 4600), 296.5 (5190), 290 (5 8 10). ~ X(cyc1ohexane) max. 1,4-Dimethylindole : n Z 5 1.5880. 295 mp (e 7510), 289 (7230), 284 (7420). 1,5-Dimethylindole: n 2 0 ~1.5910. X(cyc1ohexane) max. 295.5 mp ( e 6430), 289.5 (6510), 284 (6440). 1,6-Dimethylindole: n 2 5 ~1.5995. h(cyc1ohexane) max. 287 mp ( E 7530), 282.5 (8490), 276 (8610). 1,7-Dimethylindole: mp 77.5 OC. [Lit. (11) 78 "C]. X(cyc1ohexane) max. 294 mp ( e 4410), 286.5 (4190), 277 (10800), 273.5 (5130). 1-Ethylindole. Five hundred mg of indole was dissolved in 10 ml of xylene and refluxed with 1.3 grams of ethyl p toluenesulfonate (recrystallized from alcohol, mp 34 "C) and 600 mg of anhydrous sodium carbonate. For gas chromatographic analysis, small aliquots were taken once a day. The maximum yield of 67% was reached after seven days. The repeated chromatographic purification on alumina (activity I) resulted in 350 mg of 1-ethylindole ( 5 6 z yield). This compound, however, showed in the gas chromatogram a small impurity with the retention time of 1-methylindole. Therefore, we purified a portion of the colorless oil by preparative gas chromatography under conditions described below. The resulting 1-ethylindole had a refractive index of ( n 2 * ~ 1.5878) [Lit. (12) 1.58891. X(cyc1ohexane) max. 294 mp ( e 9025), 288 (9515), 282 (10300). Distribution. Distribution between methanol/water (4: 1) and n-hexane ; and between n-hexane and acetonitrile leads to a significant enrichment of N-alkylindoles from cigarette "tar"under separation of more than 95% of the tobacco smoke paraffins. Large excesses of the latter impede the column chromatographic enrichment of N-alkylindoles on F l o r i d , silica gel, and alumina. The distribution coefficients were determined by ultraviolet absorption spectra (Table I). Gas Chromatography. A 3-mm by 5-m column filled with 5% XE-60 on Gas Chrom P gave the most satisfactory separation of I-alkylindoles. The retention times are presented in Table I. This table also gives the retention times of indole, 2- and 3-methylindole, and 2,3-, 2,5-, and 2,7-dimethylindole. About 0.6 pg of 1-methylindole reaches the full scale of a 1-mV recorder with an attenuation of 100. Extensive investigations with various liquid phases and columns failed to separate 1,2- and 1,7-dimethylindole and 1,4-, 1,5-, and 1,6-dimethylindole. One of the columns tested was a 50-m capillary column (i.d. 0.15-mm.) coated with XE-60. For the gas chromatographic isolation of individual N-alkylindoles from the concentrate, a 4 : l glass splitter was installed. The column effluents of the gas chromatographic separation which had retention times comparable with those of the references were collected in glass capillaries and rechromatographed for mass spectral analysis. Procedures. A. ISOLATION OF ~-ALKYLINDOLES. Three thousand, two hundred cigarettes without filter tips (85 mm) were smoked in three equal portions. The standard condi(11) L. Marion and C. W. Oldfield, Can. J. Res., 25, Sect. B, 1
(1947). (12) A. Gray, H. Kraus, and D. Heitmeier,J. Org. Chem., 45, 1939 (1960).
~
~~
~
Table I. 1-Alkylindoles-Partition Coefficients and Retention Times Partition coefficients (20 "C) CH/cX"
3.3 2.9
CAICH
Retention timeb (min. after injection)
1-iblethylindole 7.9 1,2-Dimethylindole 14.5 3.1 3.5 1,3-Dimethylindole 9.9 2.5 3.7 1,4-Dimethylindole 10.7 2.5 3.7 1,5-Dimethylindole 10.7 2.5 3.8 1,6-Dimethylindole 10.8 2.8 3.6 1,7-Dimethylindole 14.5 3.6 3.2 1-Ethylindole 9.0 a CH = Concentration in n-hexane. CJI = Concentration in methanol/water (4 :1). C A = Concentration in acetonitrile. Column condition, 3-mm X 5-m stainless steel 5 XE-60. * For indoles, which were not N-alkylated, we found the following retention times in minutes: indole 18.8; 2-methylindole 22.0; 3-methylindole 20.8; 4-methylindole 23.4; 5-methylindole 22.8; 6-methylindole 22.6 ; 7-methylindole 22.0 ; 3-ethylindole 28 .O; 2,3-dimethylindole 31.2; 2,5-dimethylindole 31, 8 ; 2,7-dimethylindole 31.4. 3.7
3.6
tions and the setup for the collection of the nonvolatiles of the mainstream smoke were recently reported (3). The resulting dry residue (94.5 grams) was suspended in 750 ml of methanol/ water (4 :1) and extracted three times with 750 ml of n-hexane. The combined n-hexane layers were concentrated to 500 ml (residue 33.2 grams) and extracted three times with 250 ml of acetonitrile. The combined acetonitrile layers were evaporated and dried over paraffin in a vacuum desiccator. The residue (9.6 grams) was dissolved in 50 ml of n-hexane/ benzene (6 :1) and chromatographed on 700 grams of alumina (column 3 X 120 cm). The first 15 fractions were eluted with 100 ml of n-hexane each, fractions 16-20 with 100 ml of n-hexanelbenzene (8 :1); and fractions 21-25 each with 100 ml of n-hexaneibenzene (6 : 1). Fractions 17-22 contained compounds which gave retention times comparable to those of the references. (Non-N-substituted indoles were not present in the first 25 fractions.) The residue of these fractions (67.2 mg) was dissolved and chromatographed with 2propanol on 60 grams of Sephadex LH-20 (column 2 X 120 cm) at a column temperature of 32 "C. Every hour a fraction of about 1.8 ml was collected. Fractions 43-48 (21.3 mg) and 49-55 (1 1.4 mg) were analyzed by gas chromatography for N-alkylindoles. Column effluents with maxima comparable with those of 1-alkylindoles were collected and rechromatographed for mass spectral analysis. Column effluents with the retention times of 1,2- and 1,7-dimethylindole and 1,4-, 1,5-, and 1,6-dimethylindole were also analyzed by ultraviolet spectrometry. B. QUANTITATIVE ANALYSIS.Three hundred cigarettes were smoked under standard conditions with a CSM-10. The mainstream smoke was directed through a series of three gas wash bottles filled with n-hexane. The smoke suspensions and washings were combined and 2.3 pg of l-methylindole-214Cwere added as an internal standard. The residue was suspended in 100 ml of methanol/water (4 :1) and extracted four times with 100 ml of n-hexane. The upper layers were combined, concentrated to dryness, dissolved in 100 ml of n-hexane, extracted three times with 100 ml of acetonitrile and evaporated. The residue of the acetonitrile layers was dissolved in 10 ml of n-hexanelbenzene and chromatographed with n-hexanelbenzene on 120 grams alumina. The eluates were collected in 50-ml fractions. In general the &activity was found in fractions 8-12. These were combined, evaporated to dryness (residue 3-5 mg), and aliquots were taken for gas chromatographic analysis and liquid scintillation counting.
ANALYTICAL CHEMISTRY, VOL. 42, NO. 3, MARCH 1970
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Figure 4. Mass spectra of 1,2-dimethylindole, 1,7-dimethylindole, and a mixture of 1,2-and 1,7-dimethylindole isolated from cigarette smoke
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Figure 2. Mass spectra of 1-ethylindole Pyrolysis Experiments. Fifteen mg of DL-tryptophan (or 30 mg of DL-nicotine) were placed in combustion boats (17 X 6 X 4-mm), which were previously heated for two hours at 880 "C. The temperature stabilized and closed quartz tube (i.d. 10-mm) was filled with nitrogen (1 atm) and heated to 880 OC at a length of about 5 cm. The loaded combustion boats were placed inside the tube and in the shortest possible time advanced into the hot zone with a boat pusher with a magnet. The boat was exposed to 880 "C 368
ANALYTICAL CHEMISTRY, VOL. 42, NO. 3, MARCH 1970
for five minutes. After cooling, boat and tube were rinsed with hot methanol/water (4 :1) and n-hexane. The solvents were filtered, combined, and each layer made up to 50 ml. The quantitative determination of the N-indcles followed the above outlined method and that of indoles followed an earlier published procedure (8). 1-Methylindole-2-14C and indole2- 14C(4.08 mCi/mM) were employed as internal standards. RESULTS AND DISCUSSION
Figures 1 to 3 represent mass spectra of synthetic and isolated 1-methylindole, 1-ethylindole, and 1,3-dimethylindole,
4
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Figure 5. Mass spectra of 1,4dimethylindole and a mixture of 1 , 4 , 1,5- and/or l,6-dimethylindole isolated from cigarette smoke ~
Figure 4 those of synthetic 1,2- and 1,7-dimethylindole and of isolated 1,2- and/or 1,7-dimethylindole(s). We were unable to separate these two isomers. Their mass spectra are similar and the ultraviolet absorption spectrum of the isolated material shows only a broad band between 285 and 295 mp, as does a synthetic mixture of the two isomers (1 : 1). We conclude, therefore, that cigarette smoke most likely contains both 1,2and 1,7-dimethylindole. Although these two compounds have sufficiently different NMR spectra (1,ZDMI :l-CH3, T 6.3; 2-CH3, 7 7.61; 1,7-DMI:l-CHs, 7 6.07; 7-CH3, 77.32) we calculated that at least 20,000 cigarettes would have to be smoked for a definitive analytical result. We consider the effort unwarranted at this time. Figure 5 presents the mass spectra of synthetic 1,4-dimethylindole and of isolated mateiial with the retention time of 1,4-, 1,5-, and 1,6-dimethylindole. Again, the mass spectra for the three dimethyl isomers were similar and the ultraviolet adsorption spectrum of the isolated specimen gave only a broad band between 295.2 and 283.8 mp. For the reasons discussed above, we excluded a NMR analysis. As we experienced during this study non-N-alkylated indoles (indole, C-methylindoles and C,C-dimethylindoles) were easily separated from the N-alkylated indoles (N-methyl, N,Cdimethyl- and N-ethylindole) by column chromatography on deactivated alumina. In addition in our gas chromatographic system the retention times for indole, C-methylindoles and C,C,-dimethylindoles were significantly longer than those for N-alkylindoles (Table I). Therefore, the isolated dimethylindoles were N-substituted. Figure 6 shows a gas chromatogram of one of the Nalkylindole concentrates from the mainstream smoke of 300 cigarettes as obtained at the end of the separation of the quantitative analysis. The peak with the retention time of 12.3 minutes gave an ultraviolet absorption spectrum and mass spectrum which we tentatively assigned to represent a Nmethylbenzylpyrrole. Reference compounds are presently being synthesized for proper identification. So far, unknown I1 has not been identified. The mainstream smoke of an 85-mm U. S. blended cigarette without filter tip contains 420 ng of I-methylindole, 126 ng of
~~
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Table 11. 1-Alkylindoles in Cigarette Smoke.
(ng/Cig.)
++
1,4 1,2 1,5 1,J-Di- 1,3-Di- 1,6-DiNumber of 1-Methyl- methyl- methyl- methylanalysis indole indole indole indole I 438 37 295 111 33 277 I1 395 98 111 444 32 284 100 IV 452 32 279 97 30 277 91 V 383 422 282 Av. 32 99 31 2.7 7.6 Std. dev. 7.3 Dev. coeff. 7.3% 8.4% 2.7% 7.3Z a Calculated with the isotope dilution method using indole-2-I4Cas internal standard.
+
1-Ethylindole 138 130 126 124 112 126 9.0 7.1% l-methyl-
1-ethylindole, 280 ng of 1,3-dimethylindole, 99 ng of 1,4-, 1,5-, and 1,6-dimethylindole, and 32 ng of 1,2- and 1,7-dimethylindole (Table 11). The experimental deviation coefficients of the analysis varied between 2.7 and 8.4z. The significantly lower deviation coefficient for 1,3-dimethylindole may be explained by the lower chemical reactivityof 3-substituted indoles compared to indole and indoles unsubstituted on carbon-3 (13). The recovery rate for 1-methylindole varied between 65-74z. The analysis from several preparations of the carcinogenic subfraction BI showed that these contained 70-80 of the 1-methylindoles found in the mainstream smoke of the test cigarette. The pyrolysis of 2 X 30 mg of DL-nicotine did not yield any detectable amounts of indoles and/or N-alkylindoles. The average value calculated from two pyrolysis experiments with 2 X 15 mg of DL-tryptophan each were for 1.0 gram of the amino acid: 40 mg indole, 35.8 mg 3-methylindole, 0.9 mg 1-methylindole, and 0.58 mg 1,3-dimethylindole. In cigarette (13) P. L. Julian, E. W. Meyer, and H. C. Printy: “The Chemistry of Indoles,” in “Heterocyclic Compounds, Vol. 3,” R. C . Elderfield, Ed., J. Wiley & Sons, New York, 1952. ANALYTICAL CHEMISTRY, VOL. 42, NO. 3, MARCH 1970
369
smoke the ratio of indole (13.9 gg/cig.) to 1-methylindole (0.42 p g ) is 100:3 and that of 3-methylindole (14.0 pg) to 1,3dimethylindole (0.28 pg) is 100:2. In the pyrolyzate, the ratios are 100 :2.25 and 100 :1.6. Despite the use of different analytical methods the indole value for the tryptophan pyrolysis (880 “C) is comparable to the 3 x yield reported by Patterson et al. from the pyrolysis of tryptophan at 850 “C (14). This suggests that free and/or protein bound tryptophan in
(14) J. M. Patterson, M. LaFuse Baedecker, R. Musick, and W. T. Smith, Jr., Tobacco Sci., 13,26 (1969).
tobacco is an important precursor for indoles and N-alkylindoles in cigarette smoke. Biological data are required in order to verify whether 1-alkylindoles contribute to the carcinogenicity of the particulate matter of cigarette smoke and its most active subfraction BI. Bioassays on mouse skin are being conducted to determine the tumorigenicity of 1-methylindoles.
RECEIVED for review September 29, 1969. Accepted December 24, 1969. This study was supported by American Cancer Society Grant E-231, and in part by National Cancer Institute Grant CA-08748.
Square-Wave Polarographic Determination of Lead as a Pollutant in River Water E. B. Buchanan, Jr., Thomas D. Schroeder,’ and Bozena Novosel* Department of Chemistry, The Unioersity of Iowa, Iowa City, Iowa 52240 A square-wave polarograph has been used to determine trace amounts of lead in potable water as part of a pollution study in the vicinity of Iowa City, Iowa. By using a combination of sodium perchlorate and sodium fluoride as a supporting electrolyte under acidic conditions, as little as 2 & 0.32 ppb lead could be determined without preconcentration of the samples. The sensitivity of this technique i s roughly 100 times that of similar methods by atomic absorption.
ALTHOUGHSINE-WAVE polarography has been much more widely investigated as a means of chemical analysis, squarewave polarography is potentially more sensitive to trace quantities of reversibly reduced species in solution. The greater separation of faradaic and capacitance currents possible with the electronic gating of square-wave signals provides this improved sensitivity. Despite this, few reports have been published where square-wave polarography has been used as a n analytical tool for trace analysis. Barker and Jenkins ( I ) , who first reported the use of a square-wave polarograph, predicted a sensitivity to reducible species as low as 2 X 10-SM. Since then Ferrett, Milner, and Smales have used this technique to analyze for less than 1 ppm lead in cocoa ( 2 ) , Niki, Sirai, and Kyoya have measured 2 X lOU7Mlead and cadmium in phosphoric acid (3),Goode and Campbell ( 4 ) have analyzed uranium metal for less than 5 ppm each of copper, lead, cadmium, and zinc and Kashiki and Oshima determined 0.01 ppm free sulfur in petroleum ( 5 ) . Square-wave polarography in combination with anodic Present address, Shippensburg State College, Shippensburg, Pa. 17257 * Present address, Department of Chemistry, Faculty of Science, University of Zagreb, Zagreb, Yugoslavia
stripping has been used by von Strum and Ressel to determine copper, antimony, lead, tin, thallium, cadmium, indium, zinc, manganese, and barium in concentrations ranging down to 1 X 10-9M (6). We have employed a squarewave polarograph to analyze potable water as part of a study of lead pollution in the rural Iowa City, Iowa, area. Concentrations ranging down to 2 X 10-*M have been measured without preconcentration of samples. INSTRUMENTAL
Briefly, the instrument used in these investigations is a controlled potential device employing a variable frequency, variable amplitude square-wave signal to modulate a dc ramp voltage applied to a polarographic cell. The ac signal resulting from the oxidation-reduction process occurring at the surface of the D M E is directed to a gating circuit which passes current during a preselected portion of each half square-wave period. Since capacitance current drops off rapidly after each transition and faradaic current does not, separation of the two is achieved on the basis of time by allowing the gate to pass current only during a later portion of each half-period. This current is amplified, rectified, and sent to a recorder. The polarogram is a symmetrical peak whose height is proportional to the concentration of the electroactive species. For a more detailed discussion of the instrumentation and theory, references (7) and (8) should be consulted. EXPERIMENTAL
During the course of trace analyses performed with the instrument, the square-wave frequency was maintained at a value of 500 Hz. The gate was set such that it was open for of each half-period just before transition. The dc ramp voltage was scanned over a range of -0.1 to -0.4 volt US. a saturated silver/silver chloride electrode.
(1) G. C. Barker and I. L. Jenkins, Analyst, 77,685 (1952).
(2) D. J. Ferrett, G. W. C. Milner, and A. A. Smales, ibid., 731 (1954).
(3) E. Niki, H. Sirai, and T. Kyoya, Japan Analyst, 15, 257 (1966). (4) G. C. Goode and M. C. Campbell, Anal. Chinz. Acta., 27, 442 (1962). (5) M. Kashiki and S. Oshima, Bull. Chem. Soc. Japan, 40, 1630 (1967); Electroanal. Abstr., 6, 2180 (1968).
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(6) F. von Strum and M. Ressel, “Proc. 1st Australian Conf. Electrochem.,” Pergamon Press, Long Island City, N.Y., 310 (1965). (7) E. B. Buchanan, Jr., and J. B. McCarten, ANAL.CHEM., 37, 29 (1965). (8) J. R. Bacon, Ph.D. Thesis, The University of Iowa, Iowa City, Iowa, 1968.
