pyrene in Cigarette Smoke Condensate. - ACS Publications

The method presented in this in- vestigation for the quantitative deter- mination of BaP in cigarette smoke condensate employs a short series of separ...
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tron capture detection system and to apply this technique to the analysis of biologic materials. The potential sensitivity utilizing such a detector is appreciably greater than is achieved with the ceric-arsenite color reaction. Use of the electron capture detector for determining thyroid hormones was first suggested by Lovelock (1).

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LITERATURE CITED

(1) Lovelock, J. E., Nature

Figure 1 . Chromatogram of iodinated tyrosine and thyronine derivatives See text for abbreviations and conditions. from 2 to 0.5 inches per minute

At point A the chart speed was changed

DISCUSSION AND RESULTS

metrical with little evidence of tailing. The separation between T) and T d is especially noteworthy. The elution times are relatively short and the chemical procedure is sufficiently simple to be useful with little modification for checking the purity of iodoamino acid preparations or their qualitative identification. Further work is in progress to increase sensitivity by utilizing an elec-

Figure 1 shows a tracing obtained with the composite mixture. The aliquot injected contained 0.01 pmole of each iodinated amino acid. Peaks correspond to the retention times obtained with preparations of the individual components. Separation between the components is excellent and the peaks are sym-

189, 729 (1961). ( 2 ) Richards, A. H., M.S. thesis, University of Rochester, Department of Biochemistry, Rochester, N. Y. (1965). (3) Sandell, E. B., Kolthoff, I. M., J. Am. Chem. SOC.56, 1426 (1934). (4) Zomaely, C., Marco, G., Emery, E., ANAL.CHEM.34, 1414 (1962).

A. H. &CHARDS W. B. MASON

Department of Biochemistry, University of Rochester School of Medicine and Dentistry. Rochester, N. Y. RECEIVEDfor review July 14, 1966. Accepted September 2, 1966. Investigation supported in part b research grants AM 10244-01 and A d 08567-15 from the National Institute of Arthritis and Metabolic Diseases.

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Fluorometric Determination of Benzo(a)Pyrene in Cigarette Smoke Condensate SIR: Polycyclic aromatic hydrocarbons have been the subject of intensive analytical investigations in studies relating to tobacco smoke condensate and environmental a b mosphere. Among the polycyclics, benzo(a)pyrene (BaP) has received the most attention. In recent years, a number of methods ( I - I 3 , 1 6 , 1 6 ) on the quantitative analysis of BaP have been published which employed improved techniques and apparatus over the older, more laborious methods. These improvements resulted in better accuracy and in significant reductions in sample size. The former was accomplished by Hoffmann and Wynder (7) through the use of CWabeled BaP as an internal standard in an isotope dilution technique, thereby providing an absolute correction for losses incurred during the analysis. DeSouza and Scherbak (I),Robb et al. ( I O ) , and Oliver (8) also used Cl4-labeled BaP in their methods of analysis. The polycyclic hydrocarbon fraction enrichment scheme by liquid-liquid partitioning used by Grimmer ( 5 ) and Hoffmann 1752

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and Wynder (6) proved useful in tobacco smoke analysis because it removed the bulk of extraneous material prior to subsequent refined fractionation. Fluorescence spectral analyses, made easier by the recent availability of commercial spectrophotofluorometers, and as practiced by Sawicki, Hauser, and Stanley (11) and others (2, 8, IO) contributed to increased sensitivity and further identification of BaP. Oliver (8) recently described a shortened method of analysis employing CI%beled BaP and thin layer chromatography. In all methods of analysis for BaP in tobacco smoke, some form(s) of chromatographic separation-column, paper, and thin layer-is essential in isolating this compound for the quantitative measurement. Ayres (1) used a quantitative gas chromatographic procedure for BaP in cigarette smoke. The method presented in this investigation for the quantitative determination of BaP in cigarette smoke condensate employs a short series of separation and isolation steps with an added inactive internal standard, pery-

lene, in contrast to the use of C14labeled BaP which requires relatively expensive counting equipment such as a liquid scintillation counter. Perylene as used herein is simply and conveniently determined along with BaP by fluorescence spectral analysis. The internal standard is necessary for the normalization of test data to the 100% recovery level. Isotope dilution studies with C14-labeled BaP confirm the accuracy of data based on perylene as the internal standard. The amount of perylene added is many times greater, about 25X, than that actually found in smoke so that any applied blank correction, by comparision, is small. The blank is run separately-Le., a smoke condensate sample to which no perylene is added. Novel pre-equilibrated solvent pairs are used in the liquid-liquid partitioning step. These solvents possess lower boiling points than the solvents heretofore used ( 5 , 6) and, hence, are more easily removed under vacuum with no loss of constituents being analyzed.

EXPERIMENTAL

Reagents. €'re-equilibrated solvents: methanol/water (6: 1) and hexane; hexane and acetonitrile. Spectroquality or chromatoquality solvents are used. Liquid scintillation solution consisted of 3 grams of 2,5-diphenyloxazole (PPO) and 100 mg. of 1,4-bis-2(5phenyl-oxazolyl) benzene (POPOP) in 1000 ml. of scintillation grade tolueneall available from Packard Instrument Sales Corp. The benzo(a)pyrene (8,9 C14)was purchased from Kuclear E q u i p ment Corp. (NEC). Inactive polynuclear hydrocarbons, including benzo(a)pyrene and perylene, were obtained from Aldrich Chemical Co., Inc., Milwaukee, Wis.; K and K Laboratories, Plainview, N. Y.; and Eastman Chemicals, Rochester, X. Y. Apparatus. Fluorescence spectra were obtained with the Aminco-Bowman spect rophotofluorometer (SPF) equipped with X-Y rtlcorder, hmerican Instruments Co. The Packard Model 314-ES liquid scintillation spectrometer \vas used in the isotope dilution analysis. Procedure. The summary analysis scheme for 13aP is as follows: Smoke condensate collection from only 20 or 25 cigarettes Partitioning between solvent pairs Column chromatography Paper chromatography Fluorescence spectral analysis SMOKE COXDENSATE COLLECTIOX. Vi arettes, conditioned and smoked a t 6 0 b R H and 74" F., are machinesmoked to the specified butt length, usually 30 mm., on a smoking cycle of one 35-ml. puff every 60 seconds. A constant volume smoking machine was used. Usually 25 cigarettes are smoked and the mainstream condensate is collected in a glass trap a t about -80' V. The condensate, after being weighed, is dissolved in about 60 ml. of 6: 1 (by volume) methanollwater to which 4.0 pg. of the internal standard, perylene, have been added. A blank tar sample is carried along-i.e., one to which no perylene is added. LIQUID-LIQUIDP.4RTITIONING (5, 6). l'he methanollu-ater solution of the condensate is extracted three times with 35 ml. of hexane. The combined hexane extract is quickly evaporated to dryness in a rotary vacuum evaporator a t 30" to 35" C. and 70 to 90 mm. Hg and the reyidue is dissolved in 50-ml. of hexane previously equilibrated with acetonitrile. l'he hexane solution is extracted five times with 35 ml. of acetonitrile. The combined acetonitrile extract is quickly evaporated to dryness a t 40" C. and 70 to 90 mm. Hg. The residue is dissolved in 5 to 10 ml. of hexane. SILICA GEL COLUMN CHROMATOGRAPHY. Silica gel (Davison Grade 922) of low activity is prepared by thorough washing of the silica gel with methanol and heating under vacuum to 70" to 75" C. for about 45 minutes. h silica gel colurnn 12 mm. in diameter and 23 cm. high is eluted with hexane a t a rate of 2.5 to 3.0 ml. per minute under slight nitrogen pressure. A calibrating solution of 30 pg. of BaP and 30 pg. of

Cigarette A-12 G-15

Test filter

Table I. Benzo(a)pyrene in Cigarette Smoke Recovery, %, based on: BaP per 100 Condensate, cigarettes, fig." C14-BaP Perylene mg./cigarette 73, 73 75, 77 3 . 0 , 3.2 38 =t 2 79, 81 76, 79 3.6, 4 . 2 55 =t 1

3.1. 2 . 6

on A-12

M-1 (filter) M-2 (filter) M-3 (filter)

2.4; 2 . 2 2.1,2.2 2.2, 2 . 3

3.3,3.3

MX-1 MX-2

MX-3 GCalculation based on perylene recovery.

perylene in 5 ml. of hexane is added and the column is eluted with hexane; 30-ml. fractions are collected. The silica gel activity is satisfactory when most of the BaP comes out in the seventh fraction, with minor amounts in the sixth and eighth fractions. The distribution of BaP and perylene in the fractions is conveniently found by ultraviolet absorption analysis with a recording spectrophotometer. The condensate extract in 5 t o 10 ml. of hexane from the liquid-liquid partitioning is added to a silica gel column, which is then eluted with hexane as described above. Fractions containing the BaP and perylene are combined and evaporated almost to dryness. PAPERCHROMATOGRAPHY (2,4,7,10). The concentrated fraction is streaked on acetylated paper (S &. S Grade 2495) previously extracted with methanol. Reference spots of BaP and perylene are also applied. The chromatogram is developed a t least 16 hours (overnight) in an ascending mode with 17 :4:4 ethanollwaterltoluene mixture (12). The RaP and perylene zones are cut out and extracted separately with methanol and filtered. The solutions are evaporated to dryness, and 5.0 ml. of methanol are added t o the flask containing BaP; 10.0 ml. of methanol are added to the flask containing perylene. Hexane may be used instead as the solvent. SPECTROPHOTOFLUOROMETRIC (SPF) QUANTITATIVE MEASUREhIENT (2,9,11). The excitation and emission spectra of the two solutions are obtained with the Aminco-Bowman SPF unit. Calibrations, preferably run along with the sample solutions, permit the quantitative measurements of BaP and perylene in the sample solutions. Recoveries of the internal standard, in general, range from 60 to 80%. The appropriate recovery correction is applied to the BaP value, expressed as micrograms per 100 cigarettes. RESULTS AND DISCUSSION

The partition coefficients a t 25' C. of BaP and perylene between hexane/ methanol were 18 and 6, respectively, and, between acetonitrile/hexane, 2.2 and 2.1, respectively. No measurable losses of these compounds were observed in the overall partitioning step.

2.8, 2 . 5 2.9, 2 . 5

34 iz 2 34 iz 1 33 =t 2

31 f 1 46 1 45 2 43 iz 1

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This polycyclic fraction enrichment step yielded a fraction, based on a number of such measurements, which was about 8% of the original total condensate. Clean separations of BaP and perylene were achieved on an acetylated paper chromatogram developed 16 hours overnight or in a stop and start technique (4) in an ascending manner with Re17 :4: 4 ethanol/water/toluene. coveries of polycyclic hydrocarbons off the paper chromatogram were over 90%. The recoveries of BaP from tobacco smoke condensate based on perylene as the internal standard were confirmed by isotope dilution analyses with BaP-8,9C14 ( 7 ) . The labeled BaP, purified by paper chromatography, had a specific activity of 21,900 c.p.m. per pg. *2%. Two levels of labeled BaP additions to smoke condensate from 25 cigarettes were tested, 0.26 and 1.05 pg. The actual amount of (inactive) BaP in the smoke condensate from 25 cigarettes lies about midway between these two levels. The total amount of BaP, active and inactive, was measured in 5.0 ml. of methanol by fluorescence spectral analysis a t 376 mp (activation) and 405 mp (emission). The C14-labeled BaP was measured by liquid scintillation counting technique with a Packard Model 314 ES spectrometer. All counting was done with a standard, fixed volume, 15.0 ml., of scintillation solution (PPO POPOP) in vials of constant dimensions a t 4" C. The necessary blanks and calibrations were run along with the sample solutions. Perylene was measured by fluorescence spectral analysis a t 427 mp. (activation) and 438 mp (emission) in methanol. The fluorescence activation and emission calibration curves of BaP in methanol and perylene in methanol were linear over the concentration ranges encountered and beyond. In Table I, the recoveries based on the two internal standards, perylene and C14-BaP, are reported along with the values of BaP and condensate. All cigarettes were 85 mm. in length and were smoked a t the rate of one puff per minute to a 30-mm. butt

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VOL. 38, NO. 12, NOVEMBER 1966

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Figure 2. Fluorescence activation (solid line) and emission (dotted line) spectra of perylene recovered from smoke condensate and dissolved in 10.0 ml. of methanol, Curve 1 and standard perylene solution, Curve 2, 0.40 pg./ml.

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Figure 1 . Fluorescence activation (solid line) and emission (dotted) spectra of unknown, Curve 1, isolated from smoke condensate of 25 cigarettes and dissolved in methanol, and 3,4-benzpyrene, Curve 2, in methanol, 0.1 0 pg./ml.

length, with the exception of (3-15 which was smoked on a 30-second cycle to a 25-mm. butt length. Twenty or 25 cigarettes were used per analysis. The BaP values in Table I are in line with those reported by Wynder and Hoffmann (17) and De Souaa and Scherbak (4). The average value reported in the Surgeon General's Advisory Panel Report is about 20% lower, with the correction applied for losses as explained (14, page 57) for the nonfilter cigarettes. The recoveries based on perylene as the internal standard were confirmed by the values found with the C14labeled BaP. The agreement in Table I between the two internal standards is good. Typical A uorescence activation and emission spectra of an unknown compound isolated from the mainstream smoke of 25 cigarettes (American, A-13) and dissolved in 5.0 ml. of methanol are identical with those of authentic BaP in methanol (Figure 1). The amount of BaP found was 3.4 pg. per 100 cigarettes. In Figure 2, the activation and emission spectra of perylene standard solution and the recovered perylene, both in methanol, are shown. In this case the perylene recovery, corrected for the blank, was 6070. The small blank correction need be measured only once in studies where a standard tobacco column is used in assessing the effects of variables on BaP yield in smoke. For a number of American cigarettes the blank correction, measured as perylene with the fluorometer, was 0.6 to 0.8 pg. per 100 cigarettes. The ultraviolet absorption spectrum of the unknown compound isolated from the mainstream smoke of 50 cigarettes 1754

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Figure 3. Ultraviolet absorption spectra of unknown compound, Curve 1, isolated from smoke condensate of 50 cigarettes and dissolved in methanol, and 3,4benzpyrene, Curve 2, in methanol, 1 .O pg./ml. Cell length, 1 .O cm.

(American, A-12) by the above described procedure is qualitatively identical to that of BaP (Figure 3). Moreover, the peak height a t 384 mp permits an estimate of the BaP level, about 2.8 pg. per 100 cigarettes, which closely a p proximates the value found by the more sensitive and precise fluorescence spectral analysis, 3.1 fig per 100 Cigarettes, for cigarette A-12. The relatively simple and straightforward operations of this method, employing a convenient and easily measured internal standard, permit a

rapid, accurate analysis to be completed in less than 2 days. The relative standard deviation for the analysis is 9%, using the standard deviation computed by the range method from the data in Table I. LITERATURE CITED

(1) Ayres, C. I., Thornton, R. E., Bear. T a b a k - F o r ~ h 3, . 285 (1965). (2) Barkemeyer, von H., Ibid., 1, 325 (1962). (3) Cooper, R. L., Lindsey, A. J., Brit. J. Cancer 9, 304 (1955).

(4) DeSouza, J. E., Scherbak, M., Analyst 89, 735 (1964). (5) Grimmer, von G., Beitr. TabakForsch. 1. 107 (1961). (6) Hoffm&--D. H., Wynder, E. L., ANAL.CHEM.32, 295 (1960). (7) . . Hoffmann. D. H.. Wvnder, E. L., Cancer 13, 1062 (1960). ” (8) Oliver, B. J., Jr., “Thin-Layer ChromatoeraDhic SeDaration of Benzo(ab pyrene from Cigarette T&,” 19ih \-

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Tobacco Chemists Research Conference, Lexington, Ky. (Oct. 26, 1965). (9) Pailer, M., Hubsch, W., Kuhn, H., Fachllche Mitt. Oesterr: Tabakregie 1-11; (1965); Abstract #1795, Corresta

Information Bulletin, No. 3, p. 32 (1965). (10) Robb, E. W., Governator, G. C., Edmonds. M. D.. Bavelv. A.. Beitr. Tabak-Fokch. 3, 278 (i966j. ’ (11) Sawicki, E., Hauser, T. R Stanley, T. W., Intern. J. Air Pollut&n 2, 253 (1960): (12) Sawicki, E., Stanley, T. W., Elbert, W. C.. Pfaff. J. D.. ANAL. CHEM.36. 497 (i964). ‘ (13) Schmetz, I., Stedman, R. L., Chamberlain, W. J., Ibid., p. 2499. (14) “Smoking and Health,” report of Advisory C-ommittee to Surgeon Genera1 of Public Health Service, U. S.

Public Health Service Publication No. 1103, U. S. Govt. Print. Office, Washington, D. C., 1964. (15) Van Duuren, B. L., J. Natl. Cancer

Inst. 21, 1 (1958). (16) Ibid., p. 623. (17) Wynder, E. L., Hoffmann, D., Advances in Cancer Research 8, pp. 249453, Academic Press, New York, 1964.

HOWARD J. DAVIS A. LEE LEONARD R. DAVIDSON THOMAS Celanese Research Co. Summit, N. J.

Rapid Determination of Molecular Weight Distribution of Polyoxyethyle ne-Type Nonionic Surfacta nts by Ci rcula r Thin Layer Chromatog ra phy SIR: In the preparation of a polyoxyethylene-type nonionic surfactant by the addition of ethylene oxide to a molecule containing an active hydrogen, the product is always a mixture in terms of the number of moles of ethylene oxide added to a molecule. This phenomenon gives rise to many important problems not only in the study of the physical properties of the material but also in practical applications. Flory (2) suggested that the molecular weight distribution corresponds to a Poisson distribution. Previously, a molecular distribution (3, 5 ) was used t o obtain the molecular weight distribution of nonionic surfactants. With materials of a low polymerization degree, the distribution was compared with the Poisson distribution or Weibull’s by Nagase ( 7 ) . Kelly (4) separated a mixed para-, tert-octylphenoxy-polyoqethyleneethanol with 9.7 ethylene oxide units per phenol into its component compounds by elution chromatography and obtained a molecular weight distribution curve which corresponded to a Poisson distribution, Thus, an accurate method has been established for the determination of the molecular weight distribution of polyoxyethylene-type nonionic surfactants, However, this elution chromatographic determination requires more than 15 days for the separation of one sample, Burger (1) reported a rapid separation of polyoxyethylene-type nonionic surfactants into their component compounds by thin layer chromatography. However, a molecular weight distribution curve has not been reported. The object of this work was t o develop a method for a rapid determination of the molecular weight distribution of polyoxyethylene-type nonionic surfactants. Polyo.xyethylene-type nonionic surfactants were separated into their component compounds by circular thin

Figure ment

1.

Apparatus

for develop-

1, separatory funnel; 2, developing solvent; 3, stopper; 4, glass tube (35-mm. diameter, 80-mm. length); 5, capillary; 6, circular glass plate (200-mm. diameter) with a hole (20-mm. diameter); 7, filter paper containing deveioping solvent; 8, adhesive; 9, ring made of 5-mm. acryl-resin plate; 10, chromatoplate ( 2 0 0 X 2 0 0 mm.); 1 1 , stand

layer chromatography (CTLC) and the visualized chromatograms were photographed. The transmittance of the film was measured and the molecular weight distribution curve could be constructed readily by a simple calibration of the density distribution curve. The resultant molecular weight distribution curve is compared with the Poisson distribution curve. Successful results were obtained with polyoxyethylene nonylphenolether (p = 9 ) . EXPERIMENTAL

Apparatus. A Stahl-type apparatus (Desaga) for thin layer chromatography and 20- X 20-cm. glass plates were used. An apparatus for circular thin layer development was made in our laboratory according t o bhe procedures described by Musha (6),and is shown in Figure 1. A micro-

photometer (Shimazu Seisakusho Ltd., %type) was used for the transmittance measurements. A vapor pressure osmometer (Mechrolah Inc. Model 301A) was used for the molecular weight determinations. Reagents and Materials. A 1% iodine solution was made by dissolving 2.5 grams of iodine in 250 ml. of methanol. Silicic acid (Wako Gel B-0 suitable for thin layer chromatography) was used as an adsorbent with the addition of 0.5% carboxymethylcellulose (CMC). Special grade methyl ethyl ketone saturated with distilled water a t room temperature was used as a developing agent. Polyoxyethylene nonylphenolether (p = 9) was prepared in our laboratory. Polyoxyethylene glycols which are present as by-products were removed by a counter-current distribution extraction (8) using a n-butanol-water system. Five hundred milligrams of the refined polyoxyethylene-type nonionic surfactants were dissolved in 5 ml. of methanol and used as the sample solution. Polyoxyethylene nonylphenolethers, with 8, 9, and 10 mole ethylene oxide units, were prepared by the elution chromatographic separations and these were used as standard reagents. Minicopy film (Fuji Film Co., Ltd. ASA32) was used to take photographs of the chromatograms. Preparation of Chromatoplates. Thin layer chromatoplates (20 x 20 em.) were prepared from silicic acid by mixing 30 grams of dry powder and 150 mg. of CMC with 60 ml. of water and applying this onto the glass plates with a ~piwtderset a t a thickness of 250 microns. The plates were air-dried for 30 minutes and heated in an oven a t 105’ C. for 2 hours. After drying, the layers were stored in a dry box. Sample Application and Circular Development. Fifty microliters of the sample solution were applied through a micropipet to the center of a layer in a ring (about 20 mm. in diameter). The chromatoplate and the developing V O L 38, NO. 12, NOVEMBER 1 9 6 6

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