Copper-Binding Activity of Tobacco Smoke Condensate Leslie W. Michael, Edward E. Menden, and Harold G . Petering Department of Environmental Health, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45219
The distribution of copper between a buffered aqueous solution of the metal ion and a n organic solution of tobacco smoke condensate (TSC) in 4-methyl-2-pentanone (MIBK)has been studied. The analytical parameters, metal ion and hydrogen ion concentrations, were investigated. Metal ion concentrations were determihed by atomic absorption spectrophotometry. TSC was collected with use of a Mason Mark I11 Smoker and acetone traps maintained at 0°C. This material was transferred to MIBK by evaporation at reduced pressure. A 10-ml aliquot of an aqueous solution 0.1M in 2-amino-2(hydroxymethyl)-1,3-propanediol(TRIS)0.01M in CuCl? and p H 5.6 t o 6.1 was equilibrated with a n equal volume of the MIBK solution of TSC, which contained the equivalent of five cigarettes. Analysis of a n aliquot from the organic phase after equilibration of the two phases yielded a maximum value of 300 pg of copper per cigarette when fresh, nonfractionated TSC was used. This simple, rapid extraction technique has been used to determine variations in the chemical activity of tobacco smoke condensate.
T
his report presents initial results of our investigation o n the reaction between tobacco smoke condensate (TSC) and transition metals. Particular attention was directed toward the ligands present in the condensate, rather than metals or metallic constituents in native tobacco products or tobacco smoke. This study reports the reaction between tobacco tar and copper ions, which was demonstrated by changes in the distribution of copper between an aqueous phase and an immiscible solvent. The chemical nature of tobacco and tobacco smoke was reviewed recently by Stedman (1968), who noted, as have other investigators (Pillsbury et a/.. 1969; Swain et a/., 1969; Wartman et al., 1959), the variability of tobacco tar as a n analytical sample. Reactions using the alkylating activity (Stedman and Miller, 1967), reducing power (Hagopian, 1969), bacterial toxicity (Weiss and Weiss, 1967), and tumorigenicity (Bock et al., 1969) of tobacco tar have been reported. From a consideration of the reaction between TSC and copper ions, a procedure for assaying condensate was developed.
Experimental Apparatus. A Beckman research model p H meter was used with a Sargent combination electrode. A Perkin-Elmer Model 303 atomic absorption spectrophotometer, with recorder readout, was used for the determination of copper. The copper hollow cathode lamp was operated at 15 mA in conjunction with acetylene and air-flow rates of 8 and 10 ft3/min, respectively, and wavelength setting of 324.7 A. Cigarettes were smoked o n a Mark I11 Rotary Smoker (R.W. Mason, Cleve-
don, England). This unit smokes 24 cigarettes sequentially with a 2-sec puff per min per cigarette and a flow volume of 17.5 + 0.5 ccjsec. Both ends of the cigarettes are exposed to the atmosphere between puffs. Reagents. Stock solutions 0.2M in CuClz (Mallinckrodt, AR) and 1.OM in 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS)(Fisher, Analyzed) were prepared with deionized water (Crystalab Deminizer). The 4-methyl-2-pentanone (MIBK) (Matheson, Coleman, and Bell, 114' to 6°C) and acetone (Amsco C. P.) were used as received. Mixed metal atomic absorption standards were prepared by serial dilution of a stock 500 ppm standard solution of metal ions. This solution was prepared from 0.5 g each of cadmium, copper, iron, lead, and zinc (analytical grade metals), and 1.410 g K 2 C r 0 4(Mallinckrodt, AR). The metals dissolved in acid were made to 1 liter total volume with 10% nitric acid. Tobacco Smoke Condensate. Research cigarettes, prepared under the auspices of the Tobacco and Health Research Program of the University of Kentucky, were used. These 85-mm nonfilter cigarettes were smoked o n the Mark I11 Rotary Smoker to a 23-mm butt length. An average of 12 puffs per cigarette was required. Tobacco smoke condensate was trapped in modified 250-ml vacuum traps, which were partially filled with glass beads and contained approximately 100 ml of acetone. Two traps, cooled in an ice bath, were used in series. The efficiency of the traps was accessed by placing a Cambridge filter held in a Lucite holder (Phipps and Bird) between the flow controller of the Mark I11 and the acetone traps; less than 0.1 mg of TSC was found on the filter. The flow system in series was cigarette, acetone trap one, acetone trap two, Cambridge filter, and Mark I11 flow control console. Solutions from the traps were transferred to a 1-liter, roundbottomed flask to which MIBK was added. An excess of MIBK was used so that, upon removal of the acetone, the stock MIBK tar solution contained tobacco smoke condensate equivalent to 0.5 cigarette per ml. The acetone was removed by evaporation a t reduced pressure with a water aspirator as a vacuum source and a water bath maintained at 35°C i 5". Filtration of the resultant solution yielded 1 =k 0.15 mg of MIBK insoluble material per cigarette. The average yield of TSC collected by this procedure was 36 =I= 3 mg of tar per cigarette. Procedure. An aqueous solution 0.01M in cupric ion and 0.1M in TRIS was prepared by dilution of the stock solutions and adjusted to p H 5.6 to 6.0 before dilution to final volume. A 10-ml aliquot of the metal ion solution and an equal volume of the MIBK solution of tar (equivalent to five cigarettes) were combined in a 45-ml conical centrifuge tube fitted with a glass stopper. The resultant mixture was shaken for 1 min, and centrifuged to facilitate phase separation. A 1-ml aliquot of the organic phase was extracted with 5 ml of 10% nitric acid with use of a vortex mixer. The nitric acid solution was aspirated into the flame of the atomic absorption spectrophotometer Volume 5, Number 3, March 1971 249
Table I. Copper-Binding Activity of Tobacco Smoke Condensate
Sample
PH
5.2 5.7 5.9 5.9 6.1
Amt of copper bound/ cigarette," pg 295 315 290 305 300
a A 10-ml aliquot of a n aqueous solution 0 . 1 M in 'IRIS, 0.01M in CuCL and p H 5.6 to 6.1 was equilibrated with a n equal volume of the solution of TSC, which contained the equivalent of five cigarettes. Copper concentration was determined by analysis of aliquot from the organic phase after equilibration of the two phases.
MIBK
after removal of the organic phase in order to determine the copper concentration. Copper concentration was determined by interpolation of the nonlinear graph of standard copper solutions. Back extraction of copper from the organic phase into nitric acid was performed to place the copper in a n aqueous matrix similar to the standards used for analysis. The amount of copper extracted from the buffered aqueous solution of copper (11) in the absence of TSC and the amount of copper remaining in the organic phase after back extraction with nitric acid were less than 3 pg per cigarette. Data collected by use of the described technique are summarized in Table I. A variation of + 5 % was observed in replicates collected from the same MIBK tar solution. A maximum of 300 + 15 pg of copper per cigarette was found when recommended conditions were used.
Results and Discussion The extraction process provided several parameters for the control of the metal ion distribution. The choice of solvent was made from an evaluation of the solubility of tobacco tar and from a consideration of the data available on the distribution ratio, D, of copper complexes (Kertes and Marcus, 1969; Morrison and Freiser, 1957; Ringbom, 1963). The distribution ratio is defined as the ratio of metal ion in the organic phase to that found in the aqueous phase. The solvent of choice was 4-methyl-2-pentanone,h m K . The absorbance of chloroform soluble copper complexes of Ci-Cls acids has been used as a quantitative method for the determination of fatty acids (Ayers, 1956). Such materials are known constituents of TSC and, therefore, contribute to the distribution of copper in the proposed assay procedure; however, these acids are a small fraction of the variety of compounds in TSC with potential copper-binding activity (Stedman, 1968). Variation in the hydrogen ion and copper ion concentrations were studied by suitable alterations of the aqueous phases prior to volume adjustments and phase equilibria. The p H was measured after equilibration as tobacco condensate also exhibited some buffering capacity. Analysis of respective phases was conducted directly on the aqueous phase and on the nitric acid extract of the organic phase. The dependence of the distribution of copper in the presence of 0.1M TRIS on the hydrogen ion concentration is shown in Figure 1. These data are similar to data obtained in the absence of TRIS, although reproducibility and ease of handling were improved by the addition of TRIS. These observations are consistent with the reported equilibrium data for copper TRLS complexes (Hanlon et a/., 1966). The interaction of TRIS with Cu(I1) was small over the range of hydrogen ion concentrations which were found optimal for copper extraction with tobacco tar. 250 Environmental Science & Technolog)
Therefore, the function of TRIS is, as a metal buffer, to minimize formation of copper hydroxides and facilitate the separation of the organic and aqueous phases. An increase in the copper concentration in the organic phase was observed at higher concentrations of copper in the aqueous phase. This increase was concurrent with a decrease in the distribution ratio as shown in Figure 2. This procedure provides an additional method for the quantitative determination of certain aspects of the chemical activity of TSC as measured by the movement of copper across a phase barrier. The proposed extraction technique can be performed simply and rapidly, and the copper concentrations are readily determined by atomic absorption spectrophotometry. In contrast, both the alkylation technique (Stedman and Miller, 1967) and reduction assay (Hagopian, 1969) require a digestion period, and bioassay procedures also have extensive time requirements. Each of these techniques, however, provides assays of different aspects of the chemical activity of TSC, and a combination of these procedures is required to characterize this complex material. The assay method proposed here presents an additional parameter for correlating TSC samples and is being used in our laboratory to study the preparation, handling, and storage of TSC. Changes in fractionation schemes are known to alter the alkylating and biological activity of TSC. Comparisons of these probes of TSC with the method reported here are currently in progress.
0.0
1.0
1
ir L L - - I
1
4.5
--A
5.5
5.0
6.0
PH
Figure 1. Hydrogen ion dependence of the distribution of copper, D,between MIBK solutions of tobacco smoke condensate and an aqueous solution of TRIS
I
I
I
I
2.0
2.5
3.0
Figure 2. Copper concentration dependence of the distribution of copper, D,between MIBK solutions of tobacco smoke condensate and an aqueous solution of TRIS
Literature Cited Ayers, C. W., Anal. Chem. Acta 15,77 (1956). Bock, F. G., Swain, A. P., Stedman, R. L., Cancer Res. 29, 584 (1969). Hanlon. D. P.. Watt, D. S.. Westhead. E. W.. Anal. Biochem. 16,225 (1966). ’ Hagopian, M., ENVIRON. Scr. TECHNOL. 3,567 (1969). Kertes. A. S.. Marcus. Y.. “Solvent Extraction Research.” Wiley, New York, 1969, pp 257-80. Morrison, G . H., Freiser, H., “Solvent Extraction in Analytical Chemistry,” Wiley, New York, 1957, pp 204 and 5. Pillsbury, H. C., Bright, C. C., O’Connor, K. J., Irish, F. W., J . Ass. Off. Anal. Chenz. 52,458 (1969). Ringbom, A., “Complexation in Analytical Chemistry,” Interscience, New York, 1963, pp 373 and 4.
Stedman, R. L., Chem. Rea. 68,153 (1968). Stedman, R. L., Miller, R. L., Chem. Znd. 1967,618. Swain, A. P., Cooper, J. E., Stedman, R. L., Cancer Res. 29,579 (1969). Wartman, W. B., Jr., Cogbill, E. C., Harlow, E. S., Anal. Chem. 31,1705 (1959). Weiss, W., Weiss, W. A., Arch. Enciron. Health 14, 682 (1967).
Receiced,for reaiew February 11, 1970. Accepted July 13, 1970. This work was supported by a grant from the American Medical Association Education Research Fund and by Public Health Service Grant ES-00159. Presented at the Division of Analytical Chemistry, 159th Meeting ACS, Houston, Tex., February 1970.
COM M UN lCATl ON
An Improved Impactor for Aerosol Studies-Modified
Andersen Sampler
John Nan-Hai Hu Ethyl Corp., Ferndale, Mich. 48220
Reducing Particle Bounce-Off An Andersen sampler has been modified to extend its lower range from 0.56 to 0.17 p by operating the Andersen sampler a t 3 ft3/min instead of the designed value of 1 ft3/min. The conventional method of coating the collection surface with grease or other materials did not effectively eliminate particle reflection. Effective operation was obtained by use of glassfiber filters as collection plates. The modified Andersen sampler was calibrated with a Royco Particle Counter (Model 200A) and monodispersed polystyrene particles. The wall loss was very low in the range of particle sizes studied.
C
ascade impactors of various designs have been used for aerosol studies for many years (Andersen, 1958; Brink, 1958; Gillespie and Johnstone, 1955; May, 1945; Mitchell and Pilcher, 1959; Ranz and Wong, 1952). Among these impactors, the Andersen sampler has the advantages of being simple, inexpensive, and rugged. However, it is disadvantageous in that the lowest stage constants (50% cutoff point of the last stage) are limited to about 0.5 p (aerodynamic equivalent) because of the flow-rate limitation. For example, the manufacturer of the Andersen sampler recommends operation a t 1 ft3/min. Under this flow condition, its lowest stage constant, D50for Stage 6, is 0.56 p (Flesch et al. 1967). To extend the lower range of usefulness, many investigators have studied impactors operating under reduced pressure (Parker and Buchholz, 1968; Prins, 1965; Tomaides and McFarland, 1968). The stage constants of a n impactor can also be reduced by increasing the flow rate. This method has a further advantage in that the sampling time is reduced. However, McFarland and Zeller (1963) showed that particles begin to bounce off the collection surface when the sampling flow rate is too high. Coating the collection surface with sticky materials has been known t o reduce bounce-off (Parker and Buchholz, 1968; McFarland and Zeller, 1963). However, Liu (1969) observed bounce-off even when the collection surface had been coated with materials reported to prevent bounce-off effectively.
To reduce the stage constants, we increased the flow rate of the Andersen sampler from 1 to 3 ft3/min. While calibrating the unit, we found that even with a thin layer of grease o n the collection plates, the collection efficiency increases with particle size, reaching a maximum at a certain size and begins t o drop as the particle size increases. Theoretically, the collection efficiency should increase continuously with particle size if there is no bounce-off. Thus, the drop in collection efficiency a t large particle size was clearly the result of bounceOff.
After investigating several methods for reducing particle bounce-off, we discovered that placing a glass-fiber filter (Gelman Type A) o n top of each collection plate greatly reduced bounce-off. With this method, we were able to efficiently run the Andersen sampler at a flow rate of 3 ft3/min without appreciable particle bounce-off. The effectiveness of the glass-fiber filters in minimizing bounce-off probably results from their porous, fibrous structure. When a particle hits a flat surface and bounces, the direction of bounce is upward. When a fibrous filter is used as the collection surface, chances are that a particle will hit a cylindrical fiber, bounce downward, and eventually be collected by other fibers. A particle that bounces upward also has a good chance of being collected by the surrounding fibers if the fiber it initially hits is lower than the surrounding fibers. A question that might be asked is: Does the rough surface of the glass-fiber filter cause any diffusional losses of very fine particles? The answer is no. We have fed 0.23-p particles into the modified Andersen sampler running a t 3 ft3/min. At this rate, we did not expect any collection of particles of this size in the first few stages, and we found this to be so. In addition t o eliminating bounce-off effectively, the glassfiber filters o n top of the collection plates greatly simplify sample handling, especially for chemical analysis. The modified method could be tested at higher flow rates to further reduce the stage constants and the sampling time. This is particularly important for dilute samples, such as atmospheric aerosols. Volume 5, Number 3, March 1971 251
