An Easily Built Smoking Machine for Use by ... - ACS Publications

Laboratory Experiment pubs.acs.org/jchemeduc

An Easily Built Smoking Machine for Use by Undergraduate Students in the Determination of Total Particulate Matter and Nicotine in Tobacco Smoke Víctor González-Ruiz, M. Antonia Martín, and Ana I. Olives* S. D. de Química Analítica, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza de Ramón y Cajal s/n, 28040 Madrid, Spain S Supporting Information *

ABSTRACT: Sampling mainstream cigarette smoke is a challenging and stimulating laboratory activity for undergraduate students. In addition to the public health significance, cigarette smoke is an unusual source of analytes to examine the differences between gaseous matrices versus liquid or solid matrices. Sophisticated automated smoking machines complying with international standards are not affordable for educational purposes. However, a less expensive and simple smoking apparatus can be easily built in any laboratory that yields reproducible smoking conditions and allows several cigarettes to be smoked simultaneously. We describe the construction of such an apparatus utilizing a solid-phase extraction manifold and chamber, and how it can be used by undergraduate students to generate cigarette smoke and trap the total particulate matter (TPM). The TPM can be later gravimetrically quantified and eluted with 2-propanol containing an internal standard to quantify the nicotine content. Because a set of six cigarettes can be “smoked” simultaneously, the proposed procedure allows the comparison of TPM and nicotine content in mainstream smoke from normal and light cigarettes. KEYWORDS: Second-Year Undergraduate, Upper-Division Undergraduate, Analytical Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Consumer Chemistry, Gas Chromatography, Laboratory Equipment/Apparatus, Thin Layer Chromatography, Toxicology

T

he work of analytical chemists involved in solving forensic or public health problems is attractive to students. Reproducing this kind of work in the laboratory helps teachers convey the relevance of analytical chemistry to real-life problems of high social impact.1 Several sets of standard methods to determine different analytes in tobacco smoke have been developed by various institutions, for example, Cooperation Centre for Scientific Research Relative to Tobacco, CORESTA; International Organization for Standardization, ISO; Federal Trade Commission, FTC; Tobacco Institute of Japan, TIOJ. These methods share many similarities and can be easily adapted to a college laboratory program if the use of a commercial analytical smoking machine2 is avoided by developing an alternative smoking device. The proposed laboratory exercise shows students how complex official analytical methods can be adapted following simpler methodologies with lower costs, provided that the limitations of such methodologies are acknowledged and critically discussed. The two most relevant constituents of tobacco smoke are examined: total particulate matter (TPM) and nicotine (Figure 1). Several approaches to generate and trap tobacco smoke have been reported.3−8 Most of these systems are able to “smoke” only individual cigarettes, leading to low reproducibility. Our simple smoking apparatus avoids this drawback by using a setup that allows six cigarettes to be smoked simultaneously. This ensures that all the cigarettes in a set are smoked under identical © 2012 American Chemical Society and Division of Chemical Education, Inc.

Figure 1. Chemical structure of the alkaloid nicotine.

conditions, allowing comparison between different groups inside the set. The students become familiar with international standard methods and also have the opportunity to identify which steps of an official method are critical to the final outcome. The experiment was carried out in an instrumental analysis laboratory course (second year in the graduate studies in pharmacy) with more than 50 students per semester. The students were divided into groups of four, each group having an experienced student supervising the experimental steps and the data handling. The construction of the smoking device takes less than 2 h. The laboratory experiment is designed to take 2 to 3 h, plus the time required for the GC−MS analysis.

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EXPERIMENTAL DETAILS

Cigarettes

Lucky Strike Original Red (normal) and Lucky Strike Blue (light) filtered cigarettes were purchased at a local tobacconist. Published: April 11, 2012 771

dx.doi.org/10.1021/ed200664q | J. Chem. Educ. 2012, 89, 771−775

Journal of Chemical Education

Laboratory Experiment

A line was marked on the cigarettes indicating the length of the cigarette that must remain unburned.9 For these cigarettes, a butt length of 35 mm was set; this value must be calculated for every brand and type of cigarette as detailed by the international guidelines (indications can be found in the Supporting Information). Reagents and Solutions

Heptadecane (99%) and (−)-nicotine (free base, >99%,) were purchased from Sigma-Aldrich (Steinheim, Germany). 2Propanol (HPLC grade) was obtained from Panreac (Barcelona, Spain). All reagents were used as received. 2Propanol was added with heptadecane acting as internal standard (IS) at 0.20 mg/mL and designated as “labeled solvent”. A nicotine stock solution (1.00 mg/mL) was prepared from an appropriate quantity of nicotine diluted with labeled solvent. From this stock, the corresponding dilutions were carried out using the same labeled solvent to produce a set of calibration solutions consisting of five nicotine concentrations ranging from 0.01 to 1.00 mg/mL, plus a blank (labeled solvent only). For the thin-layer chromatography (TLC) assays, a mixture consisting of 100:1.5 (v/v) methanol and ammonia (30% aqueous solution) was used as mobile phase. All the reagents were from Panreac (Barcelona, Spain). Silica TLC plates with fluorescent indicator were used (ALUGRAM SIL G/UV254, Macherey-Nagel, Düren, Germany). Dragendorff’s reagent (ready to use) was purchased from Sigma-Aldrich.

Figure 3. Construction of the cigarette holders and picture illustrating the final appearance of the cigarette-to-Luer adapter.

puffs) is critical to the quantity of TPM trapped on the filters.11 The profile must provide enough TPM to allow its gravimetric determination in every single filter while avoiding filter saturation and TPM breakthrough. The appropriate quantity of TPM was achieved by smoking one cigarette per filter using a puff volume of 35 mL (2 s long), four times per minute. As the six cigarettes were smoked simultaneously, imprecisions in the puffing regime become negligible.

The Smoking Device

The smoking machine was set up as shown in Figure 2. A twelve-port solid-phase extraction system (Macherey-Nagel,

Determination of TPM Mass and Extraction of Nicotine

The quantity of TPM was determined gravimetrically by weighing the filters before and after the smoking process. To avoid any inaccuracy due to water adsorption, filters must be weighed and marked immediately before and immediately after smoking. Afterward, an accurately measured volume of 20.0 mL of labeled solvent was passed through the filters by using a syringe at a flow rate of 8 mL/ min to obtain an eluate where nicotine can be quantified.

Figure 2. Smoking device used to trap TPM from cigarette mainstream smoke.

Nicotine Quantitation

The nicotine in the resulting extracts can be quantified by GC− MS using a standard curve constructed from the areas of the peaks of the calibration solutions. The GC protocol suggested in the standard method12 is adaptable to any laboratory equipped with a gas chromatograph having a flame ionization detector. In our case, a Varian CP 3800 Chromatograph− Saturn 2200 ion trap mass spectrometer (Varian, Palo Alto, CA) equipped with an SLB-5 ms column, (30 m × 0.25 mm, 0.25 μm film thickness, from Supelco−Sigma-Aldrich, St. Louis, MO) was employed. More detailed experimental conditions can be found in the Supporting Information. As an alternative, and with the aim to broaden the range of techniques handled by the students, a semiquantitative analysis can be carried out by using simple TLC13 on silica plates with UV254 indicator. Nicotine was separated and identified with an Rf = 0.54 employing a mobile phase consisting on 100:1.5 (v/v) methanol/ 30% aqueous ammonia solution. The spots corresponding to nicotine absorption can be observed when the plate is illuminated with 254 nm UV light. Additionally, spraying the plates with

Düren, Germany) was employed as the smoking machine and was coupled to a vacuum pump (Eyela A3S, Tokyo Rikakika, Tokyo, Japan) to provide the necessary airflow. TPM and nicotine were collected on glass-fiber filters (25 mm diameter, 1.2 μm nominal pore size, Phenomenex, Torrance, CA). The adapters to couple the cigarettes to the filters were built using the end of a 1 mL polypropylene syringe (ICO plus3, Novico Médica, Barcelona, Spain), a common rubber septum properly modified, and a heat-resistant silicone cement (Nural 28, Henkel, Düsseldorf, Germany). The full details are summarized in Figure 3 and given in the Supporting Information. Smoking Process and TPM Trapping

Six cigarettes (three normal and three light cigarettes) were smoked simultaneously to determine whether any difference in nicotine or TPM content existed between the two groups. The cigarettes were previously conditioned according to the international guidelines10 (see the Supporting Information). The smoking profile (air flow speed, puff duration, pause between 772




plates can be also sprayed with Dragendorff’s solution (a classical reagent for identification of amines), to make the nicotine visible as red spots where the alkaloidal bismuth iodide forms. The size and intensity of the spots correlates with the concentration of the alkaloid (see Figure 5 of the student handout in the Supporting Information). Running the samples together with standard solutions having concentrations under and over the expected in the samples allows a consistent identification and semiquantitative determination of nicotine in the eluates.

Dragendroff’s reagent provides another method to visualize the nicotine spots.

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HAZARDS 2-Propanol, methanol, and heptadecane are flammable and proximity to any ignition source must be avoided. Vapors of 2propanol are irritating and exposure to high concentrations has a narcotic effect. Methanol is highly toxic and may be fatal or cause blindness if swallowed. Nicotine is hazardous in case of skin contact or ingestion and can be fatal if enough dose is absorbed (oral LD50 in rats is 50 mg/kg, dermal LD50 in rats 140 mg/kg14). Aqueous ammonia is corrosive and irritating. Dragendorff’s reagent must be sprayed in a fume hood. Flammable and corrosive reagents must be stored separately from other reagents in a cool, dry, well-ventilated hood. The mobile phase for TLC (if used) as well as the most concentrated nicotine calibration solution should be prepared by experienced undergraduate teaching assistants or instructors to minimize direct manipulation of concentrated ammonia and pure nicotine by the students. Solutions used in this laboratory work should be prepared in fume hoods, and wearing of labcoat, gloves, and goggles is necessary for full individual protection. If more information is needed, refer to reagent manufacturer’s MSDS or check Bretherick’s Handbook of Reactive Chemical Hazards.15

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RESULTS AND DISCUSSION The housing of the glass-fiber filters is often transparent or translucent, so the students can easily get a visual idea of the quantity of solid particles inhaled by a smoker (Figure 5). The

Figure 5. Three glass-fiber filters showing different appearances after used under different smoking profiles: from left to right, clean filter; filter used with the suggested smoking profile; filter used in a continuous smoking profile.

ANALYSIS OF THE DATA

TPM Quantitation

The TPM obtained from every cigarette was gravimetrically quantified as the increase in the mass of the glass fiber filter after the smoking process. An ANOVA analysis (OriginPro 8, OriginLab Software, Northampton, MA) was used to determine if significant differences existed between normal and light groups.

ANOVA analysis showed that significant differences existed between the quantity of TPM generated by the normal and the light cigarettes. To set up this comparison, two consecutive smoking runs were carried out. In each one of them, three normal and three light cigarettes were smoked simultaneously. Over 30% more TPM is produced from smoking normal cigarettes relative to the light cigarettes (Table 1).

Nicotine Quantitation from GC−MS Chromatograms

To construct the calibration curve, the ratios of the areas of the nicotine and heptadecane peaks of the six calibration solutions were plotted versus the nicotine concentration of every solution. The experimental data were fitted using a linear regression model. Later, the concentrations of nicotine in the extracts were determined by comparison to the regression line of the calibration curve (Figure 4). Comparison of nicotine

Table 1. TPM Trapped from Normal and Light Cigarettes

a

Cigarette Groupa

TPM /(mg/cigarette)

SD.

Normal Light

25.25 19.23

2.67 1.54

n = 6.

The eluates from the filters can be directly injected into the GC−MS chromatograph without any additional pretreatment. The identity of the nicotine peaks in the samples (Figure 6) can be confirmed from comparison of their mass spectra (see Figure 3 in the student handout in the Supporting Information) against the calibration solutions or a spectra library. Compared to the eluates corresponding to normal cigarettes, the quantity of nicotine in the smoke from light cigarettes was almost 20% lower (Table 2). TLC is an easier and less expensive alternative to GC−MS allowing a semiquantitative analysis of the nicotine content. If the unknown eluates are run in a single plate together with standards of lower and higher concentration, it is possible to get an approximate idea about the nicotine content of the cigarettes using the size and intensity of the spots (Figure 7). This procedure can be converted into a quantitative assay by reading the plates with a densitometer. At the end of the laboratory experiment, the students were encouraged to work at home on a short test comprising

Figure 4. Calibration curve obtained by GC−MS for the standard nicotine solutions containing heptadecane as internal standard.

contents between the normal and the light cigarettes was performed by ANOVA analysis as described previously. Analysis of the Data from TLC

After the chromatographic run, nicotine appeared as dark spots when the plates were examined under 254 nm UV light. The 773




key concepts of analytical chemistry was reinforced, so the learning process was improved. Taking into account the limitations imposed by the adaptations, the experimental results demonstrated that the apparatus is suitable for comparison of different kinds of cigarettes, yielding reproducible analytical results. Furthermore, it helped the students become familiar with the language and structure of the international guidelines and stimulated their ability to perform critical analysis and compare analytical methods. In addition, most materials employed were inexpensive and accessible in many analytical laboratories, resulting in easy implementation as part of a practical course with a lower cost compared to a commercial smoking machine.

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Figure 6. Chromatogram showing the separation of nicotine (tR = 2.89 min) and the internal standard (tR = 7.13 min). The peak at 2.5 min corresponds to 2-propanol (sample solvent).

Student handout and instructor notes. This material is available via the Internet at http://pubs.acs.org.

Nicotine/(mg/cigarette)

SD.

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1.91 1.56

0.08 0.03

Notes

Table 2. Nicotine Content from Normal and Light Cigarettes Cigarette Group Normal Light a

a

ASSOCIATED CONTENT

S Supporting Information *

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. The authors declare no competing financial interest.

n = 6.

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ACKNOWLEDGMENTS The authors are grateful to Universidad Complutense de Madrid for financial support through PIMCD UCM 77/2010 (Proyectos de Innovación Educativa 2010) and to MEC for an FPU Research Fellowship for V. González-Ruiz. The authors would also like to express their appreciation to the experienced undergraduate students Marı ́a Ortiz, Alejandro Buendı ́a, and Marı ́a del Mar Hilillo and also to the laboratory technicians Rosa Rodrigo and Purificación Bootello for their valuable work and support. The authors are very thankful to an anonymous reviewer whose careful and detailed English corrections helped us to improve the language of the manuscript.

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REFERENCES

(1) Tyson, J. Analysis: What Analytical Chemists Do; 1st ed.; Royal Society of Chemistry: Cambridge, 1997. (2) CORESTA. CORESTA Recommended Method N° 64; Routine Analytical Cigar-Smoking Machine Specifications, Definitions and Standard Conditions; CORESTA: Paris, 2005. (3) Ondrus, M. J. Chem. Educ. 1979, 56, 551−552. (4) Wong, J. W.; Ngim, K. K.; Shibamoto, T.; Mabury, S. A. J. Chem. Educ. 1997, 74, 1100−1103. (5) Wingen, L.; Low, J.; Finlayson-Pitts, B. J. Chem. Educ. 1998, 75, 1599−1603. (6) Rustemeier, K.; Piade, J. Determination of Nicotine in Mainstream and Sidestream Cigarette Smoke. In Analytical Determination of Nicotine and Related Compounds and Their Metabolites; Gorrod, J. W., Jacob, P., III, Eds.; Elsevier: Amsterdam, 1999; pp 489− 529. (7) Griffith, R. Toxicology 1984, 33, 33−41. (8) Herraiz, T.; Chaparro, C. Biochem. Biophys. Res. Commun. 2005, 326, 378−386. (9) CORESTA. CORESTA Recommended Method N° 65; Determination of Total and Nicotine-Free Dry Particulate Matter Using a Routine Analytical Cigar-Smoking MachineDetermination of Total Particulate Matter and Preparation for Water and Nicotine Measurements; CORESTA: Paris, 2010. (10) CORESTA. CORESTA Recommended Method N° 46; Atmosphere for Conditioning and Testing Cigars of all Sizes and Shapes; CORESTA: Paris, 1998.

Figure 7. TLC plates showing the spots corresponding to nicotine extracted from TPM samples obtained from normal and light cigarettes and nicotine standard solutions at two different levels of concentration: (top) plate sprayed with Dragendorff’s reagent and (bottom) plate observed under UV light (254 nm).

questions and problems (see the Supporting Information). Most of the students fulfilled the test and were able to answer and solve the proposed activities correctly. Moreover, the students were required to elaborate, in report format, on the key concepts learned during this laboratory activity as well as the main difficulties they found.

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CONCLUSIONS An inexpensive and easily built smoking machine was developed that allows students to determine TPM and nicotine in cigarette smoke by following an adapted form of the internationally available guidelines. The students were highly involved and motivated by this activity and showed great enthusiasm due to the unusual nature of the matrix where the analytes were recovered. Moreover, their knowledge of several 774




(11) Borgerding, M.; Klus, H. Exp. Toxicol. Pathol. 2005, 57, 43−73. (12) CORESTA. Coresta Recommended Method N° 66; Determination of Nicotine in the Mainstream Smoke of Cigars by Gas Chromatographic Analysis; CORESTA: Paris, 2005. (13) Moffat, A. C., Osselton, M. D., Widdop, B., Watts, J., Eds. Clarke’s Analysis of Drugs & Poisons, 4th ed.; Pharmaceutical Press: London, 2011. (14) Lewis, R. J., Ed. Sax’s Dangerous Properties of Industrial Materials; 11th ed.; John Wiley & Sons: New York, 2005. (15) Urben, P., Ed. Bretherick’s Handbook of Reactive Chemical Hazards, 7th ed.; Academic Press: Burlington, 2006.

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