In the Laboratory
Polybrominated Diphenyl Ethers in Dryer Lint An Advanced Analysis Laboratory Robert Q. Thompson Department of Chemistry and Biochemistry, Oberlin College, Oberlin, OH 44074;
[email protected] A gas chromatography–mass spectrometry (GC–MS) experiment that addresses an environmental issue of current interest was developed for an advanced analytical chemistry laboratory. Polybrominated diphenyl ethers (PBDEs) are flame retardant chemicals widely mixed with plastics in electronic devices, such as televisions and computers, and in furniture and carpeting. Bits of these materials are released over time and can be incorporated in household dust and enter a person’s airway and mouth. Chronic exposure to PBDEs may cause cancer and toxicity. The environmental impact of PBDEs has received much public attention (1–3). An entire issue of the journal Chemosphere recently was devoted to brominated flame retardants (4). This experiment in environmental chemical analysis was adapted from three articles in the research literature concerning PBDEs (5–7). No laboratory education literature on the topic is known. The experiment supports classroom material covering topics in mass spectrometry. Students gain experience with several modes of mass spectrometry: electron ionization (EI), negative chemical ionization (NCI), selected ion monitoring (SIM), tandem mass spectrometry (MS–MS), and selected reaction monitoring (SRM). Using an ion trap mass analyzer, the quantities of several PBDE congeners in clothes dryer lint are determined, using single-stage MS in NCI–SIM mode for bromide ions (a tell-tale sign of PBDEs) and MS–MS in EI–SRM mode for quantification with isotope-labeled analytes. Through the setup of the GC-ion trap MS and its instrumental parameters and through the analysis of the generated data, students learn much about mass spectrometry. The tremendous selectivity and sensitivity of MS is revealed as students gain knowledge about the local chemical environment in which they live. Experiment Reagents and Sample Preparation A mixture of nine isotope-labeled (13C12) PBDEs was purchased from Cambridge Isotope Laboratories, Inc. Each PBDE congener is designated with a number, for example, PBDE 99, that corresponds to the number and positions of the bromine atoms on the diphenyl ether. The contents of the 1.2 mL standard ampoule were diluted to 25.00 mL with acetone:hexanes solvent to prepare the internal standard solution (Table 1). The sample was prepared in the following manner. A weighed (~0.5 g) portion of dryer lint collected from a clothes dryer in a dormitory laundry room was placed into a Soxhlet extraction thimble, and the thimble was added to the Soxhlet apparatus. A 2.00 mL aliquot of internal standard solution was pipetted into the thimble. About 75 mL of acetone:hexanes solvent was heated to boiling with a heating mantle, and the lint was extracted for 12 h–18 h. The Soxhlet apparatus was then allowed to cool, and the colored sample solution was collected. The sample volume was reduced to near dryness by solvent evaporation in a fume hood with stirring and warming,
and then reconstituted with 5.0 mL n-hexane. A blank solution was prepared in exactly the same manner, except that no lint was added to the extraction thimble. The colored (polar) material extracted from the lint was removed by solid phase extraction (SPE) through a pure silica cartridge. The colored material remained in the cartridge while the PBDEs eluted with the sample load. Next, the sample was reduced to near dryness by solvent evaporation in a fume hood with stirring and warming and then reconstituted with 1.00 mL acetone:hexanes. The blank was also carried through the identical SPE procedure. Gas Chromatography–Mass Spectrometry A GC–MS with the ability to perform both EI and NCI and MS–MS is required for this experiment. Specifically, we used a Trace GC–Polaris Q ion trap GC–MS from ThermoFinnigan, equipped with a DB-5MS column of dimensions 30 m × 0.2 mm × 0.2 μm film. The temperature program for the gas chromatograph was as described by Wang et al. (6) and is listed in the online material along with the other important GC–MS parameters. The experiments involving NCI–SIM of bromide ions (m/z 79 and m/z 81) provided qualitative data that allowed the PBDEs present in the sample to be tentatively identified. The GC–MS–MS experiments confirmed the presence of the PBDEs and provided quantitative data. For the tandem MS studies, a segmented scan was made, using the parameters (retention times and m/z values) given in the online material. Hazards Solutions of polybrominated diphenyl ethers are toxic and should be handled with care in the fume hood with gloves. Any spills or waste should be collected in a container marked for halogenated organic waste. Hexane and the acetone:hexanes solvent are quite flammable, so avoid open flames.
Table 1. The Composition of the Internal Standard Solution Brominated Diphenyl Ethers (13C12, 99%)
PBDE #
Diluted Conc/ (ng/mL)
3
4-monoBDE
4.6
15
4,4’-diBDE
4.5
28
2,4,4’-triBDE
4.6
47
2,2’,4,4’-tetraBDE
4.8
99
2,2’,4,4’,5-pentaBDE
7.4
100
2,2’,4,4’,6-pentaBDE
6.9
118
2,3’,4,4’,5-pentaBDE
7.0
153
2,2’,4,4’,5,5’ hexaBDE
183
2,2’,3,4,4’,5’,6-heptaBDE
9.4 12.0
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 85 No. 10 October 2008 • Journal of Chemical Education
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In the Laboratory Table 2. Results from GC–MS–MS Analysis of a Typical Sample Peak Area (analyte)
Peak Area (labeled analyte)
Area Ratio
PBDE #
Retention Time/min
3
10.8
0
1440
—
15
17.6
0
1340
—
28
25.1
0
1700
—
47
33.8
7830
1200
6.52
100
40.7
620
550
1.13
99
42.8
4180
620
6.74
118
44.8
0
720
—
153
53.3
0
420
—
183
62.6
0
260
—
Results and Discussion The experiment was performed by two pairs or trios of students and required operations on four consecutive days (or two days on two consecutive weeks). On day 1 (or day 2, depending on the group) the Soxhlet extraction of the dryer lint was begun; each student group spent about an hour in the lab. On day 2 (or day 3, depending on the group) students removed the extract from the Soxhlet apparatus. On day 3 they evaporated the solvent from the sample extract, performed the SPE clean-up steps, and prepared the final sample extract. The instrument was set up for GC–MS (NCI–SIM) analysis and for GC–MS–MS (EI–SRM) analysis. The blank (prepared by the instructor in advance to save time) and sample runs in both GC–MS modes were completed overnight. Each student group spent about 3 hours in the lab on day 3. Finally, the students came to lab on day 4 for about an hour to clean up and to learn about the instrument software that could provide retention times, peak areas, and SRM data. Further data analysis and calculations were performed outside of the formal laboratory time. In one experiment, 0.580 g of dryer lint was treated. Nine peaks were evident in the GC–MS (NCI–SIM) chromatograms. The relative areas of three peaks increased significantly between the blank and sample runs, indicating the likely presence of three PBDEs extracted from the dryer lint. The relative retention times were correlated with literature values (7) to tentatively identify the compounds as PBDE 47, 99, and 100. Correspondence of the GC–MS and GC–MS–MS results (Table 2) confirmed the assignments. As is the practice of isotope dilution analysis, the peak area ratios from the tandem MS experiments were used to calculate directly the mass of the three PBDEs present in the dryer lint. The results (per gram of lint) were 110 ng/g, 170 ng/g, and 27 ng/g, respectively, for PBDE 47, 99, and 100. These values are similar to ones reported in the
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literature (5) for these particular PBDEs in dryer lint, ranging from 8 ng/g to 370 ng/g. Another experiment had four groups test the same sample of dryer lint. The mass fraction of PBDE 47 was 160 ng/g ± 50 ng/g, indicating a reasonably good reproducibility for the entire process. It is important to realize that only picogram quantities of the PBDEs were injected into the GC and that they were measured among all of the other materials extracted from the dryer lint and in the solvents used. The amazing selectivity and sensitivity of modern MS methods was evident. Students have shown interest and enthusiasm in this chemical analysis experiment that provides an assessment of their own environment and that involves sophisticated instrumentation and techniques. With such motivation students cannot help but be fully invested in the work. Learning of the principles of analytical mass spectrometry is enhanced by hands-on experience with a number of aspects (and acronyms) of its practice, including EI, NCI, SIM, MS–MS, and SRM. Acknowledgments The author would like to thank Oberlin students, Aviva Richman and Michael Brenner, for testing the experiment. Thanks also go to the National Science Foundation and its CCLI program for partial funding of the purchase of the GC– MS (NSF Award #DUE -0088173). Literature Cited 1. de Boer, J.; Wells, D. Organohalogen Compounds 2004, 66, 501–509. 2. D’Silva, K.; Fernandes, A.; Rose, M. Crit. Rev. Environ. Sci. Technol. 2004, 34, 141–207. 3. Covaci, A.; Voorspoels, S.; de Boer, J. Environ. Int. 2003, 29, 735–756. 4. Chemosphere 2006, 64, issue 2. 5. Stapleton, H. M.; Dodder, N. G.; Offenberg, J. H.; Schantz, M. M.; Wise, S. A. Env. Sci. Technol. 2005, 39, 925–931. 6. Wang, D.; Cai, Z.; Jiang, G.; Wong, M. H.; Wong, W. K. Rapid Comm. Mass Spec. 2005, 19, 83–89. 7. Korytar, P.; Covaci, A.; de Boer, J.; Gelbin, A.; Brinkman, U. A. T. J. Chromatog. A 2005, 1065, 239–249.
Supporting JCE Online Material
http://www.jce.divched.org/Journal/Issues/2008/Oct/abs1419.html Abstract and keywords Full text (PDF) Supplement
Student handouts
Instructor notes including answers to the student questions
Journal of Chemical Education • Vol. 85 No. 10 October 2008 • www.JCE.DivCHED.org • © Division of Chemical Education
