In the Laboratory
Got a Match? Ion Extraction GC–MS Characterization of Accelerants Adsorbed in Charcoal Using Negative Pressure Dynamic Headspace Concentration Barbara Anzivino and Leon J. Tilley* Department of Chemistry, Stonehill College, Easton, MA 02357; *
[email protected] Laura R. Ingalls Center for Green Chemistry, University of Massachusetts–Lowell, Lowell, MA 01854 Adam B. Hall Biomedical Forensic Sciences Program, Boston University School of Medicine, Boston, MA 02118 John E. Drugan Arson and Explosives Unit, Commonwealth of Massachusetts Department of State Police Crime Laboratory, Sudbury, MA 01776
Students of organic chemistry are more enthusiastic about experiments connected to real-world applications. Experiments faithfully duplicating real-world situations are interesting yet often too complicated, while simulated experiments are often oversimplified and lose their appeal. Using a Mock Forensics Case Study in the Lab Student interest in forensics led us to create an organic chemistry laboratory experiment to introduce and reinforce the concept of gas chromatography–mass spectrometry (GC–MS), a state-of-the art technique used to analyze for arson accelerants (1, 2). Using methods adapted from the American Society for Testing and Materials (ASTM) (3, 4) and the National Fire Protection Agency (NFPA) (5), students recover and compare accelerants from samples by adsorption onto charcoal-filled tubes using a negative pressure method, then extract the accelerants with pentane and analyze them by GC–MS. Our approach to data analysis is unique in that, following actual crime lab protocol, students perform ion extractions and use the resulting extracted ion profiles (EIPs) to assist in accelerant characterization. Setting Up the Problem Students are presented with this scenario. An arson suspect has been arrested. A sample of the individual’s clothing is taken (a swatch of cloth spiked with a trace amount of an accelerant) for comparison with a burned carpet sample recovered from a “crime scene”. According to NFPA 921, if a canine trained for this purpose detects the presence of an accelerant, the presence or absence of an ignitable liquid must be confirmed by analysis in a laboratory (5). Following procedures a forensic chemist would use in a crime lab, students set out to confirm the presence (or absence) of an accelerant on both samples, and to determine whether a probable match exists. The experiment is designed with a standard set of procedures that allows for nearly an endless variety of permutations. Desired Outcomes for Students When designing this experiment, we wished to pique students’ interest, expand their knowledge of organic, analytical, and forensic chemistry, and promote critical thinking. Students learn basic analytical techniques by preparing, collecting, and
extracting accelerant samples. They become familiar with operation of the GC–MS instrument by injecting their extract sample. They apply a basic knowledge of the processes of gas chromatography and mass spectrometry when analyzing the resulting total ion chromatogram (TIC) data. They gain an appreciation of the analytical power of GC–MS by investigating ion extraction and delve more into their organic chemistry knowledge by analyzing fragmentation patterns. When the time comes for students to make conclusions about the probable innocence or guilt of a suspect, they learn that real-world results are not always unambiguous. Simple comparison (such as matching TICs) is certainly not sufficient; even the use of ion extraction and the spectral library does not necessarily lead to a perfect match between samples, even of the same accelerant. This raises such critical thinking issues as combustion time, sample location, watering, weathering, and contaminants. The importance of good laboratory technique is emphasized, as students must recognize that in a real situation a falsely identified sample could incarcerate an individual wrongly accused of arson or allow an arsonist to go free. Although it may be possible to state with certainty whether or not there is consistency between the two samples, lack of an exact match could provide defense attorneys with opportunities for argument. Context for This Lab Experiments for detection of accelerants have been previously described. Elderd, Kildahl, and Berka (6) provide a GC headspace sampling method for flammable liquids. Sodeman and Lillard (7) reference several forensic experiments and describe an experiment using headspace GC–MS for an instrumental methods course. McCord (8) uses gas chromatography–flame ionization detection (GC–FID) for arson accelerant identification in a forensic chemistry laboratory course, while Smith, Warnke, and Erickson (9) use static-headspace enrichment of accelerants followed by GC–FID detection to teach students real-world application of simplex optimization techniques. Our experiment differs from others in both data analysis and sample extraction. First and foremost is the use of a multilevel GC–MS approach that includes ion extraction to help narrow down the accelerant class. For sample analysis, GC–MS provides much more information than GC–FID (8, 9). Following actual crime laboratory protocol, we use ion extraction
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 86 No. 1 January 2009 • Journal of Chemical Education
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In the Laboratory
in addition to TICs and spectral library searches (7) to match the results to a variant of the ASTM ignitable liquid classification scheme (4). To avoid blind reliance upon the library and reinforce critical thinking, students must verify compound structures by fragmentation pattern analysis. Because the primary goal of this experiment is to introduce the student to the capabilities of GC–MS, we designed the recovery portion of this experiment to be safe, reasonably authentic, fast, and convenient so that students could perform it unaided. The instructor is then able to spend more time introducing the students to the use of the GC–MS instrument and its data analysis features. In contrast to other experiments, the accelerant is recovered from cloth or burned carpeting using a modified version of an ASTM-approved negative pressure dynamic headspace method (3) in which vapors from the heated sample are adsorbed in a charcoal tube and subsequently extracted with pentane. Pentane is preferred to carbon disulfide, which is malodorous and toxic (9). Our method has been optimized to maximize recovery of compounds with both high and low volatilities and can be conducted within the space of one, three- or four-hour laboratory period. Collection of highboiling components is important because in real-world samples evaporation can change accelerant profiles and leave only less volatile substances behind. This method is more authentic and more reliable than simple collection of heated vapors in a syringe (7). It is also faster and more convenient than static headspace methods (9–11) employing charcoal packets, charcoal strips, or solid phase microextraction (SPME). Methods using charcoal packets or strips as adsorbents describe prolonged heating times for samples. The brevity of our procedure allows students to repeat or modify the experiment, as desired (see the online supplement). Additionally, charcoal strips, packets, and SPME
to aspirator
glass tubing through one-hole stopper
hose connector ORBO tube sample boiling water bath
hotplate
Figure 1. Schematic of the extraction apparatus.
56
air in
fibers are generally heated together with the sample in closed containers, using drying ovens. Large numbers of students would require prohibitively large numbers of evidence cans, occupying storage space both in and out of drying ovens. Our use of an Erlenmeyer flask in a boiling water bath to volatilize the sample is much simpler. Finally, SPME fibers are rather fragile, and only one injection is possible before a repeat extraction is necessary. Our experiment also provides flexibility in sample preparation. In the usual case, students are provided with a burned sample (6–8) to compare to the “suspect’s clothing”. Because of safety concerns, we do not recommend that students burn the samples themselves. There are several alternatives: (i) the instructor burning individual samples for the students while they watch; (ii) the instructor doing a demonstration in which a sample is burned and then providing preburned samples; (iii) or (if time or instrumentation is limited) the instructor providing part of the data as an already prepared GC–MS data packet. If students have ample time, they may wish to explore a number of variables, including the use of other accelerants, the use of other adsorbent materials for the accelerant, variation in burn times, variation in evaporation times to simulate weathering, the use of water or other extinguishers to simulate watering, and even changes in the brand of a particular accelerant used. Procedure Summary Our procedure was adapted from a variety of sources (2, 3, 6–8). The negative pressure extraction apparatus is shown in Figure 1. An adjustable ORBO tube cutter was used to uniformly break both sealed ends of a SUPELCO ORBO-32 large (400 or 200 mg) charcoal tube. Two different types of samples were used. The “suspect’s clothing” was prepared by spiking a 4 × 4-cm piece of cotton cloth with 40 μL of an accelerant (gasoline, paint thinner, charcoal lighter, or lamp oil). The use of diesel fuel as an accelerant is not recommended (see the online supplement). The sample recovered from the “crime scene” was prepared by placing a 4 × 4-cm piece of short-fiber synthetic carpeting (with no rubber backing or foam padding!) into a porcelain evaporating dish in a fume hood. Next, 2 mL of accelerant (gasoline, paint thinner, charcoal lighter) was poured over it. After several seconds, the carpet sample was ignited with a Bunsen burner flame, and allowed to burn for 30 s. The flame was extinguished either by smothering with a watch glass or using distilled water from a squirt bottle. An untreated, unburned cloth square and an untreated, burned carpet square were also used as blanks. The sample was placed into a 250 mL filtering flask. Using a short piece of rubber tubing as a connector, a charcoal tube was attached to the end of a piece of glass tubing in a one-hole stopper, such that the tube was almost touching the sample inside the filtering flask when the stopper was inserted. After assembly, the other end of the glass tubing was connected to an aspirator (via a trap). A piece of rubber tubing was also placed on the sidearm of the filter flask to prevent aspiration of steam. The apparatus was lowered into a boiling water bath, and the aspirator was turned on and allowed to run for 10 min. At the end of this time, the flask was removed from the water bath, and the charcoal tube was disconnected and allowed to cool to room temperature. The fiberglass plug was then removed using the plug puller attachment on the ORBO tube cutter. Next, 4 mL of pentane (B and J Brand High Purity Solvent) were eluted through the charcoal tube into a 5-mL screw-top vial. To ensure
Journal of Chemical Education • Vol. 86 No. 1 January 2009 • www.JCE.DivCHED.org • © Division of Chemical Education
In the Laboratory
complete extraction, the collected pentane was removed from the vial and rerun through the charcoal tube four more times. The pentane extract was analyzed by splitless injection of 1 μL of the sample into a GC. The sample was detected using a mass selective detector with 5972 MSD MSChemStation analysis software. Analysis parameters are given in Table 1.
and make conclusions about the suspect’s probable innocence or guilt. Total Ion Chromatograms Sample TICs for gasoline extracted from cloth and from a burned carpet sample are given in Figures 2 and 3, respec-
This experiment requires extreme caution because of the flammability of the accelerants and the pentane used as an eluent. All materials should be used in very small quantities contained in vials. Volatile compounds should be handled in a hood. Some potential accelerants (e.g., gasoline) are carcinogenic. Combustion of samples should be done with caution in an empty fume hood. It is recommended that the instructor either perform combustion of samples for the students, or for a demonstration, or as advance preparation. The ends of the ORBO tubes can be difficult to cut off and do not always break evenly; practice is required even with the ORBO tube cutter. It is recommended that the instructor (wearing goggles!) break the tubes in advance as pieces of glass are produced during this process.
Response / (104 Count)
Hazards
9 8 7 6 5 4 3 2 1 0 3
4
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Retention Time / min Figure 2. Chromatogram for gasoline extracted from a cloth sample.
The data were analyzed at a variety of levels. Following an initial inspection of the TIC, ion extractions were performed to assist in determining relative amounts and classes of hydrocarbons present. Mass spectral analyses of individual peaks were used to determine the molar mass range (number of carbons) for hydrocarbons in the sample. Library searches were employed to identify specific target compounds, which were further characterized by fragmentation pattern analyses. Students were given a practice data packet to assist them with their interpretations (see the online supplement). After completing the tasks in the practice packet, students then analyzed their own samples to determine the presence or absence of an accelerant, the class of the accelerant, the extent of match,
Response / (104 Count)
Results and Discussion 6 5 4 3 2 1 0 3
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Retention Time / min Figure 3. Chromatogram for gasoline combusted on a carpet sample.
Table 1. GC–MS Parameters for Analysis of Accelerants Mass Spectral Parameters
Chromatographic Parameters
Instrument: HP 5972A MS
Instrument: HP 5890 GC
Solvent delay: 2.50 min
Column: SUPELCO MDN-5 fused silica (30 m × 0.25 mm × 0.25 μm film)
Acquisition mode: scan
Oven temperatures: Initial temperature: 40 °C
MS Scan Parameters:
Initial time: 2.00 min–1
Start time: 2.50
Rate: 15.0 °C min–1
Mass Range: Low 35.0, High 550.0
Final temperature: 240 °C
Threshold: 50
Final time: 10.00 min
Sampling: 2
Injector temperature: 280 °C
Number of scans each second: 1.49
Detector temperature: 280 °C Carrier gas: Helium Flow rate: 0.900 mL min–1 Purge valve A:
Initial value: off On time: 0.08 min Off time: 0.00 min Injection: splitless
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 86 No. 1 January 2009 • Journal of Chemical Education
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In the Laboratory Table 2. Ion Extractions for Hydrocarbon Classes Class of Hydrocarbon m/z Values ions extracted
Cycloalkanes/ Alkenes Napthalenes
Alkanes
Aromatics
43, 57, 71, 85, 99
91, 105, 106, 119, 120, 134
55, 69, 83
128, 142, 156
15
m/z = 43
10 5 0 15
m/z = 57
Response / (103 Count)
10 5 0 15
m/z = 71
10 5 0 15
m/z = 85
10 5 0 15
m/z = 99
10 5 0 3
4
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Retention Time / min Figure 4. Ion extraction for abundance of alkanes in gasoline extracted from a cloth sample. 20
tively. From the TIC spectra, students can see that gasoline is a complex mixture containing many components of different abundance values. However, the two samples are not identical; it appears as though the combusted sample contains fewer of the more volatile (shorter retention time) components of accelerant. Such variation in accelerant profiles illustrates to students that simply matching TICs is insufficient. In order for students to be able to classify (or even identify the presence of ) an accelerant, they need to determine its extracted ion profile and corresponding target compounds. Ion Extractions Four sets of ion extractions were performed on the chromatogram, as seen in Table 2. The abundance values for each class of hydrocarbon were then examined to get an estimate of the relative amounts of each. Although detector sensitivity, ionization efficiencies, and extraction efficiencies may vary, all that is needed is a qualitative determination that a given class is present. A sample set of ion extractions for gasoline (Figures 4–6) shows alkanes, aromatics, and cycloalkanes, but essentially no naphthalenes. Molar Mass Range of Hydrocarbons Because the accelerants in this experiment are primarily nonpolar hydrocarbons that are expected to elute roughly in the order of their boiling points, students used the spectral library to search several early- and late-eluting peaks. The carbon range in the sample was estimated from the resulting structural matches. Students also confirmed the results visually by comparing the molecular ion and fragmentation patterns in the spectra. For example, if one or more of the earliest eluting peaks showed a molecular ion of m/z 86 (and other smaller characteristic frag-
6 4 2
m/z = 91
10
0
0 20
m/z = 105
0 20
m/z = 106
10 0 20
m/z = 119
10 0 20
m/z = 120
10 0 20
m/z = 134
10 0 3
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Retention Time / min Figure 5. Ion extraction for abundance of aromatics in gasoline extracted from a cloth sample.
Response / (103 Count)
Response / (103 Count)
10
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m/z = 55
6 4 2 0
m/z = 69
6 4 2 0
m/z = 83
6 4 2 0
m/z = 128
6 4 2 0
m/z = 142
6 4 2 0
m/z = 156
3
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Retention Time / min Figure 6. Ion extraction for abundance of cycloalkanes, alkenes, and naphthalenes in gasoline extracted from a cloth sample.
Journal of Chemical Education • Vol. 86 No. 1 January 2009 • www.JCE.DivCHED.org • © Division of Chemical Education
In the Laboratory
Conclusion We have performed versions of this lab successfully with organic chemistry students and (in condensed form) with students of general chemistry. This laboratory is very adaptable for inquiry-based courses because sample preparation allows for a large degree of variability, which students should be encouraged to explore. The negative pressure extraction method is fast and easy to accomplish, and the GC–MS analysis provides results that teach students to use ion-extraction and to analyze fragmentation patterns in a readily understandable manner. Following sample preparation and analysis, students are required to think critically about the conclusions to be drawn. Results may be consistent yet not conclusive; students learn that there is rarely complete certainty in real life. Students found the experiment, particularly its real-world aspects, to be enjoyable and to provide a clear illustration of the technique of GC–MS. Literature Cited 1. Bertsch, W. Anal. Chem. 1996, 68, 541A–545A. 2. Keto, R. O.; Wineman, P. L. Anal. Chem. 1991, 63, 1964–1971. 3. ASTM Method E1413-00 (Re-approved 2005). Annual Book of ASTM Standards; Vol. 14.02; ASTM Intl.: West Conshohoken, PA; http://www.astm.org/Standards/E1413.htm (accessed Sep 2008). 4. ASTM Method E1618-01. Annual Book of ASTM Standards; Vol. 14.02; ASTM Intl.: West Conshohocken, PA; http://www.astm.
Response / (103 Count)
Target Compounds, Determining Unknowns, Variations After determination of the abundance of various types of hydrocarbons and the carbon range, a comparison to a condensed summary of the ASTM ignitable liquid classification (4) scheme (see the online supplement) was made. The unknown was matched to one or more classes of accelerants. The specific nature of the accelerant was narrowed further using the library to find target compound(s). Students were also expected to confirm the search results by fragmentation pattern analysis. The SDBS online spectral database (12) for the target compound can also be used as further confirmation. In the case of an unknown such as gasoline, characterization is relatively straightforward. In the case of some petroleum distillates, distinguishing the particular type can be difficult, even with the help of target compounds. Ambiguity reinforces the idea that there may not always be one right (or clear) answer to a problem— indeed, such instances are encountered in the real world all the time. Even results showing consistency between two samples cannot provide definitive evidence regarding a suspect’s guilt or innocence but would merely provide once piece of the puzzle. Even in cases designed to illustrate weathering and watering, accelerants could be identified. We were easily able to detect a sample of gasoline that had been combusted on a carpet and extinguished with copious amounts of water. We were also able to detect gasoline on a sample that had been extinguished with water and allowed to stand for eight days.
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5 4 41 3 2 43 1
39
55
51
30
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40
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69
53
37
0
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84 86 75
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m/z Figure 7. Spectrum of 2.6-min. peak (see Figure 2 C6 hydrocarbon). 57
Response / (102 Count)
ments; see Figure 7) while one of the latest eluting peaks (Figure 8) showed a molecular ion of m/z 170 (with accompanying fragments), a determination could be made of the sample’s carbon range as being from C6 (MM C6H14 = 86) to C12 (MM C12H26 = 170). Students were alerted that the molecular ion may not always be present; they were also apprised that the carbon range may vary due to evaporation (see the online supplement).
8 131
43
7 6
71
5 4
85
3
146
2
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1
115 98
64
162 170
0 30
40
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90 100 110 120 130 140 150 160 170 180
m/z Figure 8. Spectrum of 9.8-min. peak (see Figure 2 C12 hydrocarbon).
org/Standards/E1618.htm (accessed Sep 2008). 5. NFPA 921 Guide for Fire and Explosion Investigations, 2004 ed.; National Fire Protection Association: Quincy, MA, 2003. 6. Elderd, D. M.; Kildahl, M. K.; Berka, L. H. J. Chem. Educ. 1996, 73, 675. 7. Sodeman, D. A.; Lillard, S. J. J. Chem. Educ. 2001, 78, 1228–1230. 8. McCord, B. Forensic Chemistry Laboratory Manual, Rev. 7.1; Florida International University: Miami, 2005; http://www.fiu. edu/%7Emccordb/Manualv7.2.doc (accessed Sep 2008). 9. Warnke, M. M.; Erickson, A. E.; Smith, E. T. J. Chem. Educ. 2005, 82, 1082–1085. 10. ASTM Method E1412-00. Annual Book of ASTM Standards; Vol. 14.02; ASTM Intl.: West Conshohocken, PA; http://www.astm. org/Standards/E1412.htm (accessed Sep 2008). 11. ASTM Method E2154-01. Annual Book of ASTM Standards; Vol. 14.02; ASTM Intl.: West Conshohocken, PA; http://www.astm. org/Standards/E2154.htm (accessed Sep 2008). 12. National Institute of Advanced Industrial Science and Technology, Japan. Spectral Database for Organic Compounds, SDBS; http://riodb01.ibase.aist.go.jp/sdbs/cgi-bin/cre_index.cgi?lang=eng (accessed Sep 2008).
Supporting JCE Online Material
http://www.jce.divched.org/Journal/Issues/2009/Jan/abs55.html Abstract and keywords Full text (PDF) Links to cited URLs and JCE articles Supplement Student handouts; Notes for the instructor; Spectra
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