Molecular-Formula Determination through Accurate-Mass Analysis: A

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Laboratory Experiment Cite This: J. Chem. Educ. 2019, 96, 1458−1464

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Molecular-Formula Determination through Accurate-Mass Analysis: A Forensic Investigation Alan Austin Doucette* and Roderick A. Chisholm Department of Chemistry, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada

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S Supporting Information *

ABSTRACT: Mass spectrometry is frequently introduced to undergraduate students as an instrument for both qualitative and quantitative chemical analysis. One of the most common uses of mass spectrometry (MS) is to deduce or confirm a compound’s chemical formula, by relying on high-resolution accurate-mass measurements. However, like all forms of instrumental analysis, qualitative characterization of an unknown entails a degree of measurement uncertainty. By extension, the conclusions of any analysis may not be definitive. Here, we describe a form of inquiry-based learning wherein students acquire scientific evidence to probe a question (“Is the person guilty?”). A laboratory-based experiment involves the use of accurate-mass analysis to characterize a trace unknown, extracted from “simulated” forensic evidence. Together with the accurate mass, the isotopic distribution, some chemical intuition, and an online molecular-formula tool (available through ChemCalc), students arrive at the suspected chemical identity of the unknown. The analysis of cocaine on circulating currency is used in this report to illustrate the lab exercise. At the conclusion of the exercise, students should realize that the evidence they generate is insufficient to support a definitive identification. The strategies employed to deduce a molecular formula may also be adopted to a classroom setting. KEYWORDS: Second-Year Undergraduate, Upper-Division Undergraduate, Analytical Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Inquiry-Based/Discovery Learning, Forensic Chemistry, Mass Spectrometry, Qualitative Analysis

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INTRODUCTION Methods to determine molecular formulas parallel the advancement of chemical analysis. Four decades after Lavoisier established the law of conservation of mass in 1789, Justus von Leibig designed an elegant piece of glassware that greatly improved chemical-combustion analysis.1 Leibig’s kaliapparat (a triangular arrangement of bulbs, recognizable from its depiction in the ACS logo) accurately trapped combustion byproducts, resulting in a reliable means to determine an empirical formula. It took several more decades and a refined analytical balance2 before Fritz Pregl established elemental microanalysis (for which he received the Nobel Prize in 1923).3 Today’s combustion analyzers remain a staple of structure and purity assessment;4 most introductory chemistry students are familiar with the associated stoichiometric calculations. However, students might be surprised to learn that accurate-mass analysis by high-resolution mass spectrometry (HRMS) is now favored by chemists to determine a molecular formula. Multiple reports have highlighted the versatility of mass spectrometry (>200 articles in this Journal alone) for both qualitative and quantitative analysis. Most of these articles utilize MS fragmentation, either through electron ionization (with GC/MS)5 or by way of tandem MS.6 MS fragments are pivotal to structural analysis and can afford enhanced © 2019 American Chemical Society and Division of Chemical Education, Inc.

selectivity and improved signal-to-noise (e.g., selected-reaction monitoring).7 By contrast, the use of accurate-mass measurements to decipher a molecular formula involves a clearly distinct set of chemical principles,8 which are now prominently applied to organic chemical analysis. Accurate-mass determination is a key parameter in reporting newly synthesized compounds and also plays critical roles in monitoring environmental pollutants,9 protein identification,10 petroleomics,11 and forensics,12 among many other fields. Only a few educational reports have incorporated HRMS for sample analysis, though none have described HRMS in the context of formula determination. Stock employed HRMS to quantify a target pharmaceutical in the presence of an interference of very similar mass.13 Alty and LaRiviere recorded the masses of digested peptides at millidalton precision to improve confidence of protein identification by peptide mass fingerprinting.14 Walsh et al. used HRMS in combination with NMR for the structural elucidation of parthenolide (C15H20O5).15 Interestingly, an assessment of their HRMS data alone would have sufficed to yield the formula, though this was not the objective of the study. Received: December 19, 2018 Revised: April 30, 2019 Published: June 12, 2019 1458

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“Atomic masses are not exactly integral multiples of a unit mass... it follows that if the mass of any ion is measured with sufficient precision, its elemental composition can be immediately deduced.” This statement is a quote from John Beynon’s 1954 article in Nature (ref 16, p 736), which was the first to employ HRMS for enhanced qualitative analysis.16 Graham Cooks writes that Beynon “is as responsible as any individual for the development of HRMS”.17 The last 20 years in particular have seen a revolution in HRMS instrumentation, with Orbitrap,18 Fourier-transform-ion-cyclotron-resonance (FT-ICR), and even benchtop time-of-flight instruments19 now capable of measuring compounds to within 5 ppm of their theoretical masses, which is sufficient for accurate-mass analysis.20 Interestingly, 5 ppm mass accuracy does not necessarily confine a compound to a single chemical formula. Fortunately, other information is available (isotope patterns, the nitrogen rule, etc.) to yield a more select candidate list.21 Described in this report is a hands-on MS-based experiment highlighting the qualitative chemical analysis of an unknown, isolated at trace levels from (simulated) forensic evidence. Students extract and purify their unknown and record an HRMS spectrum. With the aid of the online software ChemCalc,22 the observed accurate mass renders a list of possible molecular formulas, which the students further constrain to yield the compound’s suspected identity. A forensic theme lends a pedagogical approach that enforces the principles of analytical-measurement error.23 Just as von Liebig understood the limitations of his kaliapparat to generate exact chemical formulas, students appreciate that a definitive conclusion based on HRMS analysis will require additional confirmatory analysis.24

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Analyte Extraction and Purification

Students extract their analyte from the evidence and then use solid-phase extraction (SPE) to purify, concentrate, and transfer the unknown into a solvent system suitable for MS analysis. Starting with the currency, students roll and place their bill into a 15 mL plastic tube, to which 1 mL of acetonitrile is added via micropipette (see the lab manual in the Supporting Information for a photo). After 1−2 min of gentle agitation, 2 mL of water is added, and the tube is shaken an additional 1−2 min. More water is added to create a final solvent system of 10% acetonitrile (10 mL total). For purification, precisely 4 mL of the extraction solvent is slowly passed through a 1 cc hydrophobic SPE cartridge (Oasis HLB, 10 mg sorbent, 30 μm particles, part #186000383; Waters Limited, Mississauga, Canada), facilitated by the micropipette acting as a pump (flow rate of ∼1 mL/min, see the photo provided in the student lab manual). Following a wash of the SPE column with 1 mL of water, the cartridge is flushed with 400 μL of elution solvent (75% acetonitrile, 0.1% formic acid in water), which is retained for MS analysis. Acetonitrile is HPLC-grade or higher, formic acid is MS-grade, and water is purified to 18 MΩ·cm. Students also prepare a “solvent blank” in an identical fashion, with the exception of the loading of 4 mL of 10% acetonitrile/water onto their SPE cartridge, with washing and elution into 400 μL of elution solvent. Detailed protocols are outlined in the student lab manual (provided in the Supporting Information). MS Analysis

Students employ an orthogonal quadrupole time-of-flight mass spectrometer, qTOF-MS (Bruker Daltonics, micrOTOF benchtop system) equipped with an electrospray-ionization (ESI) source operating in positive mode. Students rinse and load their samples into a syringe, connect to an infusion pump, and set the flow to 2 μL/min such that they record MS signals for ∼30 s per sample. Students first infuse their solvent blank, followed by their extracted forensic sample. They are also provided with a standard containing the unknown analyte at a predefined concentration (to estimate the amount of analyte spiked on the evidence). Finally, the qTOF-MS requires calibration of the m/z scale, using an MS calibration standard (0.01 M sodium formate).26 MS spectra for all four samples are recorded as a single data file (shown in the Results and Discussion).

EXPERIMENTAL DETAILS

Organization

All experiments, including data analysis, can be completed in a 3 h lab. Students typically work in groups of two. Approximately 1 h is assigned to prepare samples for MS analysis. Data acquisition requires as little as 10 min per group, though students are first treated to a discussion of MS theory, along with a basic overview of instrument operation (plan 1.5 h of MS time for five groups). Data analysis requires ∼30 min. Custom videos are also made available to the students, which repeat aspects of MS theory as well as strategies for data interpretation.25 The lab is appropriate for second to fourth year undergraduates in analytical chemistry, forensic sciences, or a course in organic structural characterization. The concept of HRMS for molecular-formula determination has also been adapted to a classroom setting (details below).

Data Interpretation

Students calibrate the m/z scale using internal software. They then print the MS spectrum of their unknown analyte, focusing on signals that are distinct from the solvent blank. Together with an accurate m/z value, the printout also displays the corresponding isotope peaks, along with their respective intensities. Working with this data, students determine a molecular formula using an online calculator, ChemCalc,22 ultimately translating it to the compound’s suspected identity. The strategy to narrow the candidate list of formulas is described in the Results and Discussion. Finally, students use the intensity ratio of their extracted analyte relative to a standard to estimate the amount present on the forensic evidence.

Premise

Students are provided with “simulated” forensic evidence, typically consisting of circulated paper currency spiked with one (or more) unknown compounds at trace levels (low microgram levels, further details are provided with the instructor notes, included as Supporting Information). Neither the compound’s identity nor quantity is indicated to the students, and each group is provided with a different unknown. A police report accompanies the evidence to provide context to their analysis (see the instructor notes). Students employ HRMS to determine the potential identity of suspect compound on the evidence and also to estimate the amount.

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HAZARDS The “simulated” forensic evidence is spiked with certified reference materials and may include illicit drugs, explosive 1459

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3 to 11), and so the theoretical mass of each ion cluster can be calculated (Table 1). To compute the monoisotopic mass of

residues, or environmental contaminants. These stock solutions should be stored away from students to prevent direct access. Students are informed that the evidence is “simulated”, though the compounds are real, and so proper techniques should be employed when handling the evidence (e.g., wearing gloves). Students are also informed that only trace quantities (90%). Similarly, the SPE purification protocol retains and concentrates the analyte with equally high yield. Lower yields may result if the extraction−purification protocol is not precisely followed. To minimize this, the purification protocol is demonstrated at the beginning of lab using a colored dye, which stands in for the unknown analyte. Mass Calibration

Figure 1 presents typical MS data as it would be generated by the students from analysis of an unknown, together with their

error (ppm) =

observed mass − expected mass × 106 expected mass (1)

Typically, errors related to accurate-mass measurements are reported in parts per million (ppm). As an example, prior to m/z calibration, the mass error for the sodium formate cluster shown in Figure 1C is 61 ppm. After calibration, this error drops to 10 ppm. The magnitude of the mass shift appears subtle (Figure 1C), though it is essential to the process of formula determination. Students are instructed to consider their mass measurements accurate to within 20 ppm. Though conservative, this is sufficient to determine the compound formula. Employing Controls

Proper controls are pivotal to all forms of chemical analysis. Students run a “solvent blank” alongside their extracted analyte. In Figure 2, multiple peaks are observed from analysis of the solvent blank, which are also seen in the extracted forensic evidence. These matrix components were therefore ignored. By contrast, the prominent signal near m/z 304 appears as unique to the evidence.

Figure 1. (A) Total-ion chromatogram recorded though infusion of a solvent blank (i); the extracted forensic evidence (ii); a standard containing the unknown analyte (iii); and a sodium formate solution, which acts as the mass calibrant (iv). (B) MS spectrum of sodium formate, with asterisks (*) indicating ion clusters listed in Table 1. (C) Zoomed-in spectrum for the boxed ion cluster, with an overlay of the data after application of m/z calibration.

Formula Determination

ESI Charge State and Protonation. Figure 2C shows a signal obtained from analysis of the forensic evidence. Along with a peak at m/z 304.1526, one also observes the corresponding isotopic peaks (m/z 305, 306), and their spacing conveys the charge state (z). Here, a spacing of 1 m/z indicates a charge state of +1 (doubly charged ions have a spacing of 0.5). Students must realize how the charge originates in order for the ion to be detected by MS. From an earlier discussion of electrospray ionization (ESI), positive ions tend to form through protonation. This singly charged ion was detected in

associated controls. The data constitutes a total-ion chromatogram (TIC), which displays the summed MS signal plotted over time during successive infusions of the various samples (Figure 1A). Regions where samples were infused are readily identifiable in the TIC (labeled i−iv). Figure 1B shows the corresponding MS spectrum from infusion of sodium formate (iv), which acted as the m/z calibrant. The evenly spaced peaks correspond to a series of ion clusters ([HCOO−Na+]nNa+, n = 1460

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only integer unsaturations”. At this point, 21 possibilities remain. Estimating Carbon Number. Recall that the X + 2 isotope conveys information about Cl, Br, and S. The relative intensity of the X + 1 isotope in turn provides an estimate of the number of carbons in the molecule. relative intensity (%) of the X + 1 isotope (3)

= 1.1 × no. of carbons

The equation stems from the ∼1.1% abundance of C. From Figure 2C, the intensity of the signal at m/z 305 relative to that at m/z 304 is 19.4%, which suggests perhaps 17 or 18 carbons. Noting that this is an approximate calculation and that intensity ratios are influenced by background interferences, students are advised to accept a wider tolerance (i.e., ≈15−20 carbons). After this analysis, the list has fallen to five possibilities. Chemical Intuition. Students should not assume the formula with the smallest mass error is the most likely. The instrument mass accuracy dictates that all formulas within 20 ppm are possible. At this point, students may apply intuitive chemical reasoning, which brings into consideration the context of the forensic investigation. Of the five possible formulas remaining, three contain fluorine. Although fluorinecontaining compounds are encountered as pharmaceuticals, such compounds are less likely to be targets of forensic investigations. This suggests the two nonfluorinated compounds are the more likely candidates. If the molecular formula of a compound is known, one can calculate the relative intensities of the X + 1 and X + 2 isotopes as follows: 13

Figure 2. MS spectra obtained for (A) solvent blank and (B) extracted forensic evidence. Signals common to both spectra are ignored as part of the matrix background, whereas the prominent signal near m/z 304 constitutes a component that is uniquely present in the extracted forensic evidence. (C) Observed MS spectrum including the relative intensities of the isotopic peaks for the signal of interest.

the form [M + H]+ and so the mass of the neutral compound is obtained by subtracting the mass of a single proton: mass = 304.1526 u − 1.007825 u = 303.1448 u

(2)

For the purpose of accurate-mass analysis, it is essential that the “exact” monoisotopic mass of hydrogen (1.007825 u) is used. Employing its nominal mass introduces significant error (>20 ppm), which prevents formula determination by accurate-mass analysis. ChemCalc. Students now turn their attention to the Molecular Formula Finder tool, available online at ChemCalc.org. There are two options for entering the observed mass: (i) inputting the mass after subtraction of the proton mass or (ii) inputting the mass exactly as observed. In the latter case, the output formulas contain one additional hydrogen atom, which can be subtracted after the fact. With the mass 303.1448 u, ChemCalc computes all potential formulas within a defined tolerance (default ±0.5 u). With this example, almost 8000 potential formulas are found. These possibilities are confined by (i) the elements specified in the “MF range” (C, H, N, O, S, Cl, Br, F) and (ii) the degree of unsaturation, whereby a compound with n carbons cannot exceed 2n + 2 hydrogens. Focusing on those formulas within 20 ppm of the observed mass, 95 possibilities remained (summarized as tables provided in the Supporting Information). Chlorine−Bromine Isotopes. From the natural abundance of the two prominent isotopes of chlorine (35Cl and 37 Cl) and bromine (79Br and 81Br), the X + 2 isotope will show a characteristic intensity ratio of ∼3:1 (Cl) or 1:1 (Br) when one of these halides is present in the molecular formula. The low relative intensity for m/z 306 eliminated both Cl and Br, which constrained the list to 70 possibilities. Sulfur Isotopic Signature. The isotope at m/z 306 is also diagnostic of sulfur. The isotope 34S is present in ∼4.5% abundance. The observed X + 2 isotope (∼2.4% relative intensity) again eliminated sulfur from the formula, reducing the list to 41 possibilities. Nitrogen Rule. Organic compounds with odd numbers of nitrogens possess odd nominal masses. The [M + H]+ ion at m/z 304 implied an odd neutral mass, which arises from a compound with an odd number of nitrogens. Students can eliminate even nitrogen-containing formulas manually or through ChemCalc by checking the box in order to “allow

intensity of the X + 1 isotope (%) = (0.011nC + 0.00015nH + 0.0037nN + 0.0079nS) (4)

× 100% intensity of the X + 2 isotope (%)

= [0.00205nO + 0.0452nS + 0.0000596(nC)(nC − 1) + 0.3196nCl + 0.9728nBr ] × 100%

(5)

where nX refers to the number of X atoms in the formula. Students can apply these formulas by way of a spreadsheet calculation to all the remaining compounds on the list (note that the influence of a single added proton is negligible): • C17H21NO4: m/z 304 (100%), 305 (19.4%), 306 (2.4%) • C18H17N5: m/z 304 (100%), 305 (21.9%), 306 (1.8%) • observed: m/z 304 (100%), 305 (19.4%), 306 (2.3%) The formula C17H21NO4 appears to be the best fit to the data. Although many students may not recognize this formula, inputting the formula into Google (or an online chemical database) will immediately point to cocaine as a possible chemical compound matching this formula. But Are We Sure?

Did this analysis confirm the identification of cocaine? Not necessarily. Several other possible formulas can fit the HRMS spectrum, particularly if we included additional elements (e.g., F, Na, or P, which do not skew the isotope intensity ratios). The formula C17H21NO4 also matches countless other chemical isomers. Students are asked to consider presenting their “evidence” in a courtroom setting. Their data cannot 1461

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extraction efficiency. Although a followup lab assesses the extraction efficiency (using internal standards), students may simply assume a value here. For simplicity, 100% efficiency is selected and provides the minimal amount of analyte on the evidence. In this example, the mass of cocaine was calculated at ∼1.7 μg (i.e., slightly higher than normal background levels). In the context of a forensic investigation, this low level of illicit drug on circulating currency brings into question whether the subject of the investigation would be guilty. In fact, it is not for the students to decide; rather they are to articulate the results of their analysis and recognize the limitations of the experiment they conducted.

conclude that cocaine was present on the evidence, but rather there is evidence to suggest the presence of a compound with a formula identical to cocaine. The forensic nature of the experiment facilitates this learning objective. This discussion can readily extend to all forms of chemical analysis, where it is essential to recognize the limits of any form of scientific investigation. To confirm the presence of cocaine, a follow-up analysis would be required. Such an analysis is conducted using GC/ MS in a subsequent lab (details are not discussed here, though interested readers are directed to the “Day 2” portion of the lab experiment described in the student lab manual included in the Supporting Information).

Classroom Exercise

Quantitative Analysis

The use of HRMS to determine a chemical formula can also be applied in a classroom setting. An excel spreadsheet to plot a “simulated” mass spectrum is provided in the Supporting Information. A sample test question is attached, noting that students would be given online access to ChemCalc during the test. To further emphasize that a set of analytical results may lead to multiple conclusions, we also present a different test situation wherein students are provided with MS data stemming from an analysis similar to the one described in this report. The students are asked to use the data to support a suspect’s possible guilt. We then challenge the students to employ the same MS data to support their innocence. Further details are found in the Supporting Information.

Let it be assumed that cocaine was truly present on the evidence. To this point, the amount of cocaine on the bill has not been discussed. Several prominent studies have shown that trace levels of illicit drugs have been detected on U.S. currency.27 Students are asked to look up these expected levels, where they will discover a median background value of ∼1 μg of cocaine per bill. In other words, it is not out of the ordinary to detect cocaine at this level on circulating currency. To provide an estimate of the level of cocaine on the dollar bill, students begin with their ratio of ESI-MS signals from the extracted evidence relative to that of a standard (Figure 3).

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ASSESSMENT OF LEARNING OBJECTIVES A classroom survey showed that the majority of students (75%) only possessed basic knowledge of mass spectrometry prior to conducting this lab. Following completion of the lab, all students surveyed noted an improved understanding and appreciation of mass spectrometry and in particular the use of high-resolution accurate-mass measurements. From student feedback, a favorite component of this experiment included the opportunity to operate “research grade” MS instrumentation, together with completion of the “detective work” problemsolving approach to identify and quantify their forensic-themed compounds. From 5 years of experience conducting this lab, students have always managed to obtain a detectable MS signal for their extracted unknown analyte (though occasionally with lowerthan-expected intensity). During the lab, approximately twothirds of students successfully translate their HRMS spectra into plausible suspect formulas without further aid from instructors or TAs. The remaining one-third of students typically require a short discussion to remind them of key features contained within the HRMS spectrum that can be extracted to aid in compound identification. Questions on midterms and a final exam are also used to evaluate the student’s comprehension of exact mass measurements (i.e., mass accuracy, relative isotope intensities, and isotope spacing) and show that approximately 60% of students retain an understanding of these concepts and succeed to convey their understanding in a “traditional” testing environment. Quantifying the mass of the suspect compound on the evidence was a secondary objective of the experiment. As anticipated, this portion of the lab was the most challenging to students, with 70% of the class initially unable to calculate a correct mass without additional assistance from the instructors or TAs. It is at this point that we may encourage the students to generate a workflow diagram similar to that supplied in

Figure 3. Workflow for analyte extraction, beginning with the currency and concluding with a concentrated, purified solution. Such a diagram can assist students in visualizing the various dilutions involved in determining the mass of analyte present on the forensic evidence.

The intensity ratio is proportional to the concentration ratio in these samples: MS intensity (extract) concentration (extract) = MS intensity (standard) concentration (standard) (6)

The standard solution contains 1 μg/mL cocaine, which allows students to quickly work out the concentration in their analyzed extract. Students must also work through the various sample-preparation steps (Figure 3) to arrive at the mass of cocaine present on the bill. Because they are not provided with a specific equation to determine analyte quantity, we encourage the students to generate a workflow diagram similar to that supplied in Figure 3. From there, students can input volumes and work in a stepwise fashion to arrive at the final answer. The concentration of cocaine in the extract not only depends on the initial amount on the currency but also on the 1462

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Dalhousie University, for allowing the generous use of MS instrumentation, and Xiao Feng for maintaining the qTOF-MS used by the students. We are grateful to Dalhousie University for financial support related to the development and operation of this lab. The authors also thank Jessica Nickerson (Department of Chemistry, Dalhousie University) for her assistance with generating the associated artwork presented in this report.

Figure 3. The diagram aids in visualization of the many steps involved for extraction and preconcentration of the analyte. Although some students initially struggled with this concept during the lab, postlab evaluations were particularly encouraging, as 87% of students were able to conduct a similar quantitative analysis in a midterm setting. A primary learning objective was to allow students to think critically about the limitations of their analytical assessment. As noted in the associated lab report, all students successfully recognized that their results from HRMS required confirmatory analysis (by GC/MS). Furthermore, even given this confirmatory data, students properly discuss their results as a series of arguments to support the identity of the unknown (this question is framed in the context of presenting their results in a court of law).

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(1) Michaelis, A. R. Justus von Liebig, FRS: Creator of the World’s First Scientific Research Laboratory. Interdiscip. Sci. Rev. 2003, 28 (4), 280−286. (2) Corwin, A. H. Microchemical Balances: Errors of the Kuhlmann Balance. Ind. Eng. Chem., Anal. Ed. 1944, 16 (4), 258−269. (3) Karpenko-Jereb, L. V.; Shaposhnik, V. A. Fritz Pregl, Inventor of Quantitative Elemental Microanalysis of Organic Compounds. J. Anal. Chem. 2012, 67 (6), 600−602. (4) Miki, T. State-of-the-Art in Organic Elemental Micro and Ultramicro Analysis. Fresenius' J. Anal. Chem. 1990, 337, 817−823. (5) Szalay, P. S.; Zook-Gerdau, L. A.; Schurter, E. J. A MultiTechnique Forensic Experiment for a Nonscience-Major Chemistry Course. J. Chem. Educ. 2011, 88 (10), 1419−1421. (6) Sneha, M.; Dulay, M. T.; Zare, R. N. Introducing Mass Spectrometry to First-Year Undergraduates: Analysis of Caffeine and Other Components in Energy Drinks Using Paper-Spray Mass Spectrometry. Int. J. Mass Spectrom. 2017, 418, 156−161. (7) Betts, T. A.; Palkendo, J. A. Teaching Undergraduates LC-MS/ MS Theory and Operation via Multiple Reaction Monitoring (MRM) Method Development. J. Chem. Educ. 2018, 95 (6), 1035−1039. (8) Kind, T.; Fiehn, O. Seven Golden Rules for Heuristic Filtering of Molecular Formulas Obtained by Accurate Mass Spectrometry. BMC Bioinf. 2007, 8, 1−20. (9) Díaz, R.; Ibáñez, M.; Sancho, J. V.; Hernández, F. Building an Empirical Mass Spectra Library for Screening of Organic Pollutants by Ultra-High-Pressure Liquid Chromatography/Hybrid Quadrupole Time-of-Flight Mass Spectrometry. Rapid Commun. Mass Spectrom. 2011, 25 (2), 355−369. (10) Clauser, K. R.; Baker, P.; Burlingame, A. L. Role of Accurate Mass Measurement (±10 ppm) in Protein Identification Strategies Employing MS or MS/MS and Database Searching. Anal. Chem. 1999, 71, 2871−2882. (11) Marshall, A. G.; Rodgers, R. P. Petroleomics: Chemistry of the Underworld. Proc. Natl. Acad. Sci. U. S. A. 2008, 105 (47), 18090− 18095. (12) Botch-Jones, S.; Foss, J.; Barajas, D.; Kero, F.; Young, C.; Weisenseel, J. The Detection of NBOMe Designer Drugs on Blotter Paper by High Resolution Time-of-Flight Mass Spectrometry (TOFMS) with and without Chromatography. Forensic Sci. Int. 2016, 267, 89−95. (13) Stock, N. L. Introducing Graduate Students to High-Resolution Mass Spectrometry (HRMS) Using a Hands-On Approach. J. Chem. Educ. 2017, 94 (12), 1978−1982. (14) Alty, L. T.; LaRiviere, F. J. Peptide Mass Fingerprinting of Egg White Proteins. J. Chem. Educ. 2016, 93 (4), 772−777. (15) Walsh, E. L.; Ashe, S.; Walsh, J. J. Nature’s Migraine Treatment: Isolation and Structure Elucidation of Parthenolide from Tanacetum Parthenium. J. Chem. Educ. 2012, 89 (1), 134−137. (16) Beynon, J. H. Qualitative Analysis of Organic Compounds by Mass Spectrometry. Nature 1954, 174 (4433), 735−737. (17) Cooks, R. G. John H. Beynon (1923−2015): Obituary. J. Am. Soc. Mass Spectrom. 2016, 27, 561−562. (18) Hu, Q.; Noll, R. J.; Li, H.; Makarov, A.; Hardman, M.; Cooks, R. G. The Orbitrap: A New Mass Spectrometer. J. Mass Spectrom. 2005, 40 (4), 430−443. (19) Xian, F.; Hendrickson, C. L.; Marshall, A. G. High Resolution Mass Spectrometry. Anal. Chem. 2012, 84 (2), 708−719.

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CONCLUSIONS This lab introduces students to a form of instrumental chemical analysis, with a goal of attempting to determine the chemical identity of an unknown at trace levels using highresolution, accurate-mass, time-of-flight mass spectrometry. Students extract a simulated sample of forensic evidence and subject their unknown to MS analysis. Using an accurate-mass measurement, isotope distribution, and some chemical intuition, students deduce a possible chemical identification. Although cocaine was found as a potential suspect molecule on circulating paper currency, a preliminary quantitative estimate determined the amount of cocaine to be insignificantly greater than the expected background level. Furthermore, confirmatory analysis should be employed to ensure the correct identification of the unknown. In the context of a forensic investigation, this lab gives students a firm sense of the capabilities and most importantly the limitations of HRMS analysis.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.8b00949. Notes for instructors (PDF, DOCX) Student lab manual (PDF, DOCX) Sample data: tables of possible formulas from ChemCalc (XLSX) Sample test question on formula determination (PDF) Sample exam question on interpretation of MS data (PDF) Excel spreadsheet to generate simulated MS spectra (XLSX)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Alan Austin Doucette: 0000-0002-9467-1002 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Maritime Mass Spectrometry Laboratory, located in the Department of Chemistry, 1463

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Journal of Chemical Education

Laboratory Experiment

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DOI: 10.1021/acs.jchemed.8b00949 J. Chem. Educ. 2019, 96, 1458−1464