Teaching Chemical Concepts Through the Lens of Mass Spectrometry

Mass spectrometry (MS) is a desirable skill for graduates entering the chemistry workforce. Given the variety of levels at which MS can be understood, the range of analytes that can be investigated, and the experiments that can be performed, there are MS-based activities developed for various chemistry concepts across the entire chemistry curriculum. Here, we discuss the general concept of teaching chemistry through MS and highlight examples of using MS to help teach concepts beyond the characterization of synthetic products or solely learning about the technique itself. Some challenges preventing widespread adoption and some opportunities for the future are noted.

Mass spectrometry (MS) and related chromatography methods are key skills for recent undergraduate graduates entering the chemistry workforce, according to a recent survey of industry employers.¹ It is also established that spaced exposure over time is useful for memory of information,² so additional exposure to MS over the course of a degree program could better prepare students. MS is also unique among analytical techniques in that many of the core concepts can be understood with only introductory chemistry and basic physics knowledge. Mass spectrometers (especially ion traps) have previously been dubbed “complete chemical laboratories”;³ we suggest this idea can be extended from the research laboratory to the teaching laboratory and classroom (Figure 1). There have already been a few published examples of successful integration throughout a chemistry curriculum, including one focused on atmospheric pressure chemical ionization (APCI)-MS.⁴ Here we highlight examples from the current education literature on using MS to illustrate, teach, or reinforce specific topics in chemistry across the curriculum. While directly teaching basic MS instrumentation/theory and interpretation of electron ionization (EI)-MS spectra of organic molecules is also important, that is already routinely covered and is thus not included herein. For brevity, we focus on reviewing a few examples each of MS educational literature in general chemistry, organic chemistry, biochemistry, and physical chemistry, but there are also resources available, including those for inorganic chemistry, forensic chemistry, environmental chemistry, and more.

The General Chemistry of Mass Spectrometry

General chemistry covers many topics directly related to MS, from isotopes and ions to molecular masses and the Law of Conservation of Matter. And yet, MS is often introduced only as a historical curiosity, used in the discovery of isotopes and the electron, if at all. Modern MS has the potential to bridge these foundational topics and real-world applications—from forensic science to uranium enrichment to cultural heritage analysis and more—all of which may help pique students’ interest. It can be challenging to scale hands-on MS activities to introductory classes, especially at larger enrollment institutions, and at this level, instructors are not necessarily themselves experts at MS. With this in mind, an “Instructor’s Reference” on MS has been penned, which details how it ties in with the general chemistry curriculum to help facilitate MS-based activities in the classroom.⁵ In a similar vein, a data interpretation activity using bromobenzene as a case study has been published.⁶ This guided-inquiry activity, completed near the beginning of a general chemistry course, reinforces several concepts, including isotopes/isotopic abundance, molecular mass, and average atomic mass. This activity was then extended to task students with interpreting the fragmentation chemistry, using their knowledge of molecular masses, isotopic patterns, and the Law of Conservation of Matter to identify and balance a plausible chemical reaction that leads to the major product ion. Interestingly, it is noted that it is a first introduction in a departmental curriculum that integrates MS throughout, providing another example of the overarching theme discussed herein. A laboratory activity has also been presented that gives general chemistry students experience in injecting samples and using a gas chromatography (GC)–MS firsthand, to explore concepts in percent mass and atomic mass.⁷ This is a fantastic opportunity in contexts where it is feasible.

Mass Spectrometry as an Organic Chemistry Laboratory

EI-MS is routinely used, or at least taught, in organic chemistry courses for characterizing molecules based on fragmentation patterns. However, MS has a significant additional scope for teaching organic chemistry concepts. In one laboratory experiment, students explored how electrosonic spray ionization (ESSI) can be used to accelerate chemical reactions and perform online analysis of the products of the reaction.⁸ In this activity, the Hantzsch synthesis of symmetric 1,4-dihydropyridines was performed, and students evaluated how different source conditions affected the observed product yield and the possibility for observing reaction intermediates. In another activity, headspace sampling over a reaction mixture (both a nucleophilic acyl substitution and 1,4-nucleophilic addition reaction are included as examples) was performed using an in-house constructed helium-plasma ionization source with MS detection.⁹ Beyond real-time reaction monitoring, more advanced MS experiments can also be introduced to probe additional structural questions. The Cooks’ Kinetic Method was demonstrated alongside polarimetry and nuclear magnetic resonance (NMR) spectroscopy for the determination of enantiomeric excess, allowing students to gain additional experience with MS and to compare and contrast the strengths and weaknesses of different approaches.¹⁰

Biomolecular Analysis by Mass Spectrometry

Proteomics and biomolecular analysis have led to significant expansion of MS’s reach and relevance, making exposure to biological applications of MS in the undergraduate curriculum likely beneficial to students’ employability. A series of publications on incorporating biological MS into the undergraduate curriculum have been published, focusing on intact protein identification,¹¹ peptide identification based on molecular mass (including determination of charge by examining isotope spacing and understanding how it affects mass determination from m/z),¹² and de novo sequencing of peptides.¹³ In another example laboratory activity, students measured the collision-induced dissociation MS of small peptides representing a fragment of β-amyloid protein and a related sequence permutation to see how ladder sequencing allows for the identification of sequence isomers.¹⁴ In another example (in this instance aimed at graduate students, but also relevant at advanced undergraduate levels), students participated in the entire proteomics workflow, starting with “unknown” mixtures of proteins and including sample preparation, analysis, and data interpretation, notably covering concepts in both bottom-up and top-down proteomics.¹⁵ While hands-on experiences are often the gold standard in teaching laboratory sciences, virtual platforms can also be used to reach many of the same goals and expose students to topics that may not be feasible due to a lack of time, limited access to instrumentation, or other factors. Further, in the realm of biomolecular analysis especially, data handling is itself a key component of the workflow. A virtual proteomics module at the undergraduate level, using real MS data and a free online data analysis platform incorporated into the analytical chemistry curriculum, has been published.¹⁶

Mass Spectrometry for Physical Chemistry

The study of fundamentals is a critical corner of the MS community and, likewise, there are several physical chemistry concepts that can be illustrated or reinforced through the lens of MS. The kinetic method, relying on the competitive dissociation of a proton (or lithium)-bound heterodimer, can be used to investigate gas-phase basicity, with educational laboratory experiments having been published for both proton and lithium basicities.^17,18 An MS-based experiment demonstrating the degrees of freedom effect has also been published.¹⁹ In this experiment, students collect mass spectra (from GC–MS) of a homologous series of analytes to determine the effect of molecule size (vibrational degrees of freedom) on the extent of dissociation; the results are then contextualized using RRK theory.

Mass Spectrometry for the Masses: Cost and Resource Considerations

While there are many opportunities for and potential advantages of integrating MS throughout the chemistry curriculum, there are also some obvious limitations. Research-grade instrumentation is often cost-prohibitive for many educational programs. Similarly, dedicated time for student laboratory activities may be limited and/or access for larger groups of students where multiple instruments would be required may limit the scope of possible activities and/or the levels at which experiential activities can be implemented. Still, there are several ways to overcome these challenges, with the best approach depending on the class, learning objectives, and institutional environment. Examples include data interpretation activities, laboratory tours and/or class-wide demonstrations, low-cost/simplified instrumentation geared toward educational use, and virtual laboratory programs. Similarly, some instructors outside of dedicated instrumental analysis courses may be less familiar with MS; the availability of detailed resources, like those highlighted herein, can help provide the needed theoretical background and step-by-step guide to implementation to overcome expertise limitations.

Conclusions and Future Outlook

In conclusion, there already exists a plethora of experiments and activities available for using MS to teach or reinforce chemical concepts across the curriculum. While these resources exist, there are still some challenges for widespread implementation. There are also some outstanding questions about the effectiveness of these approaches to (i) increase students’ expertise and confidence in MS and (ii) increase students’ understanding of the chemical concepts over other methods of learning the same content. Future educational research to address these two questions would be fruitful toward best approaching the teaching of MS in the chemistry curriculum and the teaching of the chemistry curriculum through the lens of MS.

References

1. Hamilton, D.; Castillo, A.; Atkinson, M. B. Survey of Instrumentation Use in Industry: What Does Industry Want New Chemists to Know? J. Chem. Educ. 2024, 101 (5), 1883–1890. DOI: 10.1021/acs.jchemed.3c00990
2. Kang, S. H. K. Spaced Repetition Promotes Efficient and Effective Learning: Policy Implications for Instruction. Policy Insights from Behav. Brain Sci. 2016, 3 (1), 12–19. DOI: 10.1177/2372732215624708
3. O’Hair, R. A. J. The 3D Quadrupole Ion Trap Mass Spectrometer as a Complete Chemical Laboratory for Fundamental Gas-Phase Studies of Metal Mediated Chemistry. Chem. Commun. 2006, 42 (14), 1469–1481. DOI: 10.1039/B516348J
4. Worrall, A. F.; Campbell, C. D.; Midson, M. O.; Stewart, M. I. University Teaching of Mass Spectrometry as a Key Practical Technique within the Context of a Fully Integrated, Spiral Curriculum. Rapid Commun. Mass Spectrom. 2024, e9851. DOI: 10.1002/rcm.9851
5. Patrick, A. L. Instructor’s Reference for Integrating Mass Spectrometry into the General Chemistry Classroom. J. Chem. Educ. 2020, 97 (10), 3595–3602. DOI: 10.1021/acs.jchemed.0c00603
6. Schildcrout, S. M. No, the Molecular Mass of Bromobenzene Is Not 157 Amu: An Exercise in Mass Spectrometry and Isotopes for Early General Chemistry. J. Chem. Educ. 2000, 77 (11), 1433. DOI: 10.1021/ed077p1433
7. Pfennig, B. W.; Schaefer, A. K. The Use of Gas Chromatography and Mass Spectrometry To Introduce General Chemistry Students to Percent Mass and Atomic Mass Calculations. J. Chem. Educ. 2011, 88 (7), 970–974. DOI: 10.1021/ed900010q
8. Bain, R. M.; Pulliam, C. J.; Raab, S. A.; Cooks, R. G. On-Line Synthesis and Analysis by Mass Spectrometry. J. Chem. Educ. 2015, 92 (12), 2146–2151. DOI: 10.1021/acs.jchemed.5b00165
9. Pavlov, J.; Errabelli, R.; Xu, S.; et al. Real-Time Monitoring of Reactions by Headspace Sampling under Ambient Mass Spectrometric Conditions. J. Chem. Educ. 2023, 100 (6), 2207–2214. DOI: 10.1021/acs.jchemed.2c01150
10. Fedick, P. W.; Bain, R. M.; Bain, K.; Cooks, R. G. Chiral Analysis by Tandem Mass Spectrometry Using the Kinetic Method, by Polarimetry, and by ¹H NMR Spectroscopy. J. Chem. Educ. 2017, 94 (9), 1329–1333. DOI: 10.1021/acs.jchemed.7b00090
11. Arnquist, I. J.; Beussman, D. J. Incorporating Biological Mass Spectrometry Into Undergraduate Teaching Labs, Part 1: Identifying Proteins Based on Molecular Mass. J. Chem. Educ. 2007, 84 (12), 1971. DOI: 10.1021/ed084p1971
12. Arnquist, I. J.; Beussman, D. J. Incorporating Biological Mass Spectrometry into Undergraduate Teaching Labs, Part 2: Peptide Identification via Molecular Mass Determination. J. Chem. Educ. 2009, 86 (3), 382. DOI: 10.1021/ed086p382
13. Arnquist, I. J.; Beussman, D. J. Incorporating Biological Mass Spectrometry into Undergraduate Teaching Labs, Part 3: De Novo Peptide Sequencing Using Electrospray Tandem Mass Spectrometry. J. Chem. Educ. 2009, 86 (8), 966. DOI: 10.1021/ed086p966
14. Perera, V.; Acharya, B.; Patrick, A. L.; Mlsna, D. Hands-On Electrospray Ionization Mass Spectrometry for Undergraduate Biochemistry Students: Peptide Identification by Ladder Sequencing. J. Chem. Educ. 2020, 97 (5), 1437–1442. DOI: 10.1021/acs.jchemed.9b00800
15. Calderón Celis, F.; García-Manrique, P.; Bettmer, J.; Encinar, J. R. Exploring Proteomics Workflows: Hands-On Learning and Evaluation of Analytical Techniques in Proteomics. J. Chem. Educ. 2025, 102 (7), 2732–2742. DOI: 10.1021/acs.jchemed.4c01460
16. Kapp, K. L.; Robinson, R. A. S.; Verberne-Sutton, S.; Stepler, K. E. Incorporation of a Virtual Proteomics Module into the Undergraduate Analytical Curriculum. J. Chem. Educ. 2023, 100 (8), 3124–3131. DOI: 10.1021/acs.jchemed.3c00067
17. Ryzhov, V.; Sunderlin, L. S.; Keller, L. M. M.; Gaillard, E. R. Measuring Gas-Phase Basicities of Amino Acids Using an Ion Trap Mass Spectrometer. A Physical Chemistry Laboratory Experiment. J. Chem. Educ. 2005, 82(7), 1071. DOI: 10.1021/ed082p1071
18. Gal, J.-F.; Mayeux, C.; Massi, L.; et al. Measuring Gas-Phase Basicities Relative to the Lithium Cation by Mass Spectrometry: A Physical Chemistry Experiment. J. Chem. Educ. 2012, 89 (11), 1476–1478. DOI: 10.1021/ed300128y
19. Keifer, D. Z.; Juncosa, J. I. J. A Mass Spectrometry Experiment on the Degrees of Freedom Effect. J. Chem. Educ. 2024, 101 (9), 3975–3982. DOI: 10.1021/acs.jchemed.4c00708