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The Eastern Analytical Symposium (EAS) Award for Outstanding Achievements in Mass Spectrometry will be presented to Barbara Larsen on Thursday, November 19.

is a Nutrition and Biosciences fellow at the DuPont Experimental Station in Wilmington, Delaware. Her research focuses on the application of mass spectrometry to support the biotechnology business. She has a keen interest in ionization methods to ensure the continued relevance of mass spectrometry for biotechnology projects at DuPont. Larsen has a passion for 

—selecting the best measurement technology to provide the critical solution to a specific problem. For her work supporting biotechnology research, Larsen has developed a digestion protocol for bioproteins that provides coverage of the expressed protein sequence or deep proteomic coverage of the organism that produced the protein. In addition, these methods can be used to provide details on protein modifications that occur during host expression or during processing. Her research interests include using a systems biology approach including metabolomics, proteomics, and transcriptomics to improve enzyme production and probiotic organisms.

Larsen has been part of the DuPont Company for the past 35 years. She received her BS from the University of Santa Clara, California, and her PhD in Physical Chemistry at the University of Delaware, followed by a post-doctoral fellowship at Johns Hopkins School of Medicine. Larsen has received the Spectroscopy Society Award and the Delaware ACS Section Research Award. DuPont awarded her research with the Pederson Award, for significant scientific achievement that reflects technical excellence. 

She is currently a member of American Society of Mass Spectrometry—where she has served as the treasurer, vice president of programs, and president—and is on the advisory board for the society journal. As an active member of the American Chemical Society (ACS), she was made an ACS fellow for her service as the chair for the Delaware Valley Mass Spectrometry Discussion Group and as a counselor for the Delaware ACS section. She also serves on the editorial advisory board of 

Articles

Barbara Larsen Receives EAS Award for Outstanding Achievements in Mass Spectrometry

The New York Society for Applied Spectroscopy Gold Medal Award will be presented to Jerome Workman, Jr. and Howard Mark at the Eastern Analytical Symposium (EAS) on Wednesday, November 18. Workman is scheduled to deliver a talk on “The Present and Future of Chemometrics in the Analytical Sciences” while Mark is scheduled to deliver a talk on the “History of Calibration Transfer.” This year marks only the second time the committee has selected two winners for the Gold Medal Award since its inception in 1952. Mark and Workman were recognized for their contributions, individually and as a team, to the field of near-infrared (NIR) spectroscopy and chemometrics, leading to a wider adoption of the technology in the spectroscopy community.

Jerome Workman, Jr. is currently the Senior Technical Editor for 

. He is also a Certified Core Adjunct Professor at National University in San Diego, California, and a principal at Biotechnology Business Associates. He was formerly the Executive Vice President of Research and Engineering for Unity Scientific and Process Sensors Corporation. Workman has more than 75 U.S. and international patent applications since 1998 with 25 issued U.S. and international patents and multiple trade secrets. He has more than 500 technical publications, including 22 reference books on a broad range of spectroscopy, chemometrics, and data processing topics.he also has received many awards, including the 2002 Eastern Analytical Symposium Award for Achievements in NIR, the 2002 ASTM International Award of Merit, the 2009 Coblentz Society Williams-Wright Award, as well as awards from the Society for Applied Spectroscopy, National Institute of Standards and Technology (NIST), Frost and Sullivan, and other organizations. He is a fellow of the American Institute of Chemists, ASTM International, and the Royal Society of Chemistry.

Howard Mark serves on the Editorial Advisory Board of 

and runs a consulting service, Mark Electronics, in Suffern, New York. Mark has authored 243 papers including 75 installments of the "Statistics in Spectroscopy" column and 123 installments of "Chemometrics in Spectroscopy," four books, numerous book chapters, and seven patents. He was a Student Awardee to the 1964 Eastern Analytical Symposium, and has received the 2003 Eastern Analytical Symposium award for Outstanding Contributions in the Field of Near Infrared Spectroscopy, an Honorary Life Membership to the Council for Near Infrared Spectroscopy, awarded in 2010, and the 2011 Coblentz Society's Williams-Wright award for Outstanding Contributions to the Field of Industrial Vibrational Spectroscopy. Mark’s interests focus on application of electronics and especially, computers, to chemical Instrumentation and analysis, with emphasis on gas chromatography and spectroscopy, particularly mid- and near-infrared spectroscopy.

The New York Society for Applied Spectroscopy Gold Medal Award will be presented to Jerome Workman, Jr. and Howard Mark at the Eastern Analytical Symposium (EAS) on Wednesday, November 18.

Jerome Workman, Jr. and Howard Mark Receive the 2020 New York Society for Applied Spectroscopy Gold Medal Award 

The Eastern Analytical Symposium (EAS) Award for Outstanding Achievements in Magnetic Resonance will be presented to Arthur G. Palmer III on Monday, November 16.

received a BA in chemistry magna cum laude in 1980 from Haverford College. At Haverford, he conducted undergraduate research in the photochemistry of carbonyl carbene with Professor Colin F. Mackay. After two years working as an analytical chemist and two years teaching high school chemistry and physics, Palmer entered graduate school, first receiving his MS in Industrial Health from the University of Michigan in 1986 and then completing his PhD from the University of North Carolina at Chapel Hill in 1989. His doctoral research, conducted under the direction of Professor Nancy L. Thompson, developed the technique of high order autocorrelation in fluorescence correlation spectroscopy.

Palmer made the transition to nuclear magnetic resonance (NMR) spectroscopy during his work as a National Science Foundation Postdoctoral Fellow with Dr. Peter E. Wright at the Scripps Research Institute from 1989 to 1992. There, Palmer codeveloped, with Wright, Mark Rance, and John Cavanagh, sensitivity-enhanced heteronuclear single quantum coherence spectroscopy (HSQC) and began his first developments and applications of NMR spin relaxation to characterize macromolecular dynamics. Palmer moved to the Department of Biochemistry and Molecular Biophysics at Columbia University as an assistant professor in 1992 before being promoted to associate professor in 1998 and then to professor in 2001. Palmer currently holds the Robert Wood Johnson junior chair title and serves as interim Chair of the Department of Biochemistry and Molecular Biophysics and as Associate Dean for Graduate Affairs at Columbia University Medical Center. He is also the Director of NMR Spectroscopy at the New York Structural Biology Center.

Palmer is the author of 150 papers and the coauthor, with John Cavanagh, Wayne J. Fairbrother, Nicholas J. Skelton, and Mark Rance, of the book 

Protein NMR Spectroscopy: Principles and Practice

, now in its second edition. Palmer uses NMR spectroscopy and molecular dynamics simulations to elucidate the coupling between conformational dynamical properties and biological functions of proteins. His research interests include the development of novel methods in NMR spectroscopy, computational and theoretical analyses of protein dynamics, and applications to protein folding, molecular recognition, and catalysis. He has contributed in particular to the use of generalized order parameters for characterizing conformational entropic effects in molecular recognition, and the use of CPMG and R1r relaxation dispersion measurements for characterizing microsecond-millisecond time scale processes.

Palmer is interested in the conformational dynamic differences between mesophilic and thermophilic enzymes that contribute to differences in activity, using the ribonuclease H superfamily as a model system. Other recent applications include the mechanism of strand swapping in dimerization of cadherin cell adhesion proteins, the role of local dynamics in DNA recognition by the GCN4 bZip transcription factor, and dynamic control of the order of substrate addition in the DNA-repair enzyme AlkB.

Arthur Palmer Receives EAS Award for Outstanding Achievements in Magnetic Resonance

The Eastern Analytical Symposium (EAS) Award for Outstanding Achievements in Vibrational Spectroscopy will be presented to John A. Reffner on Tuesday, November 17. Reffner will also give a talk on “Advancing Our Knowledge Through Studies of Vibrational Spectra.”

Reffner’s scientific interests focus on uniting spectroscopy with microscopy and applying novel technologies to advance materials and forensic science. He pioneered the development of infrared micro-spectrometers, accessories, and innovative applications in infrared microprobe technology. Reffner’s scientific accomplishments are recognized by his receiving the 2016 American Society of Trace Evidence Examiners’ Edmond Locard Award, the 2015 Society of Applied Spectroscopy’s Gold Medal Award, the 2004 American Academy of Forensic Sciences award, the 2004 Paul L. Kirk Award, the 2002 New York Microscopical Society’s Abbe Memorial Award, the 2002 Georgia Microscopical Society’s Honorary Achievement Award, the 2000 Coblentz Society’s Williams-Wright Award, and the 1993 Illinois State Microscopical Society’s Emile M. Chamot Award. In 2011, Reffner was named a fellow of the Society of Applied Spectroscopy (SAS).

Reffner has authored 90 papers and four book chapters while inventing ten patents. He served as a consultant to the Connecticut State Police for over 25 years and has testified as an expert witness in criminal, civil, and intellectual property litigations. He is also on the advisory board of the Dr. Henry C. Lee Institute at the University of New Haven. His employment, since graduation from Akron University, includes B. F. Goodrich, Walter C, McCrone Associates, the University of Connecticut, American Cyanamid, Spectra-Tech, Nicolet, Thermo Scientific, SensIR, and Smiths Detection. Reffner is currently a professor of forensic science at John Jay College, which is part of the City University of New York (CUNY) in New York, New York.

The Eastern Analytical Symposium (EAS) Award for Outstanding Achievements in Vibrational Spectroscopy will be presented to John A. Reffner on Tuesday, November 17.

John Reffner Receives EAS Award for Outstanding Achievements in Vibrational Spectroscopy

Jerome Workman Jr. serves on the Editorial Advisory Board of Spectroscopy and is currently with Unity Scientific LLC. He is also an adjunct professor at Liberty University and U.S. National University. He can be reached at jworkman04@gsb.columbia.edu.

Recent collaborative research demonstrates the use of Kerr-gated Raman spectroscopy to study catalysts and catalytic reactions for investigating renewable energy sources. The approach overcomes fluorescence interference problems normally encountered using Raman spectroscopy of catalysts and can facilitate the development of renewable energy sources, such as producing gasoline from methanol.

 (1), was performed through a collaboration between the Central Laser Facility (CLF) of the Science and Technology Facilities Council (STFC), University College London, Ghent University, and the Catalysis Hub, The Catalysis Hub is a United Kingdom consortium aimed at leading the world in catalytic science.

Catalysis is an extremely important field of study in chemistry and involves the materials and processes of increasing the rate of a chemical reactions by the use of catalysts. Catalysts are the materials used to increase reaction rates, by lowering activation energies for reactions, and can be repeatedly used while not being consumed in the catalyzed reaction. Different classification types of catalysts include electrocatalysts, organocatalysts, photocatalysts, enzymes, and biocatalysts. Catalytic reactions occur in nature as well as in synthetic chemical reactions, such as in the production of petroleum products (alkylation, catalytic cracking, catalytic converters); bulk chemical production (catalytic oxidation, hydrogenation, and hydroformylation); fine chemical production (Heck reaction, and Friedel–Crafts reactions); and in food processing (hydrogenation and biocatalysis).

Natural and synthetic zeolite crystals (Figure 1) are essential catalysts, and represent the most-used catalysts by weight in the world. Zeolites are crystalline alumino-silicate, porous materials with regular molecule-sized nanopores—having a specific physical structure of channels and cavities, which affect their catalytic properties and efficiencies (their adsorption, diffusion, and reaction properties). Understanding zeolite structure and reaction properties is an important area of research in the field of catalysis.

Understanding the detailed structure and function of zeolites in chemical reactions has been an analytical challenge. The pores of zeolites may be contaminated with heavy hydrocarbons over time that get trapped within them—as contamination occurs over time the catalytic activity will decrease and eventually cease. When the zeolite is deactivated it must be replaced or reactivate using an expensive process. This entire activation and deactivation process is of keen interest to catalysis researchers.

One of the most common techniques used to investigate zeolite structure and catalysis is Raman spectroscopy. However, the fine structural information that might be available using the Raman technique is often obscured due to background fluorescence. CLF’s expertise in Kerr-gated Raman spectroscopy is providing the means for detailed spectra to be obtained without the fluorescence background problems.

Kerr-gated Raman spectroscopy uses the Kerr gate optical effect to remove most of the fluorescence background from the Raman signal when using Raman spectroscopy. The Kerr gate effect functions by plane polarizing the combined Raman scattered signal and fluorescence signal (resulting from a 1 ps laser pulse) by using a plane polarizer. Then by passing that combined signal through a 45

 containing cell of 2 mm pathlength (Kerr medium), the resulting signals are separated into a plane polarized fluorescence signal and a perpendicularly polarized Raman signal. By then passing the signals through a cross polarizer, the Raman scattering will pass (is transmitted) to the detector, while the fluorescence signal is blocked. Therefore the fine Raman spectral structure may be observed relatively free from the fluorescence background. The Kerr gate effect takes advantage of the time delay between the instantaneous Raman scattering signal and the delayed background fluorescence that occurs a few picoseconds or nanoseconds later.

“Kerr-gated Raman is remarkable because it makes it possible to detect tiny Raman signals from zeolite reactions that would otherwise be lost in a sea of fluorescence noise, said Professor Mike Towrie of CLF “The Ultra Laser Facility at the CLF allowed us to achieve a signal quality approximately 100 times better than if we’d used standard Raman. For me, this work has been exciting because it demonstrates the potential of Kerr-gated Raman to help answer many more questions in catalysis and in other areas such as battery science.”

Kerr-gated Raman spectroscopy is being applied to gain understanding of the potential for catalytic reaction chemistry to produce gasoline (mostly octane) from simple hydrocarbons, such as methanol. Better understanding of this reaction could result in producing gasoline from biomass fermentation as a renewable energy source—reducing both fuel production costs and environmental impact. Methanol is considered a clean energy option that can be produced from many renewable sources. A methanol molecule has only half the energy density of gasoline and, in the 1970s, the chemistry was developed to convert methanol into gasoline using a catalytic reaction involving zeolites. The size of the zeolite pores determines the type of hydrocarbon produced by the catalytic reaction—small pores produce light hydrocarbons—medium pores produce gasoline.

Using Kerr-gated Raman spectroscopy the CLF team were able to track all the stages of the reaction, in increments of three to four picoseconds, and observe a conversion of methanol to octane as longer chains of hydrocarbons were being formed. This allowed detailed observations of the entire reaction process. The Raman technique also provides information of the activation and deactivation of the zeolite catalysts. This rapid analysis technique has provided insight into the formation of polyenes as the cause of deactivation of the zeolite catalysts—a new and unexpected discovery. “I think that this will change the way people think about how these materials deactivate because, as soon as you know who the culprits are, you are able to design a strategy, said team leader Professor Andy Beale of University College London. He said, “There is a huge energy cost, as well as time cost, associated with reactivating catalysts frequently, so anything that can keep catalysts doing their job for longer will save money, energy and reduce the environmental impact resulting in greener fuels. The study could be significant for industry especially as methanol can be produced from biomass stocks meaning that gasoline may not have to come from fossil fuel stocks in the future.”

Reference
(1) I. Lezcano-Gonzalez, E. Campbell, A. E. J. Hoffman, M. Bocus, I. V. Sazanovich, M. Towrie, M. Agote-Aran, E. K. Gibson, A. Greenaway, K. De Wispelaere, V. Van Speybroeck, and A. M. Beale, “Insight into the effects of confined hydrocarbon species on the lifetime of methanol conversion catalysts,” Nature Materials 19, 1081–1087 (2020). (

https://doi.org/10.1038/s41563-020-0800-y

Recent collaborative research demonstrates the use of Kerr-gated Raman spectroscopy to study catalysts and catalytic reactions for investigating renewable energy sources.

Kerr-Gated Raman Spectroscopy is Being Used to Study Catalysis Reactions for Investigating Renewable Energy Sources 

In your paper (1), you state that single-cell inductively coupled plasma mass spectrometry (SC-ICP-MS) is an emerging methodology that holds promise to enable multielemental analysis of low-volume samples in the future. Can you explain why you believe this?

In my view, SC-ICP-MS has become a hot topic in the field of elemental analysis, and rightly so: Scientist working in the field of bioanalytics applied this approach to look into the smallest units of life. As a matter of fact, most biochemical processes take place in cells. Elemental bioanalysis in cells makes it possible to obtain deep insights into cellular processes and the biological status of specific cells. In comparison, the analysis of bioliquids mainly provides insights on a systemic level of the corresponding organism. Over the last decades, many scientists applied ICP-MS to study the elemental composition in all kinds of biofluids and tissues. Due to the latest technical developments it is now possible to achieve ever lower detection limits. Thus, it is now possible to examine individual cells and their elemental composition—at least partially. This may open a completely new field of application for ICP-MS and there are many scientists who are about to prove this.

Why is ICP-MS generally considered being the method of choice for multielemental analysis as compared to other ICP techniques (AES or OES) or more traditional atomic absorption (F-AAS or GF-AAS)?

ICP-MS is unarguably the gold standard for elemental and especially multielemental analysis. This is mainly due to the fact that this technique is extremely versatile compared to ICP-OES/AES and atomic absorption spectroscopy. Most AAS devices apply element-specific radiation sources (hollow cathode lamps) that enable the detection of a single element at the same time. Although there are some commercially available AAS instruments that allow the detection of 10-20 elements simultaneously, sensitivity limitations hamper the analysis of most trace and ultratrace elements. ICP-OES allows multielemental analysis, is relatively easy to setup, and well-suited for high-throughput analysis—however, spectral interferences can be challenging. ICP-MS allows high-throughput, multielemental analysis with the lowest-possible detection limits. It offers a linear detection range of up to 10 orders of magnitude, enables isotopic analysis (isotope dilution analysis for quantification, control of spectral interferences), and it is possible to analyze any type of sample material (gases, liquids, solids).Furthermore, high-resolution separation techniques such as HPLC, GC, CE etc. can be hyphenated to the ICP-MS. Overall, this makes ICP-MS the most powerful and versatile technique for elemental analysis.

What motivated you to focus on this specific application?

Several times, we have encountered situations, where limited sample volume was available. For example, limited volume of biofluids (blood serum or plasma) might be available in human sample cohorts for interdisciplinary clinical studies. In such cases, the principal investigator often selects the panel of analytical tests according to the required sample volume. Sample volume restrictions can also arise from the donor itself, even if there is no competing analysis. This is the case for animal models, especially for studies with mice where only a few µL of blood serum or plasma are available per animal. Since most of the published multielemental profiling methodologies require relatively large sample volumes, our aim was to provide a tool that overcomes this challenge. For this reason, in 2016 we have developed an ICP-MS based method, which allows the analysis of 29 elements in a 150 µL blood serum sample. However, we soon realized that there is a need for multielement analysis in even smaller sample volumes. This is for example the case when only small amounts of serum and sample are available (for example, in precious clinical cohorts or preclinical studies with rodents). We accepted the challenge and found that it is possible to detect 20 to 30 elements in 3–10 µL of biological fluids. These positive results have motivated us to optimize and validate the method. Finally, we have successfully applied the method in a preclinical model. The method turned out to be very easy to use, it does not require expensive or complicated changes in the experimental setup, and, above all, it has shown very good analytical performance. We then decided to publish the results of our study as well as the experimental setup in order to encourage other readers to perform multi-element analyses in the smallest sample volumes in a straightforward way.

What are some cancer-specific multielemental profiles that you have identified? What type of data analysis techniques were used to determine these profiles? Are there a few predominant elements that seem to be most often present in the mice cancer cells?

In the mouse cancer modes under evaluation, the presence of the tumor influences the serum concentration levels of various elements. Some elements, such as sulfur, calcium, manganese, zinc, strontium and molybdenum, were not affected significantly by the presence of the tumor. On the other hand, elements such as phosphorous and copper were increased in the serum samples of tumor bearing mice, independently of the tumor type. Iron, on the other hand, was depleted. These results seem to indicate that the presence of tumor systemically influences the concentration levels of some serum elements, independently from the tumor type. On the contrary, elements such as vanadium, chromium, bromine, rubidium, cesium and cobalt show trends for tumor-specific concentration changes. Furthermore, our results show that different immune T cells (CD4+ and CD8+) have specific mineral profiles reflecting their specific immune function. Moreover, the presence of a tumor seems to have an impact in the mineral contents of T cells. Indeed, we observed elemental modulations within CD8 cells that could be associated with presence of cancer, such as higher levels of magnesium and manganese and lower levels of iron, cobalt and rubidium. These findings might indicate an increased metabolic need of immune cells for Mn and Mg in the presence of a tumor.

What are the biggest challenges that you encounter in this analysis method? Are there any limitations to using this technique? What options or alternatives are available to overcome these challenges?

While the analysis of small amounts of biofluids is quite straightforward (centrifugation to remove particles and a simple dilution step), the analysis of cells is more challenging. In fact, one hurdle we faced during method development was to remove the cell sorting buffer (containing a high concentration of various elements of interest) from the cells. As large volumes of this buffer are required for a single cell-sorting experiment (roughly 10 liters in our case), it was not possible to prepare a buffer solution based on reagents which are suitable for trace elemental analysis. For a washing step, we applied filters or considered osmosis for buffer removal, but none of these sample preparation strategies proved to be suitable. Finally, applying a mild centrifugation step to form a cell pellet, followed by removing the supernatant with a pipette, provided satisfying results. However, the removal of a few microliters from an Eppendorf tube, and at the same time maintaining the integrity of a tiny cell pellet, remains the Achilles heel of the method and requires some finesse and experience.

With such small samples, is contamination an issue, and how have you overcome this potential problem?

Contamination is always a challenge in ultratrace elemental analysis. Acid-washing, working in a clean environment and under a fume hood are the minimum requirements to obtain high quality results. However, contamination is not more challenging in this method, as compared to most other ICP-MS based methods dealing with trace and ultratrace analysis. This is mainly due to the fact that a relatively moderate sample dilution of (1:10) and few sample preparation steps are applied. Contamination could arise, for example, from plastic materials. We mitigate this risk by applying an acid-washing step up front. Another, often underestimated source of contaminants are chemicals and reagents applied during sample preparation. They often contain elements (for example, iron) that enter the sample in this way and thus mix with the analytes. Due to the inherent properties of elemental mass spectrometry, it is then no longer possible to distinguish easily between endogenous and exogenous elements. This can lead to an overestimation of the element concentration. In our study we only apply buffer solutions and nitric acid during sample preparation. Thus, the overall contamination risk is relatively low.

Can you briefly summarize the results that you have achieved so far?

We have developed a quantitative analytical strategy that enables the analysis of 20 biologically relevant elements in low-volume samples (10 µL) of serum. By applying an optimized workflow, the method is also applicable for the quantification of 12 elements in a low number of sorted cells (250,000 cells). In our paper, we give detailed descriptions of the experimental setup. This enables scientist to apply the approach to study biological-relevant elements (major trace and ultratrace elements) in very small sample volumes. Applied to tumor cells, we could demonstrate that the presence of certain cancer types influences differentially the elemental composition of serum concentrations. Furthermore, we could show that different immune T cells (CD4+ and CD8+) have specific mineral profiles reflecting their specific immune function. Last but not least, the presence of a tumor effects the mineral composition of T cells.

Is there anything that you have done in your study that is different from what other researchers have done as they have examined the same problem?

Most of the ICP-MS-based approaches aiming to detect major trace and ultratrace elements in cells are challenged by the media or solvent in which the cells are transported into the plasma ionization source. If the cells are present in Milli-Q water, the “elemental background” is very low. This facilitates the detection of very small quantities of cellular elements. However, cells are prone to lyse in Milli-Q-water. This makes the cellular membranes permeable for ions and small molecules, which are eventually leaking out of the cell and changing the elemental composition of the cell. Fixation strategies, aiming to stabilize cellular membranes, can prevent lysis. Unfortunately, fixation methods tend to increase permeability of the cell membranes and consequently alter the endogenous elemental composition of the cell. Moreover, aggregation of cells is often seen after fixation and needs to be monitored. In fact, the presence of cells in the monodisperse state has to be confirmed by complementary analysis. Many of the reported SC-ICP-MS methods are overcoming this issue by using yeast cells. The use of yeast cells has two advantages: first, the cells are relatively big, meaning higher absolute quantity of elements per cell. Second, these cells are extremely robust compared to immune cells. Re-suspended in Milli-Q water, yeast cells can stay alive for a couple of hours without undergoing lysis. This timespan allows conducting SC-ICP-MS experiments with intact cells while maintaining an extremely low elemental background of the solvent. Suspensions of cells in buffer solutions, on the other hand, allow keeping the cells alive and preserve the integrity of the cell. However, such buffer solutions contain “contaminants”; for example, major or trace elements which are present in the raw materials. This makes it difficult to distinguish between “cellular” elements and elements from the buffer solution. We were able to overcome this limitation by using a sample preparation strategy that includes a washing step with a suitable buffer solution (containing a relatively low elemental background).

What are your next steps in this work? Do you know how well the discoveries of multielemental profiles in mice cancer cells may extrapolate to human cancer cells?

I am now working in a company which is rather focused on elemental analysis is food and pharmaceutical products. However, my former colleagues are continuing our research work and other scientist are working on different approaches to study trace- and ultratrace elements in cells. I am confident that rather sooner than later we will see new findings in this field, both in pre-clinical and clinical studies.

Tobias Konz is currently leading the Elemental Analysis group at UFAG Laboratorien (in Sursee, Switzerland), specializing in quantitative analysis of elements in food and pharmaceutical products (GMP and ISO environment). He studied biomedical chemistry at the Johannes-Gutenberg University (2000-2007), and completed his diploma thesis and PhD in Analytical Chemistry in the research group of Alfredo Sanz-Medel, under the supervision of Maria Montes-Bayón (University of Oviedo, Spain). In 2014, he started a professional career as ICP-MS specialist at the Nestlé Institute of Health Sciences (Lausanne, Switzerland) in the Nestlé Research organization. The main focus of his work has been to develop quantitative methods for multielemental analysis in low volume samples (biofluids) in a GLP and ISO regulated environment.

(1) T. Konz, C. Monnard, M. Rincon Restrepo, J. Laval, F. Sizzano, M. Girotra, G. Dammone, A. Palini, G. Coukos, S. Rezzi, J.-P. Godin, and N. Vannini, 

https://dx.doi.org/10.1021/acs.analchem.9b05643

Tobias Konz of Nestlé Research, Lausanne, Switzerland and various associates have developed and validated what they describe as a reliable, robust, and easy-to-implement quantitative method for multielemental analysis of low-volume samples. The ICP-MS-based method comprises the analysis of 20 elements (Mg, P, S, K, Ca, V, Cr, Mn, Fe, Co, Cu, Zn, Se, Br, Rb, Sr, Mo, I, Cs, and Ba) in 10 μL of serum and 12 elements (Mg, S, Mn, Fe, Co, Cu, Zn Se, Br, Rb, Mo, and Cs) in less than 250,000 cells, and involved the analysis of elemental profiles of serum and sorted immune T cells derived from naıv̈e and tumor-bearing mice. The results indicate a tumor systemic effect on the elemental profiles of both serum and T cells. Konz and his colleagues believe their approach highlights promising applications of multielemental analysis in precious samples such as rare cell populations or limited volumes of biofluids that could provide a deeper understanding of the essential role of elements as cofactors in biological and pathological processes. Konz spoke to us about this work.

Quantitative Methods for Multielemental Analysis in Low Volume Biofluids 

 Magazine is pleased to announce the addition of seven new members to its editorial advisory board: Karl S. Booksh, John Coates, Paul J. Gemperline, Barry K. Lavine, Igor K. Lednev, Yukihiro “Yuki” Ozaki, and Barry M. Wise.

Karl S. Booksh is a professor in the Department of Chemistry and Biochemistry at the University of Delaware. He holds a BS in Chemistry from the University of Alaska and a PhD in Analytical Chemistry from the University of Washington. He is an American Chemical Society Fellow (2015) and a Society for Applied Spectroscopy (SAS) Fellow (2014), and is the president elect of SAS for 2021. His research group develops small chemical sensors capable of reliable measurements in the field and process chemical sensors for environmental, biomedical, and industrial process monitoring. His group also conducts chemometric analysis of collected data and multivariate sensor calibration. In terms of analytical techniques, Booksh is focusing largely on Raman, laser-induced breakdown spectroscopy (LIBS), and fluorescence sensors with a bit of Raman imaging for fun. He is the author of over 100 peer-reviewed articles.

John Coates was educated and started his career in the United Kingdom. He first worked as an analytical chemist for Castrol Oil Company. He graduated from the Royal Society of Chemistry (RSC) and obtained his PhD in analytical chemistry at Brunel University. After moving to the United States as a senior staff scientist at Perkin Elmer Corporation, Coates accepted positions at Spectra-Tech (Stamford, Connecticut) and then at Nicolet Instruments (Madison, Wisconsin) before returning to a position at PerkinElmer’s Real-Time Systems Division (Wilton, Connecticut) a joint-venture with Dow Chemical Company (Midland, Michigan).

In 1996, Coates opted to leave the corporate world and formed his own company, Coates Consulting LLC, a consulting company focused on applied and industrial instrumentation, optical spectroscopy, and analytical instruments and sensors for dedicated applications, with a primary focus on instrument miniaturization and spectral sensors. Coates has been involved in the development of more than 100 different instrument and sensor products for dedicated applications and received the Williams-Wright Award for Industrial Spectroscopy, presented by the Coblentz Society, in 2013. Coates has 50 years of industrial experience.

Paul J. Gemperline has been the dean of the graduate school at East Carolina University (ECU) since 2009 and a faculty member in the department of chemistry since 1982. He earned his BS and PhD in chemistry from Cleveland State University. Gemperline’s area of research is chemometrics with more than 60 publications and $1.8 million in external grant funds from government and industrial sources. Gemperline's research focuses on the development of new computer algorithms and software tools for analysis of multivariate spectroscopic data. His work has led to advances in methods for monitoring, understanding, and controlling batch chemical and pharmaceutical processes. He has developed methods of cluster analysis and classification for rapid non-destructive testing of raw materials and finished products. His work with collaborators led to the development of a licensed patent for designing optical filters for chemical calibration.

Gemperline also served as the editor-in-chief of the 

Barry K. Lavine is a professor of chemistry at Oklahoma State University (OSU). Lavine’s research interests encompass many aspects of chemical analysis including optical sensors, vibrational spectroscopy, infrared and Raman imaging, and chemometrics. Lavine has published more than 115 research papers, 21 book chapters, and 16 review articles, along with being the editor for three ACS monographs. Lavine is on the editorial board of several journals including the 

. He has served as chair of the Northern New York (1997–2004) and Oklahoma (2006–2008) sections of the ACS. Lavine has also been the program chair for several meetings including SciX (1992), the Northeast Regional ACS Meeting (1999), and the Pentasectional Meeting of the local Oklahoma Sections of the ACS (2005). Lavine is the recipient of the 2015 Kowalski prize from the 

and the 2017 Chemometrics Award sponsored by the Eastern Analytical Symposium. He is also a SAS fellow.

Igor K. Lednev is a professor at the University of Albany, State University of New York. He received his PhD from the Moscow Institute of Physics and Technology and worked in several leading laboratories around the world in the United Kingdom, Canada, Germany, and Japan. Lednev accepted a faculty position at the University at Albany in 2002 and was promoted to full professor in 2013. His research focuses on the development and application of novel laser spectroscopy techniques for medical diagnostics and forensic purposes. Lednev is a cofounder of two startup companies commercializing his patented technology. He served as an advisory member on the White House Subcommittee for Forensic Science and is a fellow of SAS and the Royal Society of Chemistry. Lednev also launched a new annual National Institute of Justice (NIJ) Forensic Science Symposium at Pittcon in 2018.

He has co-authored about 240 publications in peer-reviewed journals and seven patents.

Yukihiro (Yuki) Ozaki received his PhD in 1978 from Osaka University. He joined Kwansei Gakuin University in 1989. From 1993 to March 2018, he was a professor in School of Science and Technology. Currently, Ozaki is a professor emeritus of the university. He has been a guest professor or scientist at Kobe University, Fukui University, and Riken and Toyota Physical and Chemical Research Institute. Ozaki is involved in studies of a wide range of molecular spectroscopy techniques, ranging from far-ultraviolet to far-infrared to terahertz and Raman spectroscopy. His research focuses on both electronic and vibrational spectroscopy.

Ozaki has been a member of SAS for more than 30 years and fellow since 2013. He received several awards including the Bomem-Michelson Award (2014), Chemical Society of Japan Award (2017), The Medal with Purple Ribbon (2018), Pittsburgh Spectroscopy Award (2019), and Charles Mann Award (2020).

Barry M. Wise received BS degrees in chemistry and chemical engineering from the University of Washington in 1982. After three years with Battelle Pacific Northwest National Laboratory, he returned to the University of Washington, where he received MS (1987) and PhD (1991) degrees in chemical engineering. Wise has authored more than 50 scientific publications, patents, and book chapters. He has also created a popular chemometrics software package, PLS_Toolbox, used by scientists and engineers worldwide. Wise is president and cofounder of Eigenvector Research Inc., which provides chemometrics software, training, and consulting. Thousands have attended Wise’s chemometrics short courses at conferences and at Eigenvector University.

Wise was named the winner of the Eastern Analytical Symposium Chemometrics Award for 2001 in recognition of his significant contributions to the field. He also received the Herman Wold Medal in Gold in 2019 for his deep commitment to the proliferation of chemometrics.

Spectroscopy Magazine is pleased to announce the addition of seven new members to its editorial advisory board.

Seven New Members Join Spectroscopy's Editorial Advisory Board

Of course, this year’s meeting is different because of our response to the presence of COVID-19. 

 There will be LIVE on-line sessions each day during the week of 

 Additionally, the EAS presents pre-recorded ON-DEMAND sessions available at any time through December 31 to those who register for the Symposium. And, be sure not to miss the on-line virtual exhibition for the latest innovations from the vendors you have come to rely on.

This year, the EAS Awards for achievements in spectroscopy recognize the outstanding contributions of Barbara S. Larsen (mass spectrometry), Arthur Palmer (magnetic resonance spectroscopy), and John Reffner (vibrational spectroscopy), and you can tune in to these award sessions, LIVE. Also live are the sponsor awards such as the SAS Gold Medal honoring Howard Mark and Jerome Workman, as well as the Ernst Abbe Award to Brian J. Ford. But there also are many pre-recorded on-demand sessions and posters on spectroscopic analysis of materials, forensic analysis, biological and pharmaceutical analysis, microscopy, Raman imaging, LIBS, and many more.

Two highlights of the EAS in years past have been the keynote lecture and the plenary lecture, and this year’s lectures should be exceptionally entertaining for attendees. The keynote lecture by Eric Green of the National Institutes of Health is titled “The Human Genome Project was just the Beginning: Research Opportunities at the Forefront of Genomics.” The plenary lecture, “The Fascinating Impact of Nanoscale Structure on Chromatography and Mass Spectral Ionization,” will be given by Susan Olesik, this year’s winner of the EAS Award for Outstanding Achievements in the Fields of Analytical Chemistry. Her lecture combines two themes that should interest a wide audience. In addition, this year there is a special lecture presented by Adam Lanzarotta of the USFDA on the “FDA’s Role in the 2019 Vaping Crisis.” These three lectures focus on important issues in science and how it affects the world around us.

The Eastern Analytical Symposium is known for its large suite of short courses tailored to the needs of analysts. This year we are proud to continue the tradition of short courses on a wide range of traditional and novel topics in analysis. Whether it’s gas or liquid chromatography, hyphenated methods, spectroscopic methods including imaging, NMR spectroscopy, FT-IR spectroscopy, or topics like communication or process analysis, there is a course at EAS for you!

These will be presented in a LIVE video format that allows interaction with the instructor, over several weeks, allowing interested persons to attend multiple short courses.

, for the latest news and registration info for this year’s virtual EAS. Be sure to take advantage of the discounted pricing we offer to those who register early!

Once again, the Eastern Analytical Symposium takes the spotlight November 16–19, providing analysts with the latest developments in areas ranging from biopharmaceutical analysis to materials characterization, from quality assessment to green analysis, and from environmental contaminant determination to antibody analysis, to name a few of the many areas covered.

The 59th Eastern Analytical Symposium

R.C. McDowall is the principle of McDowall Consulting and director of R.D. McDowall Limited, and "Questions of Quality" column editor for LCGC Europe, Spectroscopy's sister magazine.

Universally available on desktop computers, easy to use, and universally loved by analytical chemists, spreadsheets are used to collate lists of instruments and perform a multitude of calculations in many regulated laboratories. But spreadsheets also present an auditing and inspecting opportunity to find abundant instances of noncompliance and can lead to problems and unnecessary risk. Why is the use of spreadsheets heavily discouraged in a regulated laboratory?

If you turn on any workstationin your laboratory, you will probably find a shortcut to a spreadsheet program. When presented with this icon of temptation and seduction, a willing analyst is happy to open Pandora’s spreadsheet box and navigate between Scylla and Charybdis. Charybdis is the whirlpool of quality assurance where you have the challenge of specifying, building, and validating each spreadsheet template. Alternatively, steer the course to Scylla, the six-headed monster of regulatory noncompliance. With Scylla, if the unvalidated spreadsheet you have written and used is found, you are dead.


Spreadsheets are often used to perform GMP–related calculations, trend data, plan calibration and qualification work, and manage laboratory assets. I have even seen spreadsheets used to document the test steps for validating software—and the least said about that the better. This article discusses why spreadsheets should be eliminated as much as possible from performing GXP work to increase business efficiency and reduce regulatory risk. Note that business efficiency came before reducing regulatory risk in the last sentence. This column is dedicated to all spreadsheet lovers, or in the words of that great analytical chemist, William Shakespeare, “I come to bury spreadsheets, not to praise them.”

One of many companies that have sailed into Scylla and been cited for noncompliant spreadsheets is Tismore Health, as seen in a Federal Drug Administration (FDA) warning letter in December 2020 (1). I hope this does not happen in your laboratory. Citation 3 is against 21 

211.160(b) (2), although I would have raised the citation against 211.68(b) for failing to verify formulas. However, that is the problem with the FDA’s last century GMP regulations:

Your firm failed to establish laboratory controls that include scientifically sound and appropriate specifications, standards, sampling plans and test procedures designed to assure that components, drug products conform to appropriate standards of identity, strength, quality, and purity (21 CFR 211.160(b)).

Your firm failed to validate the spreadsheet used to perform the assay calculation for your <redacted>. Your procedures lacked guidance on how to check and manually verify the calculation sheets. During the inspection, our investigator identified a calculation error within the spreadsheet. The incorrect formula for averaging the Internal Standard peak area was used (1).

If you really want to descend into the abyss of compliance hell, let an inspector find an error in a spreadsheet. You will receive a comment like this: “There is no assurance that the associated assay results recorded are reliable and accurate.”

It is not the best situation having an inspector that thinks that all of your data are garbage. Also, if there is one calculation problem in unvalidated spreadsheets, then there are bound to be more, because there is a total lack of control when using spreadsheets.

During the review, you identified another error within your spreadsheets. The assay test result for <redacted> batch <redacted> was incorrect due to a transcription entry error for active peak area. Your firm used a new spreadsheet and entered the correct active peak area. The result was recalculated, and the final result was reported. The product had already been released with test results using the incorrect calculation although the recalculated test result was still within specification. You have committed to manually check calculations until the spreadsheet has been validated (1).

In the last sentence, we see a way to resolve the problem: You should manually check all calculations until the spreadsheet is validated. How useful are the unvalidated spreadsheets now? Read the full warning letter to see the extent of the remediation required by FDA (1). However, the approach taken by the company to manually check calculations (which is error prone in itself) misses the point about spreadsheets: They are a hybrid system that has major business process efficiency issues as well as regulatory compliance problems, as we shall see later in this column.

Here goes a list of the spreadsheet screw-ups. That well-known pharmaceutical company, Enron Corporation, used spreadsheets for a number of activities, and after the company went bankrupt, more than 15,000 spreadsheets were analyzed from the archive of 65,000 e-mails by two academics, Felienne Hermans and Emerson Murphy-Hill (3). Their main conclusions were:

Out of all the spreadsheets used, 76% of them used the same 15 functions.

In regards to spreadsheets with at least one formula, 24% of them contained an error.

Spreadsheets were a frequent topic of e-mail conversation with 10% of emails either referring to or sending spreadsheets and frequently discussing errors in and updates to spreadsheets.

Just like in your laboratory! In fact, if spreadsheets are well embedded into your culture, a spreadsheet developed in the morning could be the department standard by the afternoon. If so, I’m assuming you are steering a course for Scylla rather than Charybdis.

In a study that had a major impact on governments, two eminent economists, Carmen Reinhart and Kenneth Rogoff, published an article “Growth in a Time of Debt

which concluded that counties with a high amount of debt to Gross Domestic Product (GDP) (>90%) have slow economic growth. This resulted in austere policies to reduce debt and simulate growth in some countries, including the United Kingdom. Unfortunately for the two intrepid economists, they published in a non-peer reviewed part of a journal and used a spreadsheet to perform their calculations. And guess what happened? There were a few problems with the spreadsheet and the major conclusion was reversed (see Wikipedia for more details).

If you really want to understand the scope of spreadsheet disasters, take a look at Ray Panko’s website (www.panko.com). Panko is a University of Hawaii academic who has studied spreadsheets and errors associated with them for about 25 years. On his website, there is a page on spreadsheet errors that states the following (4):

Humans are very accurate when we work. When we create a spreadsheet formula, we are correct 95 to 99% of the time.

Unfortunately, we are not as good at finding errors. When we look diligently for errors, we only find 40 to 90%, and finding 90% is rare.

We would like to know how effective we are at finding errors when we inspect a spreadsheet. This is not just an “academic” concern. If our spreadsheets have as many errors as they seem to have, then we must test them more. One way to do this is to inspect them cell by cell. Many people believe that if we are careful enough, and if we spend enough time, we can find all of the errors in a spreadsheet. However, this ability is not realistic. If detection rates are too low, then a team inspection is necessary to catch 80 to 90% of the errors (4).

The inability to find and resolve spreadsheet errors is a worrying problem. How should we resolve it? The obvious one is that more resources need to be put into specifying the spreadsheet and how the formulas are built and tested. As Panko’s website suggests, more effort should be placed in checking the formulas. However, there is a simpler way, which is explained later on in this article.

In a section guaranteed to ensure that many readers will want to stick large pins into my effigy, I am advocating that spreadsheets be exterminated from use in a regulatory laboratory. This is not because a spreadsheet application is bad per se, but in a regulated environment it is a recipe for potential disaster, for many reasons, as outlined in Table I.

I’m not interested in your personal life, but look at your business process in the laboratory when you are using a spreadsheet. Figure 1 shows the tasks of a general quantitative analytical process in the middle in green. If you use a spreadsheet, you’ll follow the left hand side of Figure 1: Prepare the sample, analyze it along with standards, interpret the data in the spectrometer data system, print the results, manually type data into your (hopefully) validated spreadsheet, print the results out, and then type the result into a laboratory information management system (LIMS) or similar informatics application. But just look at the mess of the process and the number of records that you have created. You are a laboratory masochist.

Job done? No, enter the poor sap who is the second person reviewer. Just look at the mess this person has to review: the number of transfers between computerized systems, and the need to check for transcription errors each time data are entered manually into a different computerized system with checks of both electronic records and paper printouts. Welcome to death by compliance: transcription error checking by the reviewer, which is also an error-prone process. If you are really unlucky, the second person review takes longer than the analysis (10)!

Guess what? Instead of the mess on the left-hand side of Figure 1, many calculations you have spent time incorporating into a spreadsheet can be done by the instrument data system—except few people bother to read the manual. You can avoid the need to print and manually enter data into a spreadsheet followed by the consequential transcription error checks. Take the time to specify and validate the calculations, and you can save much time and effort in performing the analysis and the subsequent second person review. Look at the right-hand side of Figure 1 and compare with the left-hand side and immediately you’ll see my point. You only have the sample preparation records, the manual input of data into the data system, and the electronic records in the data system. Add electronic signatures and your process is sped up immeasurably, and you only have a single location to check records. Why would you want to do anything differently?

As mentioned earlier, let us discuss two of the main problems with spreadsheets in more detail, which are hybrid systems and single accounts and passwords.

A spreadsheet macro or template is a hybrid system—an electronic record of the saved and completed file with a hand-signed paper printout. Poor design means that data are typically input manually, and there is a large transcription error checking burden placed on both the analyst and the second person reviewer. One typical omission in laboratories I have audited is the lack of record signature linking between the paper printout and the electronic record that generated it. It is not an option to state that the paper is the GXP record; the spreadsheet file is a key component of the record set of any analysis. Please read question 12 of the FDA data integrity guidance to understand what is required:

12. When does electronic data become a CGMP record?

When generated to satisfy a CGMP requirement, all data become a CGMP record. You must document, or save, the data at the time of performance to create a record in compliance with CGMP requirements, including, but not limited to, §§ 211.100(b) and 211.160(a) (11).

This is very easy to understand and follow: Save the spreadsheet file or book an appointment with Scylla.

One of the issues with spreadsheet security is that there is only a single account and password for a template. Once the developer has locked the spreadsheet (this can be individual worksheets as well as the whole template) with one or more passwords, what are you going to do? This is one case when you will write the password down and follow the steps below:

The developer needs to write the spreadsheet name and version along with the passwords, and place the paper into an opaque envelope, write the spreadsheet and version name on the front, seal the envelope, then sign and date over the seal. This is to indicate if the envelope has been opened.

The envelope should be placed in a secure location, typically a fireproof safe, until the spreadsheet needs to be updated.

The envelope is unsealed, the spreadsheet maintained and revalidated, and the new passwords start the process over again.

If you find this a bit of a pain, then get rid of the spreadsheet.

Having spent all this time saying that you should eliminate spreadsheets, I must acknowledge that there are some exceptions to this rule. There are situations where calculations across analyses cannot be performed by instrument data systems and, in the absence of a LIMS, a spreadsheet may be the only option for calculating results. However, do you want to input data manually, and what about tracking changes to data? Ideally, a validated spreadsheet should be used within an electronic lab oratory notebook (ELN) or equivalent environment. Here, data should be imported and loaded electronically with a validated process to eliminate transcription error checking, and any changes are monitored by the audit trail of the informatics application. Calculated results should be transferred electronically to the next part of the process for speed and to eliminate transcription error checking.

When it comes to persuading laboratories to use alternative ways of calculating other than spreadsheets, I feel like King Cnut, an 11th Century King of England and Denmark, who commanded the sea to retreat. This was an obvious exercise in abject failure. However, never mind regulatory risk. Look at your business processes and see what they look like; it may not be a pretty sight. Incorporate calculations into instrument data systems or use informatics applications such as ELN or LIMS to move away from spreadsheets into an electronic way of working. Slaughter spreadsheets, please. You know it makes sense.

Warning Letter Tismore Health and Wellness Pty Limited

, Part 211 (FDA, Silver Spring, Maryland, 2008).

F. Hermans and E. Murphy-Hill, “Enron’s Spreadsheets and Related Emails: A Dataset and Analysis,” paper presented at the IEEE/ACM 37th IEEE International Conference on Software Engineering, Florence, Italy, 2015.

Good Automated Manufacturing Practice (GAMP) Guide

 (International Society for Pharmaceutical Engineering, Tampa, Florida, 2008).

WHO Technical Report Series No.996 Annex 5 Guidance on Good Data and Records Management Practices (World Health Organisation: Geneva, Switzerland, 2016).

, Part 21.11 (Food and Drug Administration, Washington, DC, 1997).

European Commission Health and Consumers Directorate-General, 

The Rules Governing Medicinal Porudtcs in the European Union

Good Manufacturing Practice (GMP) Guidelines, Annex 11: Computerised Systems 

Guidance for Industry Data Integrity and Compliance With Drug CGMP Questions and Answers

 is the director of R.D. McDowall Limited and the editor of the “Questions of Quality” column for LCGC Europe, Spectroscopy’s sister magazine. Direct correspondence to: 

Spreadsheets are often used to perform GMP-related calculations, but can lead to serious problems and unnecessary risk. We explain why the use of spreadsheets is heavily discouraged in a regulated laboratory environment.

Spreadsheets: A Sound Foundation for a Lack of Data Integrity?

Our final look at the infrared spectra of organic nitrogen functional groups will be of the nitro, or NO

, group. This group has two strong and characteristic infrared features, making it easy to identify. However, attaching a NO

 group to a molecule scrambles its electronic structure, which is why these compounds are explosive, and why nitro groups make some of the infrared interpretation rules we learned irrelevant.

Over the several years that I have been writing these articles, there have been large sections of material devoted to the spectroscopy of functional groups that contain specific chemical bonds. When I began discussing organics containing nitrogen, I stated that the stretching and bending of N-H bonds would be important in determining whether or not there was nitrogen present in a molecule (1). We subsequently used N-H stretching and bending peaks to analyze the spectra of amines (2,3), amides (4,5), imides (6), and urethanes (7).

The nitro group is going to be the odd man out in this discussion. This functional group consists of a nitrogen atom with no hydrogens, but with two oxygens and a carbon attached, as seen in Figure 1.

, and that the carbon atom singly bonded to the nitro nitrogen is called the 

. Depending on whether the alpha carbon is saturated, or part of an aromatic ring, nitro molecules can be divided into saturated and aromatic nitro compounds.

 group is unusual. Normally, oxygen atoms form two chemical bonds (8). However, there is also a C-N bond in the nitro group, as seen in Figure 1. Given that nitrogen normally forms three bonds (8), how do we apportion the electrons in a nitro group to keep these compounds from falling apart?

There are three bonding electrons shared between the two oxygens in the nitro group, giving what are essentially two “bonds and a half,” as seen in Figure 1. The dashed lines in Figure 1 represent the half bonds. These NO bonds are similar to the carboxylate C-O bonds and a half discussed in a previous column (9). Neither oxygen atom has its full complement of two full chemical bonds, making the nitro groups unstable. Saturated nitro compounds such as nitroalkanes, otherwise known as rocket fuel (8), are rarely analyzed by infrared spectroscopy because they are liable to detonate during analysis. We will not study them further.

However, nitro groups attached to benzene rings can be relatively stable, assuming not too many nitro groups are attached. The chemical structure of trinitrotoluene, a commonly used explosive known as 

The presence of the three nitro groups destabilizes the benzene ring, resulting in TNT’s explosive properties. Di-nitrotoluene and mono-nitrotoluene are stable. We will restrict our discussion to non-explosive aromatic nitro compounds.

The Infrared Spectroscopy of the Nitro Functional Group

The infrared spectrum of an aromatic nitro compound, meta-nitrotoluene, is seen in Figure 3. Recall that highly polar bonds have intense infrared features due to the large change in dipole moment with respect to bond length, 

, during a vibration (10). Oxygen is more electronegative than nitrogen; therefore, the N-O bonds in the nitro group are relatively polar, and as a result, their asymmetric and symmetric stretching peaks are unusually large. The details of these vibrations are shown in Figure 4.

 stretch typically falls from 1550 to 1500 cm

, and is seen in Figure 3 labeled A at 1527 cm

 units, even if not labeled as such). The symmetric stretch is seen in Figure 3 labeled B at 1350 cm

, and, in general, this peak appears from 1390 cm

. Note how peaks A and B in Figure 3 are the two most intense peaks in the spectrum, and they stick down like eye teeth in the middle of the spectrum. The combination of a pair of intense peaks in these wavenumber ranges is unique, making the presence of a nitro group in a sample easy to spot.

The nitro group also exhibits a scissors bending vibration, similar to that of the methylene group (11). This gives rise to a medium intensity peak from 890 cm

. It can be seen in Figure 3 labeled C at 881 cm

The good news about the nitro group is that it has two strong infrared bands that are easy to spot. The bad news is that whenever the nitro group is attached to a benzene ring, it makes it difficult to determine the substitution pattern on the benzene ring. Recall that the benzene ring out-of-plane C-H bend band, in combination with the presence or absence of the aromatic ring-bending band at 690 cm

, can be used to determine the substitution pattern on a benzene ring (12). The presence of a nitro group makes it difficult to apply these rules. This is caused by the unique electronic structure of the nitro group, and how it interacts electronically with the benzene ring. Suffice it to say, one may need to use an analytical technique other than infrared spectroscopy to determine the substitution pattern on nitro-substituted aromatic rings.

Note that the structure of meta-nitrotoluene does contain a methyl group. We have learned that the diagnostic pattern for a methyl group includes saturated carbon asymmetric and symmetric stretches near 2962 cm

 (13,14). Note in Figure 3 that the saturated C-H stretches fall at 2926 cm

. Under normal circumstances, we would interpret these two peaks as the asymmetric and symmetric stretches of methylene groups, because they have peaks at 2926 cm

 (14). In this case, that interpretation is wrong, because the nitro groups scramble the electronic structure of the molecule, throwing off these peak positions. If you notice the nitro peaks first, it will give you a heads up that the saturated C-H stretches may be troublesome. Normally we can depend on the methyl group umbrella mode to indicate that the saturated C-H stretches could be an issue. However, in this case, the intense nitro symmetric stretching peak at 1350 cm

 is sitting on top of it. Unfortunately, then there is nothing in this spectrum clearly showing the presence of the methyl group. As stated above, the nitro group throws off some of the interpretation rules we have learned.

The diagnostic pattern for the presence of a nitro group in a sample then is a pair of intense peaks at about 1550 cm

, along with the scissor peak around 850 cm

. Table I summarizes the group wavenumbers for the nitro group.

The nitro group consists of a nitrogen atom with two oxygens and one carbon attached. The two nitrogen-oxygen bonds are “bonds and half” that destabilize nitro compounds, making their analysis an explosive proposition. The large dipole moments for the NO bonds give two strong peaks around 1550 cm

 as a result of the asymmetric and symmetric stretching of the NO

functional group. This is an unusual pattern, and is easy to spot. There is also a scissoring peak around 850 cm

. Nitro groups tend to scramble the electronic structure of molecules, making the interpretation of their spectra problematic.

Infrared Spectral Interpretation: A Systematic Approach

Your Next Infrared Spectral Interpretation Challenge

Using everything you have learned in this and previous columns, determine the functional groups present in the Figure i spectrum and try to determine the chemical structure of this compound. Remember that inclusion of a peak position in the table does not necessarily mean that it will be useful in the structure determination.

Because this is a particularly tricky problem, answer these questions about the spectrum in this order to guide you. In all cases, be sure to justify your answer.

2. If so, is there a benzene ring present?

3. Are there any other functional groups present?

is the founder and CEO of Big Sur Scientific, a maker of portable mid-infrared cannabis analyzers. He has over 30 years experience as an industrial infrared spectroscopist, has published numerous peer-reviewed papers, and has written three books on spectroscopy. As a trainer, he has helped thousands of people around the world improve their infrared analyses. In addition to writing for 

 Dr. Smith writes a regular column for its sister publication

and sits on its editorial board. He earned his PhD in physical chemistry from Dartmouth College. He can be reached at: 

We review one final organic nitrogen functional group, representing explosive compounds. This is the nitro group, which requires a different set of interpretation rules.

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