From a Non-Majors Course to Undergraduate Research: Integration of

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Chapter 10

From a Non-Majors Course to Undergraduate Research: Integration of NMR Spectroscopy across the Organic Chemistry Curriculum at Ashland University Perry S. Corbin* and Robert G. Bergosh Ashland University, 401 College Ave., Ashland, Ohio 44805, United States *E-mail: [email protected]

In recent years, strides have been made to enhance the coverage of NMR spectroscopy within the chemistry curriculum at Ashland University. This chapter focuses on the use of NMR in two varied settings: within a course for students with majors outside of the natural sciences and within the two-semester, organic chemistry laboratory sequence for science majors. Spectral interpretation and acquisition in the non-majors course and the introduction of 2D-NMR spectroscopy (COSY, HMQC, and HMBC) within the organic chemistry lab courses will be described in detail. In addition, the use of NMR spectroscopy in organic chemistry research is briefly outlined.

Introduction Ashland University is a private institution located in northeast Ohio that serves an undergraduate student population of approximately 2200 students. In addition to an undergraduate major in chemistry, the Department offers programs in biochemistry, forensic chemistry, and chemistry education. Heavy emphasis is placed on student/faculty interactions, data analysis, and hands-on training with instrumentation in all chemistry courses and research opportunities at Ashland. As a result, faculty are continually investigating ways to better incorporate the use of NMR spectroscopy and spectral interpretation throughout its curriculum. This effort is part of a broader goal to promote active learning and to enhance the scientific reasoning skills of students. © 2016 American Chemical Society Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

As suggested in the abstract, the use of NMR in a non-majors course, Molecular Architecture, and within the two-semester organic chemistry sequence taken by science majors will be the focus of this chapter. Further use of the spectrometer in undergraduate, organic chemistry research is discussed. The activities described are readily adaptable and illustrate the effective incorporation of NMR spectroscopy into the educational experiences of a variety of students, ranging from non-science majors to advanced undergraduate research students.


NMR Spectrometer Description Over a period from 1998-2009, a continuous wave, 60 MHz NMR spectrometer equipped with an FT NMR upgrade served the department well for curricular uses. However, with a desire to expand undergraduate research opportunities and to better facilitate coverage of NMR spectroscopy within its curriculum, a JEOL ECS 400 MHz spectrometer was purchased in 2010. When selecting an instrument, the authors were mindful of the extensive use the instrument would have in both courses and research. In addition to standard probes, the instrument is equipped with an autotuning unit, a chiller unit for facile variable temperature work, and an invaluable 24-position sample changer. These features along with an acquisition software that facilitates automated gradient shimming and allows spectra to be acquired with either full control of parameters or fully automated “point and click” operation, have allowed the instrument to be used in a variety of settings. Practical aspects of the spectrometer’s use in organic chemistry courses are included in subsequent sections, in addition to descriptions of specific problem-solving activities and the authors’ pedagogical approach to teaching NMR spectroscopy.

A Non-Majors Course Focusing on Spectroscopy Molecular Architecture—Spectroscopy and Its Role in a Liberal Arts Core Curriculum Chemistry faculty at Ashland have a long-standing commitment to develop and deliver an innovative curriculum for non-science majors that serves a vital role in the liberal arts core curriculum of the University. Beginning around the year 2000, a concerted effort was undertaken to shift from traditional survey courses for non-science majors to courses that were more focused in content. In turn, faculty sought to develop courses that would provide in-depth problem-solving opportunities and direct insight into the process of science, as well as facilitate the development of critical thinking and communication skills. Chemistry of Crime Scene Investigation, for instance, relates real world applications of both qualitative and quantitative chemical analysis to the investigation of crimes (1). Lead and Civilization examines the role lead has played in the history of civilization, with emphasis on how the uses and toxicity of this metal are related to its chemical properties. A principle laboratory component of the course provides students with direct experience in instrumental quantitative analysis (2). Chemical Perspectives on Life has a biochemistry 162

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focus that examines the structure and function of organisms from a chemical perspective. Energy, Matter, and Change explores the role of chemistry in the production, storage, and utilization of energy. In this course, the underlying chemical principals of batteries, fuels, and nutrition are examined in a hands-on setting (3). Finally, Molecular Architecture is a course that has NMR spectroscopy as a central focus. Within the course, organic chemistry is explored by seeking answers to three specific questions: 1) What is a molecule? 2) How are molecules constructed? and 3) How are molecules characterized? Answers are sought by investigating molecules that are either encountered in daily life or are, in part, critical for sustaining life (4). With regards to specific content, Molecular Architecture examines models of molecular structure, basic ideas of chemical synthesis, molecular shape, chirality, and intermolecular forces. The course ends by examining the relationship between molecular structure and material or biochemical properties (e.g., by investigating polymers, medicines, etc.). The anchoring, primary content of the course though is spectroscopy and its use in structural elucidation. One might ask: Why offer a course for non-science majors that focuses on spectroscopy, and, in particular, NMR spectroscopy? The authors would like to suggest that such investigations are ideal and aid students in developing problem-solving skills that are often not fostered in traditional introductory science courses that have broad and unfocused content coverage. By studying NMR spectroscopy students also receive direct exposure to a critical technique that is used by practicing chemists; and, thus, students are invited to step into the operational world of a “real scientist.”Most importantly, experience is provided in the collection of scientific data, as well as the interpretation of data and its subsequent use in modeling the unseen—in this case, molecular structure. Students are exposed to both the power and limitations of scientific data. Such an experience is valuable for a general audience that is, at times, leery of technical data and its interpretation. Examination of Spectroscopy within Molecular Architecture Notably, Molecular Architecture does not use a standard textbook, but instead relies on brief instructor lectures and discussion to introduce topics. A compilation of essays by Roald Hoffman (The Same and Not the Same) (5) has also been used to present basic ideas of chemistry to the non-scientist audience of the course. Individual and cooperative problem-solving activities, including laboratory investigations, allow students to learn in a hands-on manner. Face-to-face sessions of the course are typically taught in a laboratory equipped with instructional technology and requisite seating to allow efficient movement from lecture and discussion to group problem solving, laboratory activities, and instrument use. Adaptations for a hybrid (partially online) and “flipped” learning environment are discussed in a subsequent section. Prior to the study of spectroscopy within Molecular Architecture, students are introduced to simple, yet fundamental, ideas of chemical bonding within organic molecules and become comfortable with various symbolic representations of 163



molecular structure, including the use of hand-held models. Students are also introduced to the idea of structural (constitutional) isomerism and functional groups early within the course. As a step towards exploring NMR spectroscopy, IR spectroscopy is examined first. As is the case with NMR, the focus of this study is data interpretation. Students are, however, introduced to key ideas concerning electromagnetic radiation and its interaction with matter—especially the concept that light of varying energy causes disparate changes within a molecule that can be linked to an associated molecular structure. A brief experiment using visible spectroscopy to study a series of organic dyes is often carried out prior to more in-depth IR and NMR spectroscopic analysis. This investigation provides students with, perhaps, a less abstract example of light interacting with matter and allows students to directly observe the result (color) of visible light interacting with and being absorbed by highly conjugated molecules. Although theoretical descriptions of IR and NMR spectroscopy are simple, students are made well aware that signals in IR and NMR spectra arise from bond vibrational enhancements and nuclear changes (aided by a magnetic environment) that are induced, respectively, by varied frequencies of impinged infrared and radio waves.

Structural Isomerism, IR Spectroscopy, and NMR Spectroscopy To link spectroscopic analysis with previous discussions of structural isomerism, students are first provided IR spectra of molecules that have multiple constitutional isomers. They are, likewise, provided the corresponding formulas of the molecules represented by the spectra. Upon examining these spectra and identifying constituent bonds, students are asked to draw possible structures that fit the IR data and formulas, as well as structures that do not fit a given spectrum. As such, the power of IR spectroscopy in revealing functional groups is quickly realized, but its limitation in distinguishing isomers that bear identical functional groups is also exposed. This recognition serves as a segue into 13C-NMR spectroscopy. A study of constitutional isomers with a molecular formula C4H10O (see Figure 1) is one example that has been effectively used in combined lecture/discussion and in-class problem solving to transition between the topics of isomerism, IR spectroscopy, and 13C NMR spectroscopy (6, 7). Based upon the presence or absence of an –OH stretch within an individual IR spectrum of a molecule with a formula of C4H10O, a student can classify the given compound as one of four possible alcohols or three ethers shown in Figure 1. Of course, the exact structure cannot be determined solely by IR spectroscopy. Thus, 13C NMR spectroscopy is introduced, with a primary focus on examining the structure of a molecule, with the aid of molecular models as needed, and predicting its corresponding number of equivalent carbons. Students are also introduced to the concept of a chemical shift and are provided a chart that summarizes the expected chemical shift ranges for carbons in different environments. Using this basic knowledge of carbon equivalency and chemical shifts, students predict, for example, what the individual spectra (number of signals 164



and expected chemical shift ranges) of the three ether isomers in Figure 1 would look like. Upon comparing these predictions to actual spectra, the three isomers can be distinguished based upon the unique number of signals observed in the proton-decoupled 13C NMR spectra. When making similar predictions and comparisons for the alcohol isomers, it becomes apparent that isobutyl alcohol and tert-butyl alcohol can be readily distinguished from n-butyl alcohol and sec-butyl alcohol because of their differing number of unique carbons. However, the latter two compounds cannot be readily differentiated by their 13C NMR spectra because they have the same number of signals in similar chemical shift ranges. This observation allows a transition in discussion from 13C to 1H NMR spectroscopy.

Figure 1. Differentiation of the isomers of C4H10O using IR, 13C NMR, and 1H NMR spectroscopy. 1H NMR spectroscopic investigation within Molecular Architecture, again, focuses, on predicting the number of expected signals for a given molecule, along with the anticipated chemical shift ranges. Simple signal splitting is also introduced, while signal integration is not directly addressed. Integration is instead introduced on a case-by-case basis in the course’s spectroscopy unknown experiment (vide infra). It should be noted that the majority of molecules analyzed have flexible alkyl chains and, thus, coupling constants for non-equivalent

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neighboring hydrogens that are essentially identical (8). As a result, complex splitting is not typically observed in spectra that are analyzed. Returning to the example in Figure 1, although n-butyl and sec-butyl alcohol cannot be readily distinguished by their IR and proton-decoupled 13C NMR spectra, the compounds can be distinguished by first predicting the expected number of signals, the corresponding chemical shift ranges, and signal splitting that would be observed in the 1H NMR spectra of the molecules. Subsequent comparison of the predictions to the actual spectra allows the two alcohols to be identified. For instance, the 1H NMR spectrum (in CDCl3) of sec-butyl alcohol includes a doublet corresponding to the methyl group closest to the alcohol functional group, but the spectrum of n-butyl alcohol does not contain any doublets. Students might also recognize that the signal splitting for the hydrogen(s) adjacent to the alcohol functional group in each molecule is distinct—an observed triplet for the CH2OH hydrogens in n-butyl alcohol, and an apparent sextet for the methine hydrogen in sec-butyl alcohol. The authors have found that a systematic introduction to IR, 13C, and 1H NMR spectroscopy using examples such as the one outlined, in a combined lecture/ cooperative-learning environment, allows students to cultivate problem-solving skills that are necessary for examining spectral unknowns. A description of an experiment of this type is described in the next section.

Spectral Unknown Experiment The highlight of spectral examination in Molecular Architecture is an experiment in which groups of students (two to three per group) are provided an unknown liquid and a list of approximately 40 potential compounds. IR, 13C, and 1H NMR spectral data are collected to enable the identification of the unknown. During the time in which data is being collected, students make predictions concerning the number of equivalent carbons and hydrogens within each of the possible compounds on the candidate list. The groups then obtain an IR spectrum and are required to narrow their list of possibilities based upon the IR data, prior to examining the molecule’s NMR spectra. Several articles have been published that include suggestions for spectroscopy unknowns (9, 10). The molecules used in Molecular Architecture are thoughtfully selected, as are additional distractor molecules included on the candidate list. In short, the various unknowns and candidates contain differing functional groups. As a result, the possible identity of the unknown can be narrowed by using the compound’s IR spectrum. Analysis of a proton-decoupled 13C NMR spectrum then allows the probable identity to be narrowed further. Organic compounds are, however, chosen that require a reasonably detailed examination of the 1H NMR spectrum for proper identification. Two molecules that have been successfully used as unknowns are isopentyl acetate and butyl acetate. When using these molecules, a related compound, propyl propionate, is added to the unknown candidate list as a distractor, along with several other esters. The structures of isopentyl acetate, butyl acetate, and propyl propionate are not readily distinguished by their IR or proton-decoupled 166



13C

NMR spectra. Nonetheless, IR and 13C NMR spectroscopy allows the three molecules to be differentiated from compounds on the candidate list that contain functional groups other than an ester, as well as from esters on the candidate list that do not have six unique carbons. Thus, students must use 1H NMR spectroscopy to distinguish the three esters. In the case at hand, students can distinguish isopentyl acetate, butyl acetate and propyl propionate by first carefully predicting the expected splitting and corresponding chemical shift range for each unique type of hydrogen within the three molecules. The prediction and observation of a singlet in the unknown spectrum then allows the two acetates to be distinguished from propyl propionate. Subsequent prediction and observation of a doublet in the spectrum of the unknown, for instance, provides clear indication that isopentyl acetate is the unknown; whereas, the lack of a doublet reflects the structure of butyl acetate (see Figure 2). After determining the identity of the unknown, subsequent assignment of all signals can be readily made.

Figure 2. 1H NMR spectra of isopentyl acetate and butyl acetate in CDCl3. Another compound that has been successfully used as an unknown in the structural determination activity is diethyl malonate. In addition to the esters mentioned within the previous paragraph, ethyl acetate is also a distractor on the candidate list. The combination of diethyl malonate as an unknown and ethyl acetate as a potential candidate is intentional because the two molecules are virtually indistinguishable by IR and 13C NMR spectroscopy. Despite subtle differences in the 1H NMR chemical shifts and signal areas for the hydrogens in the alpha position relative to the carbonyl group(s) in the two molecules, the two esters are not easily distinguished by 1H NMR spectroscopy either. Given this minimal contrast (see Table 1), groups that have diethyl malonate as an unknown are introduced to signal integration and/or provided a mass spectrum 167 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

(mass spectrometry is covered briefly within the course) to aid in determining the compound’s identity. However, prior to being provided this additional information, students must have narrowed their list of possibilities to diethyl malonate or ethyl acetate.


Table 1.

13C

and 1H NMR Chemical Shift Assignments for Diethyl Malonate and Ethyl Acetate (spectra obtained in CDCl3)

In addition to a spectral unknown that is from a list of possible compounds, each student group is also provided data and the corresponding molecular formula for a second unknown that is not found on a candidate list. For this unknown, the authors obtain and distribute IR, 13C NMR, 1H NMR, and mass spectral data from the SDBS structural database (organized by the National Institute of Advanced Industrial Science and Technology, Japan) and the NIST Chemistry WebBook (11, 12). As an alternative, students may be provided simulated NMR spectra that are generated in ChemDraw or related structural drawing programs via chemical shift increment calculations and signal splitting predictions.

Writing Assignments Involving NMR Spectroscopy The completion of the two unknown structural determination activities in Molecular Architecture typically culminates in individual students writing detailed papers (10-12 pages) that have as a primary focus the description of the logic behind the structural determinations. Students are also required to present their data in tables, as well as within figures. For the non-science majors who take the course, this assignment is the first occasion in which many of them have been required to use data, text, tables, and figures in concert to coherently explain their reasoning in solving a problem, as opposed to simply stating an answer. Although tasks of this nature are a central component of scientific investigations and are frequently emphasized in courses for science majors, valuable writing experiences 168 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


of this type are, unfortunately, absent in many traditional chemistry courses for non-science majors. One additional opportunity to address NMR spectroscopy in writing is afforded in the final assignment for the course. Throughout the semester students are asked to research various aspects of a molecule of interest—e.g., a natural product, medicine, etc.—and then write a paper and present their research orally. As part of this assignment, students predict the number of expected 1H and 13C NMR spectral signals and corresponding chemical shift ranges for the molecule they have studied. For less complex molecules, students may also be asked to interpret spectra that have either been simulated or found within a spectral database or the chemical literature. Practical Issues, Adaptation, and Effectiveness of the Course In regards to practical issues concerning the collection of NMR spectra by non-science majors, the autosampler and automated acquisition mode of the instrument software, along with automated gradient shimming have allowed use of the NMR spectrometer in Molecular Architecture to be both safe and efficient. Students typically prepare their own samples, are introduced to the instrumentation in small groups, and then queue their samples via “single button” automation. Samples typically run during the course of a class period, and instructors workup the resulting data prior to the next class session. Multiple class sections (two to seven) have been offered each semester for the past several years and are easily accommodated. It should be pointed out that Molecular Architecture was offered prior to the acquisition of a high-field NMR spectrometer. Although having a high-field instrument in house has greatly facilitated coverage of NMR within the course, due to ease of instrument use and diminished time needed to acquire spectra, Molecular Architecture was effectively offered using the department’s FT-NMR enhanced 60 MHz instrument, along with spectra obtained from freely available databases and from ChemDraw simulation. The coverage of spectroscopy, along with the remaining content of the course, has been adapted for a variety of settings including a traditional three-hour course, a once-per-week evening course, a six week summer course, a course within the Ashland University’s Honors’ Program, a course in the Ashland University in Germany program, and as part of a week-long summer intensive course for gifted high school students. Readers should also note that Molecular Architecture and, in particular, a treatment of IR and NMR spectroscopy as described is fully appropriate for use in a “flipped” classroom and in a hybrid, partially online environment. Recorded video lectures are used for courses with these alternative delivery formats to introduce the theoretical aspects of spectroscopy and to provide guidance in interpreting spectra in a manner that is analogous to that used in the traditional, face-to-face sections. Likewise, spectra and spectral interpretation activities are presented in a digital format that is accessed through the course learning management system. Students in the hybrid course are required to participate in several lab activities, including spectral data collection, on campus. 169 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


NMR spectroscopy is an excellent candidate for digital activities that utilize the various question types (multiple choice, multiple select, fill in the blank, etc.) that are available for creating assignments in most learning management systems. For example, when asking questions that require students to predict the expected number of signals for a molecule in a 13C NMR spectrum, fill in the blank type responses work very well, because there is only one correct numeric answer for a question. As another example, when predicting the splitting of signals in a 1H NMR spectrum, a labeled structure is presented, and the multiplicity for a given hydrogen type is selected from a multiple-choice list. In most of these initial learning activities, students are allowed multiple attempts to arrive at the correct answer. For the non-majors who are taking the course, this limited mastery style is critical for building confidence and understanding of the material, yet imposing a sense of accountability. Response to the course has been very positive. Many students enjoy the “puzzle-solving” nature of structural elucidation, and as one student responded in a course evaluation: “It (the course) allowed me to learn science in a new and different way.”In a way that the authors would suggest truly promotes the development of scientific reasoning skills within an important audience.

NMR Spectroscopy in the Introductory Organic Chemistry Course Sequence for Science Majors Although chemistry students at Ashland University are briefly introduced to NMR spectroscopy as part of a concluding organic unknown identification experiment in General Chemistry I lab, science majors undertake their first in-depth study of NMR spectroscopy within the two-semester organic chemistry sequence. The investigation of NMR (introductory theory, development of data interpretation skills, and instrument use) within Organic Chemistry I and II is carried out primarily within the laboratory portion of the courses, which convenes for three hours each week. Coverage in this manner allows not only lecture and discussion, but also permits group problem solving (similar to that described for Molecular Architecture) and instrument use within the same class period and in a workshop-like setting. Prior to investigating NMR, IR spectroscopy is introduced in week three of Organic Chemistry I lab. An introductory 1H and 13C NMR spectroscopy workshop then follows in week nine of the course. This initial introduction focuses on NMR spectroscopic theory, along with spectral prediction activities—i.e., determining the number of signals and corresponding chemical shift ranges expected in the 1H and 13C NMR spectrum of a molecule, along with the predicted splitting of signals in the 1H NMR spectrum. A follow-up problem-solving session is then held in week 12 that focuses on the analysis of unknown compounds by 1H and 13C NMR spectroscopy. An introduction to 2D NMR spectroscopy (COSY and HMQC) is also included during week 12 and culminates with the characterization of the product from a Fisher esterification reaction. Use of COSY (correlation spectroscopy) and HMQC (heteronuclear multiple quantum coherence) spectroscopy to characterize reaction products then extends 13C


throughout the second-semester lab course. HMBC (heteronuclear multiple bond coherence) spectroscopy is also introduced in Organic Chemistry II. The incorporation and corresponding utility of 2D NMR spectroscopy within the introductory organic chemistry sequence is described in subsequent sections.


Introduction to 2D NMR Spectroscopy in Organic Chemistry I Upon first encounter, many students in organic courses struggle with interpreting 1H and 13C NMR spectra. Combating this struggle has been a long-standing goal of the authors. Indeed, it may seem counterintuitive to introduce COSY and HMQC spectroscopy shortly after a student’s initial encounter with NMR. However, it has been the experience of the authors that early exposure to 2D NMR spectroscopy actually helps students to develop the skills and intuition needed to interpret 1D spectra. Theoretical aspects associated with 2D NMR spectroscopic techniques are beyond the scope of the authors’ courses and are not discussed in detail. To provide students general guidance in interpreting COSY and HMQC spectra in Organic Chemistry I lab, simple examples are initially used in a lecture/discussion format. For example, the spectra of dibutyl ether are examined in detail (see Figures 3 and 4). Prior to analyzing the COSY and HMQC spectra of dibutyl ether, students are required to predict what the 1H and 13C NMR spectra for the molecule should look like. Students are then instructed to make tentative assignments of signals by comparing their predictions to the actual 1D spectra. Next, a COSY spectrum of dibutyl ether (see Figure 3) is provided. Instructions for appropriately labeling the spectrum are given, and students are shown that the signals for coupled protons along the spectral diagonal are connected via cross peaks. Specifically, the coupling of hydrogens four to three, three to two, and two to one are revealed in the COSY spectrum of dibutyl ether. Likewise, students are presented an HMQC spectrum of dibutyl ether (Figure 4), which reveals the corresponding one bond proton-carbon coupling pairs within the molecule. For example, the connection of hydrogens one to carbon one, hydrogens two to carbon two, etc., is revealed. For this introductory example, the assignment of carbon signals that are made using the HMQC spectrum typically match those that students have predicted based upon the relative position of the various carbons with respect to the electronegative oxygen in dibutyl ether. After additional introduction of COSY and HMQC spectra using ethyl benzene as an example, students are provided NMR spectra of 2-heptanone and work in small groups to assign all signals in the molecule’s 1H and 13C NMR spectra. As previously reported by Alonso and Warren (13), the COSY and HMQC spectra (HETCOR was used in Reference (13) instead of HMQC) of 2-heptanone and related ketones are especially useful in illustrating the function of the two techniques (see Figures 5 and 7). In particular, the assignment of overlapped signals for hydrogens five and six in the 1H NMR is facilitated by the COSY spectrum of 2-heptanone. As shown in Figure 5, the signal for hydrogens seven are linked to the left-hand side of the overlapped signals for hydrogens five and six. In turn, the signals for hydrogens four are connected to the right-hand side of the overlapped signals. 171 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 3. COSY spectrum of dibutyl ether in CDCl3 with signal assignments.

Figure 4. HMQC spectrum of dibutyl ether in CDCl3 with 1H and 13C NMR signal assignments. 172 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 5. COSY spectrum of 2-heptanone in CDCl3 with signal assignments.

Figure 6. HMQC spectrum of 2-heptanone in CDCl3 with signal assignments. 173 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Subsequent interpretation of 2-heptanone’s HMQC spectrum allows assignment of all signals in the molecule’s 13C NMR spectrum (Figure 6). Several of the 13C NMR assignments revealed differ from what students typically predict based upon the position of 2-heptanone’s alkyl carbons relative to the electron-withdrawing carbonyl group. As suggested by Alonso and Warren, this observation lends itself to a discussion of diamagnetic versus paramagnetic contributions to 13C NMR chemical shifts (13). The authors, however, do not pursue this discussion within this introductory course. Though by analyzing the HMQC spectrum of 2-heptanone and assigning the 13C NMR signals, students become aware, through experience, that 13C chemical shifts do not always parallel those predicted by simple electron density/inductive effect considerations.

Characterization of the Product of a Fisher Esterification Having introduced COSY and HMQC spectroscopy with examples like those described in the preceding section, the first semester organic chemistry lab course ends with a project involving the synthesis of an ester via a classic Fisher esterification reaction (see Figure 7) (14). In the experiment, student pairs prepare an ester and characterize the resultant product using GC/MS and IR spectroscopy, along with 1H, 13C, COSY, and HMQC NMR spectroscopy.

Figure 7. A Fisher esterification reaction and the subsequent products produced in Organic Chemistry I lab.

The NMR spectra of esters are somewhat ideal for introductory COSY and HMQC analysis and complement the investigation of 2-heptanone’s NMR spectra. The utility of COSY and HSQC (heteronuclear single quantum coherence) spectroscopy to characterize esters in an undergraduate laboratory was illustrated within the first edition of this ACS symposium series (15). HSQC, provides similar information as HMQC. Several of the esters synthesized have overlapping signals in their 1H NMR spectra or resonances in their 1H and/or 13C NMR spectra that are not readily assignable. COSY and HMQC aid in signal assignments. Resultant spectra from one of the molecules studied, butyl propionate, is shown in Figures 8 and 9. COSY analysis of butyl propionate allows predicted assignments to be confirmed and allows a definitive assignment of the signals for hydrogens one and seven, which both appear as triplets in a similar chemical shift range (Figure 8). Moreover, the 174 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


assignment of signals for carbons one, two, and five through seven are fully aided by the HMQC spectrum of butyl propionate and again do not match those predicted by simply considering inductive effects (see Figure 9).

Figure 8. COSY spectrum of butyl propionate in CDCl3.

Figure 9. HMQC spectrum of butyl propionate in CDCl3. 175 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

2,3-Disubstituted Pyridines—Complex Splitting Patterns and HMBC


Derived from a larger research project of co-author Bergosh that involves the preparation of nicotine analogs, students undertake the synthesis and analysis of 2,3-disubstituted pyridines in Organic Chemistry II lab. A study of these molecules serves as an excellent platform for introducing complex splitting patterns within 1H NMR spectra, as well as long-range coupling within aromatic molecules. As an example, a 1H NMR spectrum of 2-chloro-3-(2-trimethylsilylethynyl)pyridine is shown in Figure 10.

Figure 10. 1H NMR spectrum of 2-chloro-3-(2-trimethylsilylethynyl)pyridine in CDCl3 with corresponding coupling constants indicated. A signal for residual protio chloroform is denoted by an asterisk. Before examining the 1H NMR spectrum of the pyridine in Figure 10, students predict the splitting pattern for hydrogens one through three in the molecule. With limited exposure to complex splitting and long-range coupling, students propose that the signals for hydrogens one, two, and three will be a doublet, triplet, and doublet, respectively. Examination of the spectrum in Figure 10 illustrates that this is not the case. The resonances for hydrogens one and three exhibit both ortho coupling, with coupling constants that are relatively large (J1,2 = 4.9 Hz, J2,3 = 7.6 Hz), and meta coupling with a smaller coupling constant (J1,3 = 2.0 Hz). As a result, the signals for both hydrogens one and three appear as a “doublet of doublets”. The significant difference in the ortho coupling constants, J1,2 and J 2,3, also leads the signal for hydrogen two to appear as a doublet of doublets. Analysis of the 1H NMR spectrum in Figure 10 facilitates an introduction to splitting diagrams and their utility in understanding complex splitting patterns. It should be pointed out that students synthesize 2-chloro-3-(2trimethylsilyethynyl)pyridine via a Sonagashira coupling reaction (see Figure 11) (16). This synthesis is the first exposure that students have with palladium-catalyzed coupling reactions. Thus, they are not aware that the reaction is selective, with alkynyl coupling occurring at the pyridyl carbon bearing the iodine, as opposed to the carbon attached to the chlorine. Thus, a key task/challenge of the experiment is for students to use NMR spectroscopic data to determine if 2-chloro-3-(2-trimethylsilylethynyl)pyridine or 3-iodo-(2-trimethylsilyethynyl)pyridine is the product of the Sonagashira reaction. 176



Figure 11. A Sonagashira coupling reaction used to produce 2-chloro-3-(2-trimethylsilylethynyl)pyridine. A hypothetical product of the reaction, 3-iodo-2 (2-trimethylsilylethynyl)pyridine is shown in the box to the right. Note: Numbers are included to label the unique carbons in each molecule.

To identify the product, students collect and analyze 1H, 13C, COSY, and HMQC spectra. Students are also introduced to HMBC spectroscopy. HMBC reveals coupling between hydrogens and carbons that are two and three bonds away. In the case at hand, the 1H NMR spectrum is consistent with both of the potential products in Figure 11. Although there are projected differences in the chemical shifts of the signals for carbon five in 2-chloro-3-(2-trimethylsilylethynyl)pyridine and carbon four in 3-iodo-2-(2-trimethylsilylethynyl)pyridine, the 13C NMR spectra of both compounds would also be similar. Thus, HMBC is critical in unequivocally identifying the product of the reaction and allows definitive signal assignments to be made. The HMBC spectrum of the Sonagashira reaction product is shown in Figure 12. Students make initial assignments of the 1H NMR signals for hydrogens one through three using the observed splitting patterns, as described earlier. The signal for hydrogen one is speculated to be further downfield than the signal for three due to its closer position relative to the inductively-withdrawing pyridyl nitrogen. In the 13C NMR spectrum, carbons one through three are assigned using the HMQC spectrum. However, the HMBC spectrum is needed to assign the signals for the quaternary aryl carbons (four and five) and alkyne carbons (six and seven). When examining the structure of 3-iodo-2-(trimethylsilylethynyl)pyridine (the boxed molecule in Figure 11), it becomes apparent to students that the compound’s HMBC spectrum would not have signals corresponding to coupling between any of the aryl hydrogens and alkynyl carbons, because alkyne carbon six is four bonds away from the nearest aromatic hydrogen. In contrast, aromatic hydrogen three in 2-chloro-3-(trimethylsilylethynyl)pyridine is three bonds away from alkyne carbon six. A contour is, indeed, observed in the HMBC spectrum of the product (Figure 12) that corresponds to the coupling of hydrogen three and carbon six. This observation allows a conclusive assignment of the structure of the product as the chloro alkynyl pyridine. Moreover, complete analysis of the HMBC spectrum allows the initial 1H NMR spectral assignments to be verified and the assignment of signals for the four quaternary carbons in the 13C NMR spectrum to be made. 177



Figure 12. The HMBC spectrum of 2-chloro-3-(2-trimethylsilylethynyl)pyridine in CDCl3. Note: The numbers in parentheses represent coupling of hydrogens to carbons that are three bonds away. Whereas, the bracketed numbers represent coupling of hydrogens to carbons that are two bonds away.

Practical Issues, Adaptation, and Effectiveness As is the case in Molecular Architecture, students in Organic Chemistry lab acquire spectra using an automated point and click acquisition that allows single button collection of 1H, 13C, COSY, HMQC, and HMBC spectra. Students are trained to properly load and queue their samples for acquisition. This minimal training allows student pairs (typically eight per section with two to four sections per semester) to readily acquire spectra with minimal supervision. Acquisition times for the suite of techniques requires approximately 20 minutes per sample, when analyzing a relatively concentrated solution (app. 40 mg mg/mL). Lab sections are scheduled in a manner that the 2D NMR spectral acquisition does not interfere with use of the instrument in other classes and in research. With regards to data workup, students are able to specify that a data file be automatically emailed to an address of their choice during acquisition setup. Students are also able to download copies of the instrument software to their own computers. Likewise, a second data station is available in the NMR lab for data analysis. Thus, upon brief training, students are able to workup their own data outside of the class and prepare figures for inclusion in laboratory reports. As mentioned previously, all NMR spectra are acquired using the software’s standard acquisition parameters. One limitation of acquiring COSY, HMQC, and HMBC spectra in this manner is that spectral quality, at times, is not as good as would be obtained if acquisition parameters were optimized for the specific sample being studied and the corresponding analyte concentration. However, the 178



authors have found the standard software experiments to be highly suitable for use in organic chemistry lab and in most of the department’s organic chemistry research efforts. Overall, student response to coverage of 2D NMR spectroscopy has, again, been positive. In addition to aiding students in learning to interpret basic NMR spectra, introduction of 2D NMR spectroscopy in the organic chemistry sequence, along with the inclusion of research-type projects like the aforementioned pyridine synthesis and characterization, has led to greater student interest in organic research. It should be pointed out that incorporation of 2D NMR spectroscopic analysis into organic chemistry courses does not require having access to a high-field instrument. Basic 2D NMR spectra may be acquired using FT-upgraded continuous wave instruments, as well as more recently developed benchtop NMR spectrometers. Examples of 2D NMR spectral problems are also available via printed resources (e.g, see Reference 8).

Use of NMR Spectroscopy in Undergraduate Organic Chemistry Research Undergraduate students at Ashland University who develop an interest in organic chemistry through their coursework are encouraged to carry out collaborative research with a faculty member. As such, NMR spectroscopy is used in a variety of projects. Because a fairly in-depth introduction to NMR spectroscopy is provided in Organic Chemistry I and II, students readily transition from using the NMR spectrometer in lab courses to using the instrument in research. Although students collect spectra in a fully automated mode in the introductory organic courses, research students utilize a semi-automated mode that allows selected instrumental and spectral parameter adjustments. A complete description of the use of NMR spectroscopy within undergraduate research at the University is beyond the scope of this chapter. However, a few examples involving the research efforts of the authors are provided. One specific area of organic/polymer chemistry research at Ashland involves the synthesis and study of new calixarene and resorcinarene-core star polymers, and related amphiphilic star block copolymers (17). Beyond using NMR spectroscopy to characterize the macrocyclic compounds that are required to initiate production of the desired star polymers, students involved in the aforementioned project are also exposed to the use of NMR in determining average degrees of polymerization via end-group analysis. Because of the conformational mobility of the resorcinarene cores of the star polylactides (PLAs), students are also introduced to the use of variable temperature NMR spectroscopy to afford sharpened 1H NMR signals that correspond to an average of signals for rapidly interconverting conformers. As an example, a 1H NMR spectrum of a four-armed resorcinarene-core star PLA is shown in Figure 13 (17). When obtaining the spectrum of the polymer in acetone-d6 at 50 °C, relatively sharp signals corresponding to the resorcinarene-core are visible. Subsequent integration of the signals for the internal methine signal, e, of the PLA chains compared to the end-group methine hydrogens e, allows the average number of repeat units of the star polymer 179



arms to be determined. For the molecule in Figure 13, the average degree of polymerization per arm was approximately 37 repeat units/arm, yielding a PLA arm molecular weight of 2.7 kDa. Because there are four polymer arms per molecule, the putative molecular weight of the star polymer is 12.2 kDa.

Figure 13. 1H NMR spectrum of a four-armed resorcinarene-core polylactide star polymer. Note: Adapted from reference (17). Copyright (2012, Royal Society of Chemistry).

Recently, students have used no-deuterium (No-D) NMR spectroscopy to monitor and optimize the coupling of a water soluble polymer to the hydrophobic PLA chain ends of the aforementioned star polymers using an automated kinetic experiment that can be set up within the spectrometer software (18). No-D NMR spectroscopy involves spectra being acquired in non-deuterated solvents (19). The utility of No-D NMR spectroscopy has been discussed in the literature and has significant potential for use in the undergraduate chemistry curriculum (20). The NMR spectrometer has also been used extensively in the characterization of products from student research projects involving the synthesis and study of nicotine analogs that are derived from compounds similar to the substituted pyridines presented herein (21, 22). Moreover, the spectrometer has been used in the characterization of a natural product isolated from the invasive plant, Phragmites australis (23, 24).

Conclusions The study and use of NMR spectroscopy has a central role in organic chemistry courses and research at Ashland University. Whether the spectroscopic investigation is in a course that introduces non-science majors to data interpretation and the process of scientific reasoning or is within the laboratory experiences of science majors, the study of NMR spectroscopy plays a valuable part in the education and development of undergraduate students. The authors look forward to the continued advances that are certain to come in this area. 180 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Acknowledgments The authors gratefully acknowledge the NSF for funds used to purchase the NMR spectrometer used for research and coursework at Ashland University (CHE/MRI-0922921). The NSF is also acknowledged for funds used in the polymer research described herein (CHE/0910566). Prof. Matthew Arthur is also acknowledged for his extensive contributions in the development and teaching of Molecular Architecture.


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From a Non-Majors Course to Undergraduate Research: Integration of

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