NMR Spectroscopy in the Undergraduate Curriculum - American

Chapter 8

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Using Benchtop NMR in Undergraduate Organic Courses S. M. Dimick Gray* Natural Sciences Department, Metropolitan State University, 700 E. 7th St., St. Paul, Minnesota 55106, United States *E-mail: [email protected]

This chapter describes the successful integration of the new benchtop NMR instruments into undergraduate organic laboratory curriculum. Benchtop NMR instruments can provide hands-on accessibility for sophomore-level undergraduate classes with minimal adjustment to established curriculums. The experiments described herein, performed with a picoSpin 1H 45 MHz spectrophotometer, rely on neat liquid samples with well-dispersed chemical shifts.

Introduction Many faculty in chemistry departments across the country—in fact, globally—are required to make a choice that places logistical concerns above the best active learning pedagogy: they would like to grant hands-on nuclear magnetic resonance (NMR) instrument access to the majority of their sophomore-level organic students, but they do not have sufficient capital outlay for instrument purchase or technical staff to ensure the developing scientists in these classes maintain the instruments to departmental satisfaction. Students are often required to give samples to teaching assistants who will actually procure the data on a research-grade instrument. Coupled with the significant space requirements of a traditional NMR, as well as escalating costs of deuterated solvents and shortages of liquid helium, many organic chemists find themselves searching for alternatives to a standard high-field NMR for the first two semesters of organic chemistry laboratory classes.

© 2016 American Chemical Society Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: First Year and Organic Chemistry Courses Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


The introduction of benchtop NMR instruments in the past five years has increased the possibilities for chemistry departments to allow students hands-on experience without some of the stated drawbacks. The instruments currently available include the 60 MHz Pulsar by Oxford Instruments (1), 60 MHz NMReady by Nanalysis (2), 43 MHz Spinsolve by Magritek (3), and the 45 MHz or 82 MHz picoSpin marketed by Thermo Scientific (4). The experiments described in this chapter utilized a 1H 45 MHz picoSpin instrument which our department purchased four years ago. The picoSpin instrument, containing a permanent magnet, is approximately the size of a shoebox and requires only an Ethernet connection to a laptop computer as the user interface. We were able to place the instrument in a location adjacent to our laboratory, making sampling during the class session a possibility without the addition of ancillary teaching staff to monitor the instrument use. Instrument set-up was achieved in an hour and did not require additional hard wiring of the lab or the scheduling of a technician’s visit. Shimming is performed once a week on a water sample. A spectrum is then taken of an ethyl acetate sample to confirm that the appropriate adjustments have been made. In our hands, sound shimming is achieved with an hour or two even after four months of inactivity. Rather than preparing the standard 5 mm sample tube, samples are injected via a 1 mL syringe into a flow cell, with only 40 μL of sample required for complete displacement of the previous sample. The sequential sample displacement serves as a way to clean the instrument at the conclusion of the lab period, where water is the cleaning solvent. The sample cell is temperature controlled at 42 °C, ensuring stable, consistent shimming and attenuating field drift of the magnet. Data is imported from the instrument into Mestrelab Mnova software to render a standard spectrum. Our experience with the instrument has been that students can inject, acquire, import, and work-up the data in about 10 minutes, thus enabling us to get through an entire lab section worth of students within the allotted lab period. The question of what kind of samples to use in order to get robust results is a good one, and the types of experiments that can be done using these instruments are the subject of this chapter. In order to incorporate a benchtop NMR spectrometer without major restructuring of an already established program we sought experiments that fulfilled three practical criteria: 1) the analyte was a neat liquid, 2) the analyte had a boiling point above the sample cell temperature of 42 °C, and 3) the analyte had distinctive, well-resolved proton resonances. We had several learning objectives that our experiments would demonstrate: 1) using the four main aspects of a spectrum to identify a structure (number of equivalent protons equals the number of NMR signals, chemical shifts are indicative of a proton’s electronic environment, the number of protons in a signal correlates with the signal integration, and signal splitting can give information about the neighboring hydrogens), 2) NMR spectroscopy can assist in determining whether a sample is pure, 3) NMR spectroscopy is central to successful analysis of a student-synthesized product in the lab, and 4) NMR spectroscopy can be used to identify structures of natural products. We chose to explore experiments relying on neat liquids only. This required a fresh examination of our standard organic curriculum which relied heavily on crystallization to purify products (with analysis achieved via melting point, thin108

Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: First Year and Organic Chemistry Courses Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


layer chromatography (TLC), and even gas chromatography-mass spectrometry (GC/MS)). By contrast, only extraction and distillation are required for purification of the products below. In order to achieve a reasonable signal to noise ratio in the fewest number of scans, neat liquids are preferable with adequate signals being achieved in only eight scans and 1.5 minutes. By contrast, if a solid sample is to be used a 1 M solution and 256 scans are typically required to generate a sufficient signal-to-noise. This necessitates a much longer acquisition time. Furthmore, creating a 1 M solution of many small organic molecules is a challenge. For example this might translate into dissolving 300 mg of solid into 1 mL of solvent for a molecule with a molar mass of 300 g/mol. Unfortunately, this is beyond many compounds’ solubility limit. Solvents such as dimethylsulfoxide (DMSO) can dissolve many organic compounds, but because of its ability to rapidly permeate through the skin there are significant hazards associated with using this solvent. The sampling of neat liquids in NMR spectroscopy means that exchangeable protons will exhibit spin-spin splitting patterns and these can be integrated. Furthermore, tetramethylsilane is often not soluble in the neat liquid being investigated because of the concentrations used and the cell temperature (42 °C). This makes internal referencing challenging. As we have been working with previously characterized products, external referencing was accomplished by comparing our spectra with the Spectral Database for Organic Compounds (SDBS) (5).

Identification of a Small Molecule One of the first experiments we did was to try a series of different solvents found in our chemistry stockroom. Our aim was to discern which solvents could be easily identified by our students using the picoSpin. Our learning goal for this experiment was to use the four main aspects of an NMR spectrum to determine the structure of an unknown molecule. A low-field magnet, like the picoSpin, generates lower resolution spectra than a more traditional high-field NMR spectrometer. We found that smaller, densely functionalized molecules with well-dispersed signals worked best in this experiment. Successful solvents that were used included methanol, ethanol, n-propanol, isopropanol, 3-methyl-1-butanol, t-butanol, acetone, ethyl acetate, methyl benzoate, toluene, acetophenone, ethyl benzene, and dimethylformamide. n-Propanol is shown in Figure 1. Longer chain alcohols, such as n-butanol and n-pentanol, have overlapping signals for the alkyl chain protons that we deemed too ambiguous for identification. Small molecules such as para-methylbenzaldehyde and ortho-diethylphthalate also proved to be robust samples for the picoSpin.

109 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: First Year and Organic Chemistry Courses Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 1. 1H NMR spectrum of n-propanol using the picoSpin 45.

Determining Sample Purity Following a simple identification lab, we further developed students’ understanding of integration with mixtures of solvents in a single sample. Many of the example spectra students see in textbooks are of a pure sample of a single molecule, and the concept that NMR spectroscopy can assist in determining whether a sample consists of one entity versus two is a significant learning objective. We combined two solvents with non-overlapping peaks in predetermined molar ratios and then asked students to 1) identify the solvents in the mixture, and 2) determine the relative ratios of the solvents in the mixture. For the development of this portion of the experiment we needed to first create solvent pairs without overlapping proton signals. We found dimethylformamide (DMF) to be useful in this regard since the signals were distinct from the simple alcohols in the set listed above. Some sample combinations that also worked were toluene and ethanol, isopropanol, or n-propanol; acetone and methanol or n-propanol; acetophenone and methanol; ethyl acetate and dimethylformamide (Figure 2); methanol, ethanol, n-propanol, or isopropanol and dimethylformamide. Students were then challenged to use the relative integrations to determine the composition of the mixture. Variations of this experiment have been described previously (6). We found that students could identify the aldehydic singlet at 8.08 ppm as belonging to dimethylformamide and representing one hydrogen, and then base all of their calculations for the other signals’ integrations on that assumption. Students would then conclude that the mixture in Figure 2 represents a molar ratio of 2:1 ethyl acetate: dimethylformamide using this process. 110



Figure 2. 1H NMR spectrum of ethyl acetate and dimethylformamide in a 2:1 mixture.

Analysis of a Synthesized Product Our third learning objective with NMR in our curriculum was to use NMR spectroscopy as a primary method of analysis for synthesized products. We examined a variety of synthetic reactions that represented chemistries learned during the sophomore organic chemistry sequence. Herein we present examples of esterification, elimination, addition, oxidation, and reduction below. All of these products were analyzed as neat liquids with the picoSpin. The well-known synthesis of isoamyl acetate or “banana oil” from acetic acid and 3-methyl-1-butanol, as outlined in Figure 3, introduces students to extraction and distillation as purification techniques and was easily analyzed by the picoSpin NMR spectrometer. We also used FTIR as a second form of analysis. Banana oil has a well-separated triplet for students just beginning to use NMR spectroscopy for analysis, along with a more complicated overlapping set of signals between 1.5 – 1.8 ppm. In our Organic Chemistry 1 curriculum, this overlapping set of signals represents the first instance in which students are asked to interpret a signal that is not concisely resolved. A second synthesis, compatible with the undergraduate laboratory time frame and picoSpin analysis, was esterification of salicylic acid to form methyl salicylate or “oil of wintergreen” from salicylic acid and methanol (7), as shown in Figure 4. This reaction starts with the solid salicylic acid and concludes with a liquid product.



Figure 3. Synthetic scheme and picoSpin 45 1H NMR spectrum of isoamyl acetate or “banana oil”.

Multistep reactions can also be analyzed using a picoSpin NMR spectrometer. The elimination and addition reactions below, starting with cyclohexanol, are consistent with the chemistries learned by sophomore level organic students. Rather than complete identification of every peak of each molecule, which was challenging due to the overlapping signals of the alkyl protons in the cyclohexyl ring, students were instructed to focus on the major changes observed between starting material and product and several differentiated NMR signals. For example, dehydration to cyclohexene using H2SO4 or K10 montmorillonite clay (8) proceeded within the time frame of an organic chemistry lab period (H2SO4, 30 min; K-10 clay, 1.5-2 h) with an easier and cleaner work-up noted for the K10 clay reaction. With the dehydration of cyclohexanol, students observed the disappearance of the alcoholic proton at 4.00 ppm and the shift of the proton bonded to the carbon bearing the alcohol from 3.58 ppm to 5.67 ppm. This occurred along with a new relative integration of the alkenyl protons to the alkyl protons as 1:4 in the cyclohexene. The bromination of the cyclohexene using in situ Br2 generation from HBr and H2O2 completes the second step of this synthesis as shown in Figure 5 (9). Students observed the alkenyl protons shifting to 4.46 ppm once the halide was added.



Figure 4. Synthetic scheme and picoSpin 45 NMR of methyl salicylate or “oil of wintergreen”.

Cyclohexanol was also oxidized to cyclohexanone using sodium hypochlorite and acetic acid (10), as shown in Figure 6. Students could monitor the disappearance of the alcoholic proton and the proton bonded to the carbon bearing the alcohol. Another synthesis involved the oxidation of R-carvone, an inexpensive pleasant smelling oil with well-dispersed NMR signals, to the aromatic carvacrol using K10 montmorillonite clay (11). This spectrum showed distinctive signal changes that were easily distinguished using the picoSpin NMR, as shown in Figure 7. Students could easily observe the changes from an aliphatic compound to an aromatic one, including the disappearance of the vinyl protons at 4.79 ppm and the appearance of an isopropyl functional group at 1.08-1.23 ppm and 2.83 ppm. We also used the R-carvone to do a selective reduction of the conjugated alkene using zinc and potassium hydroxide (12), as shown in Figure 8. Disappearance of the downfield vinyl proton at 6.76 ppm was easily monitored with the picoSpin.



Figure 5. Two-step reaction scheme to produce 1,2-dibromocyclohexane from cyclohexanol. PicoSpin NMR spectra of cyclohexanol (top left), cyclohexene (top right) and 1,2-dibromocyclohexane (bottom).



Figure 6. Reaction scheme to produce cyclohexanone from cyclohexanol and the correlating picoSpin spectra for the product cyclohexanone.

Figure 7. Reaction scheme for oxidation of R-carvone to carvacrol from carvone and spectrum of carvone (left) and carvacrol (right). 115 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: First Year and Organic Chemistry Courses Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 8. Reaction scheme for the reduction of R-carvone and picoSpin spectrum of the reduced product.

Natural Product Identification In addition to small molecule synthesis, another application utilizing 1H NMR analysis is the determination of structure of a natural product extract. In our curriculum we routinely extract limonene from oranges (13) and analyze the essential oil using the picoSpin; the three vinyl protons at 5.41 ppm and 4.72 ppm are well-separated from the alkyl signals. One large navel orange, generating at least 10 g of peel, yields 0.5 mL of limonene within a standard lab period. This is plenty of material for NMR analysis (Figure 9), FTIR analysis, and qualitative tests such as bromine addition. We have also extracted cinnamaldehyde from ground cinnamon and eugenol from ground cloves (14) for analysis with the picoSpin NMR spectrometer. The structures of both these extracts have signals that are well-separated, but the amount of essential oil generated was deemed insufficient for routine use in the classroom.



Figure 9. Extraction scheme for limonene and correlating picoSpin spectrum.

Conclusions In conclusion, we have demonstrated a progression of applications for the new generation of benchtop NMR instruments in the undergraduate organic chemistry classroom. We have shown that this instrument can be used for identification of unknown molecules, determination of whether a sample was a single component or a mixture, analysis of a variety of synthetic transformations compatible with sophomore level organic chemistry, and identification of natural product extracts. Ultimately, we found that experiments that utilized neat liquids with well-dispersed signals were well-suited for this instrument.

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Oxford Instruments. http://www.oxford-instruments.com/products/ spectrometers/nuclear-magnetic-resonance-nmr/pulsar (accessed November 9, 2015). Nanalysis. http://www.nanalysis.com/60mhznmr.html (accessed November 9, 2015). Magritek. http://www.magritek.com/products/spinsolve/ (accessed November 9, 2015). 117


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Thermo Scientific. http://www.thermoscientific.com/en/products/nuclearmagnetic-resonance-nmr.html (accessed November 9, 2015). Spectral Database for Organic Compounds, SDBS. http://sdbs.db.aist.go.jp/ sdbs/cgi-bin/direct_frame_top.cgi (accessed November 9, 2015). Cramer, J. D. Using NMR Spectroscopy to Promote Active Learning in Undergraduate Laboratory Courses. In NMR Spectroscopy in the Undergraduate Curriculum; Soulsby, D., Anna, L. J., Wallner, A. S., Eds.; ACS Symposium Series 1128; American Chemical Society: Washington, DC, 2013; pp 57−68. Nguyen, K.; Weizman, H. J. Esterfication of salicylic acid: The synthesis of cool-smelling molecules. J. Chem. Educ. 2007, 84, 119–121. Nagendrappa, G. Organic synthesis using clay catalysts: Clays for ‘green chemistry’. Resonance 2002, 7, 64–77Cyclohexanol (10 g, 0.10 mol) was placed in a round-bottomed flask and K10 montmorillonite clay (1 g) was added. A simple distillation apparatus was assembled and the reaction mixture was refluxed at 83-90 °C, with careful monitoring of the temperature to ensure the reaction mixture did not continue heating to 100 °C. The product was then distilled and collected in a receiving flask as it was formed. Following distillation, the product was separated from water byproduct using a separatory funnel, and the organic layer was dried over anhydrous MgSO4 prior to analysis. Ho, T.-L.; Gupta, G. B.; Olah, G. A. Synthetic methods and reactions; 39. Phase transfer catalyst promoted halogenation of alkenes with hydrohalic acid/hydrogen peroxide. Synthesis 1977, 10, 676–677Cyclohexene (1.95 g, 0.024 mol) and 30% H2O2 (4.8 mL) were combined in a round bottom flask. Concentrated HBr (3.2 mL) was added to the reaction solution and the reaction was allowed to reflux for 1 h. The solution was allowed to cool to room temperature and neutralized by washing with 10% aqueous sodium bicarbonate solution. The organic layer was then dried over anhydrous MgSO4. Zuczek, N. M.; Furth, P. S. Oxidation of cyclohexanol to cyclohexanone using sodium hypochlorite. J. Chem. Educ. 1981, 58, 824. Kjonaas, R. A; Mattingly, S. P. Acid-catalyzed isomerization of carvone to carvacrol. J. Chem. Educ. 2005, 82, 1813–1814. Kelly, L. F.; Deeble, G. J. Selectivity in organic synthesis: Chemo-and regiospecific reductions of carvone. An undergraduate experiment. J. Chem. Educ. 1986, 63, 1107–1108. Greenberg, F. H. Natural products isolation—orange oil: An undergraduate organic experiment. J. Chem. Educ. 1968, 45, 537–538. Ntamila, M. S.; Hassanali, A. Isolation of oil of clove and separation of eugenol and acetyl eugenol. An instructive experiment for beginning chemistry undergraduates. J. Chem. Educ. 1976, 53, 263.


NMR Spectroscopy in the Undergraduate Curriculum - American

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