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Chapter 6

Overcoming Problems Incorporating NMR into the Organic Chemistry Lab Downloaded by UNIV OF OKLAHOMA on March 20, 2013 | http://pubs.acs.org Publication Date (Web): March 19, 2013 | doi: 10.1021/bk-2013-1128.ch006

Luke A. Kassekert and J. Thomas Ippoliti* Department of Chemistry, University of St. Thomas, 2115 Summit Avenue, St. Paul, Minnesota 55105 *E-mail: [email protected]

Organic chemistry laboratory courses present exceptional opportunities to teach students how to utilize NMR to its fullest potential. When implementing experiments that use NMR to analyze products in the undergraduate teaching lab a number of practical problems arise, even with the use of an automated instrument. The pertinent issues in our situation involve sample submission timing, data transfer, and spectra processing. This chapter addresses what can be done to alleviate those problems using a newly designed experiment incorporating flow hydrogenation that has been incorporated into our organic laboratory sequence. Solutions include having a teaching assistant present by the NMR spectrometer room as students are submitting their samples, using Dropbox as a versatile approach to distributing and accessing data, and that iNMR reader is a flexible solution for students to process their data.

Introduction In 1992 the Department of Chemistry at the University of St. Thomas purchased a Bruker AM300 NMR spectrometer with the help of an NSF- ILI grant (1). We fully incorporated this instrument into our curriculum, but by 2009 problems associated with the instrument were becoming increasingly time consuming. Therefore, in the summer of 2009, we wrote an NSF Major Research Instrumentation grant for a new NMR spectrometer. This proposal was funded in 2010 (2), and in the summer of that year a new JEOL ECS 400 MHz NMR spectrometer was installed. The most important factor considered when © 2013 American Chemical Society In NMR Spectroscopy in the Undergraduate Curriculum; Soulsby, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.

Downloaded by UNIV OF OKLAHOMA on March 20, 2013 | http://pubs.acs.org Publication Date (Web): March 19, 2013 | doi: 10.1021/bk-2013-1128.ch006

deciding on a new spectrometer was automation. Implementing NMR into our organic labs with our old instrument was problematic because taking spectra and working up data were complex. In particular, placing the samples manually in the magnet, shimming the magnet, and working up the data were sufficiently difficult that some of the faculty teaching our labs (which included many adjunct faculty) were hesitant to learn how to do these things. This made using the NMR in our organic teaching laboratories challenging. From this experience we knew that an automatic sample changer and an instrument capable of automated shimming were a necessity. The JEOL ECS 400 spectrometer we purchased has an auto sample changer that holds 24 samples and has fully automated tuning and shimming. The Delta™ Software (3) that controls the spectrometer runs on Apple’s OSX system software and is capable of fully automated data acquisition. The instrument is also capable of running No-D NMR (4), which enables spectra to be recorded without the use of deuterated solvents. Use of the sample changer, gradient shimming and auto-tuning can automate the entire process of routine analysis, making it ideal for the undergraduate teaching laboratory. Although the new NMR spectrometer is fully automated, there are still a number of practical problems associated with teaching the students how to use the new instrument, as well as how to access and work-up the associated data. To illustrate the strategies used to overcome these problems, we describe a new experiment, Hydrogenation of Eugenol, we designed and implemented in our Organic Chemistry II laboratory. Continuous-flow reactors are a modern solution to the inherent safety hazards of hydrogenation (5–12). Specifically, we used an instrument made by ThalesNano called the H-Cube Continuous-Flow Reactor (13). In this instrument, hydrogen gas is generated from the electrolysis of deionized water and is never stored in large quantities. It is immediately combined with the solution containing the unsaturated compound, pressurized, and passed through a pre-packed cartridge of catalyst that can be heated if required. The pressure, temperature, and flow rate are all digitally controlled. The pre-packed cartridges are especially convenient because they eliminate the need to weigh out flammable catalysts. This instrument is state-of-the-art and is starting to be used routinely in research labs around the world. The incorporation of the H-Cube Continuous-Flow Reactor into the undergraduate curriculum is an ideal way to demonstrate how to safely carry out the hydrogenation of an alkene. To this end, we designed an experiment to perform the hydrogenation of eugenol (Scheme 1) in an undergraduate organic chemistry lab using the H-Cube Reactor.

Scheme 1. Hydrogenation of Eugenol 84 In NMR Spectroscopy in the Undergraduate Curriculum; Soulsby, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


There are 16 students in each lab section and students work in pairs. Each pair of students hydrogenates an 80 mg sample of eugenol using the H-Cube hydrogenator. 1H NMR spectroscopy is the primary tool for determining if, and to what extent, hydrogenation has occurred. This experiment exemplifies how important NMR spectroscopy is for structure determination, and in particular for following a functional group transformation. As with any experiment involving analysis by NMR spectroscopy, students must be taught how to prepare samples and how to properly insert NMR tubes into the spin collar. This is most easily accomplished using handouts, listing detailed steps with illustrative pictures. The practical problems of timing, data transfer, and data work-up are addressed below.

Experimental Materials and Instruments Eugenol was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). The H-Cube® Continuous-flow Hydrogenation Reactor was purchased from ThalesNano (Budapest, Hungary).

Typical Experimental Procedure 80 mg of eugenol is added to a 25 mL Erlenmeyer flask. 5.0 mL of methanol is added to the same flask using a 10.0 mL graduated cylinder. A second 25 mL round-bottom flask is weighed and the weight of that flask recorded. This second flask serves as the collecting flask. The outlet tube from the H-Cube ContinuousFlow Reactor is placed into the 25 mL round-bottom collection flask. Within two seconds the sample inlet line is then placed into the 25 mL Erlenmeyer flask containing the eugenol seconds so that no air bubbles travel into the reactor. The sample is then pumped through the reactor and collected in the second roundbottomed flask. Throughout the collection period, the bottom disk located under the cylinder of the sample inlet line remains submerged in the sample solution. When about 1 mL is left in the Erlenmeyer flask, 3.0 mL of methanol is added to the flask using a plastic syringe. Once the solution has dropped to about 1 mL, the collecting capillary is transferred back to the clean methanol vial it was previously stored in. Methanol is transferred for several more minutes to wash out any remaining product in the system.The solvent is then removed from the round-bottomed flask using a rotary evaporator. Finally, the mass of the flask is recorded and the weight of the empty flask subtracted to obtain the actual yield in grams. Using the theoretical and actual yields, the percent yield for the reaction is determined. For NMR analysis about 5 mm of product is sucked into a clean Pasteur pipette via capillary action. This is then placed into an NMR tube. Approximately 1 mL of deuterated chloroform is added to the top of the pipette containing the sample to wash the sample into the NMR tube. 85 In NMR Spectroscopy in the Undergraduate Curriculum; Soulsby, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Results and Discussion Our initial approach to analyzing student samples was to bring the 24-sample carousel to the lab, wait until all of the students finished the experiment and loaded their tubes in the carousel, and then bring the fully loaded carousel back to the instrument. This approach was quickly discontinued because students finished the experiment at different times causing many to wait extended periods of time for their data. Our revised approach was to have each pair of students bring their freshly prepared NMR samples to the NMR room as soon as they finished. The hydrogenation reaction takes about 8 minutes total (5 minutes to run the eugenol solution through and a 3 minute rinse), and the solvent evaporation takes about 7 minutes. This means that all of the students (8 pairs) can finish the experimental part of the laboratory in about 2 hours. The actual time it takes to get the NMR data, from the time the tube is dropped into the magnet to the point the data file appears, is 7 minutes. Although we assumed the fully automated sample shimming and tuning would alleviate the need for a teaching assistant (TA), we soon found this not to be the case. In fact, we found it necessary for a TA to oversee the students in a number of steps, including placing their tubes in the spin collar, checking to see if the depth was properly adjusted, and placing the sample tube in an empty slot. One problem we did not anticipate was that the students would often lean against the magnet while placing their tube in the sample changer. Under the guidance of a TA, students would type in their filename and slot number, then click on the appropriate NMR experiment, in this case the 1H NMR experiment. At this point, data transfer and manipulation were our remaing concerns. The problem of data transfer is certainly platform and institution dependent. At St. Thomas, we use Apple computers. The JEOL instrument control software (Delta™) runs on an Apple iMac computer and the organic lab utilizes Apple MacBook Pro laptop computers. The iMac hard drive can be shared on a local network, but only five computers can connect at one time which is problematic. One solution we considered was to install Apple Server software on the computer that stores the data. Apple Server software is not expensive; however, one needs to erase the entire hard drive to install it. We chose not to do this because we would then need to reinstall the NMR software as well. If an institution is installing a new NMR spectrometer and using an Apple Macintosh system to run it, this is certainly something that should be considered. We recently discovered an innovative way around this issue that also allows access to the NMR data from anywhere on or off campus. We used a Dropbox account and created a symbolic link (sym link) for the lab NMR data folder. This is because aliases do not work with Dropbox (14). The sym link folder has the same name as the original folder and is placed in the Dropbox so that as soon as a file is created on the hard drive it is automatically copied to the Dropbox folder with the same name. Students can access the data from any type of computer as long as they have a Dropbox account and we have shared access to the folder with them. In our case, we put Dropbox folders on all of our lab computers, which provides a one click link to all of the NMR data. Once the students have access to their data they need to be able to manipulate it using NMR processing software. For the Apple platform the choices are Delta™, 86 In NMR Spectroscopy in the Undergraduate Curriculum; Soulsby, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


MestReNova (15), and iNMR (16). Out of these three we chose iNMR because, in our opinion, it has the cleanest and easiest interface to use. The version of iNMR we chose was iNMR Reader because site licenses are available at reasonable cost. Although iNMR Reader does not allow one to save the worked up spectrum in the program, one can print it or copy and paste it into a report. One can also save a pdf of the spectrum by choosing “Save as PDF” in the print menu. iNMR can also overlay multiple spectra and adjust the vertical offset to any value. This was important to us because it makes it very easy to see differences between starting material and product (see example in Figure 1). Overlaying spectra in this manner illustrates very clearly a functional group transformation. One can also overlay solvent spectra, and providing a folder containing spectra of various solvents is useful for this type of analysis. iNMR automatically processes the data (Fourier transform and phasing), and the students simply open the NMR data file to see the spectrum. There are only three operations the students need to learn: expansion, integration, and overlay. These spectra manipulations can be shown to the students in lab, using a computer attached to a projector, in less than 10 minutes at the beginning of the lab period. To help carry out the comparison of starting material to product and confirm functional group transformation, we provided them with the 1H NMR data of the starting material (eugenol). Then we had the students integrate all the peaks of their spectrum and overlay the spectrum of the starting material. The students quickly learned how to carry out these operations and commented on the ease of use of the iNMR software. The experiment was successfully completed by all of the students in the four hour time frame allotted for the lab. The outcome of the experiment was that nearly all of the students achieved quantitative yields, and through NMR analysis of their products, found that complete hydrogenation had occurred at 20 bar pressure. We have found that it is quite instructive to look at the change in splitting patterns of the benzylic methylene in the starting material and the product. In the starting material it is a broadened doublet with a coupling constant of 6.7 Hz (labeled S2 in Figure 1). It is interesting to note that the broadened nature is due to the long range coupling to the terminal olefin protons (see Figure 2). After the double bond is hydrogenated, the benzylic methylene is now a triplet with a coupling constant of 7.7 Hz (labeled P2 in Figure 1), typical for vicinal alkyl protons. This notable change in coupling, as well as the change in chemical shift of the methylene protons, is the subject of multiple questions in the pre-lab and lab report (17). The lab report also asks the students to compare data obtained from carrying out the hydrogenation at two different pressures (10 and 20 bar). The overlay given in the report of pure eugenol (starting material) with the product isolated from the reaction carried out at the two different pressures is shown in Figure 1. The overlay clearly shows how the olefin peaks (at 5.0 and 5.9 ppm; S4, S5 and S3 respectively) are not present when the reaction is carried out at 20 bar but are present at 10 bar, demonstrating that eugenol is only partially hydrogenated at 10 bar. In the spectrum obtained at 10 bar, the product peaks (benzylic CH2 at 2.5 ppm; P2) are integrated and compared to the starting material (benzylic CH2 at 3.3 ppm; S2) and the students are asked to determine the extent of hydrogenation from the integration data. 87 In NMR Spectroscopy in the Undergraduate Curriculum; Soulsby, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Figure 1. Overlay of 1H NMR (CDCl3) spectra of eugenol with isolated hydrogenation products(aliphatic protons assigned).

Figure 2. Partial expanded 1H NMR (CDCl3) spectrum of eugenol. 88 In NMR Spectroscopy in the Undergraduate Curriculum; Soulsby, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Several changes are being considered for future implementation in this lab. One change would be to utilize the No-D feature of the JEOL spectrometer so that the reaction can be directly analyzed in the solvent used to carry out the reaction (in this case methanol). This change would save time in that the solvent would not need to be removed by evaporation and would decrease costs since deuterated solvents would not be needed (4). Also, at 20 bar all of the students saw complete hydrogenation. In future experiments we plan on having groups of students carry out the hydrogenation at different pressures, 5, 10, 15 and 20 bar where we know complete hydrogenation does not occur. Students can then compare their results, allowing us to introduce a more “guided inquiry” approach to this experiment.

Conclusions In summary, the three most pertinent problems of incorporating NMR with an automated instrument were timing, data transfer, and data work-up. Having a TA in the NMR spectrometer room allows students to run NMR experiments on their products as soon as they are finished. Using Dropbox to share data provided a flexible solution to NMR data transfer and access issues. A signficiant advantage of using Dropbox is that any computer connected to the Internet, even if it is removed from the campus network, can access the NMR data, giving the instructor the option of assigning data workup and analysis as homework. Finally, data workup is platform dependent. Students using a PC have the option of using the free NMR processing software by ACD labs or MestReNova (there is also a beta version of iNMR for the PC available). Students using a Mac have two choices: iNMR or MestReNova. Both of these software companies now offer site licenses at reasonable costs to educational institutions (15, 16). We have found that the overlay feature in iNMR is most instructive for determining functional group transformation. In summary, all of these problem-solving strategies have been implemented in the newly developed Hydrogenation of Eugenol experiment described above and serve as an example of how to take full advantage of NMR spectroscopy in the undergraduate organic laboratory.

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NSF Grant: 9251450 “Integration of High Field, Multinuclear NMR into the Undergraduate Chemistry Curriculum.” NSF Grant: CHE-0959322 “MRI-R2: Acquisition of 400 MHz Nuclear Magnetic Resonance (NMR) Spectrometer.” http://www.jeol.com/PRODUCTS/AnalyticalInstruments/ NuclearMagneticResonance/DeltaNMRSoftware/tabid/395/Default.aspx (accessed November 2012). Hoye, T. R.; Eklov, B. M.; Ryba, T. D.; Voloshin, M.; Yao, L. Org. Lett. 2004, 6, 953–956. Amoa, K. J. Chem. Educ. 2007, 84, 1948–1950. De, S.; Gambhir, G.; Krishnamurthy, H. G. J. Chem. Educ. 1994, 71, 992–993. 89 In NMR Spectroscopy in the Undergraduate Curriculum; Soulsby, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.

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Hanson, R. W. J. Chem. Educ. 1997, 74, 430–431. Mohrig, J. R.; Hammond, C. N.; Schatz, P. F.; Davidson, T. A. J. Chem. Educ. 2009, 86, 234–239. Navarro, D. M.; Navarro, M. J. Chem. Educ. 2004, 81, 1350–1351. O’Connor, K. J.; Zuspan, K.; Berry, L. J. Chem. Educ. 2011, 88, 325–327. Plummer, B. J. Chem. Educ. 1989, 66, 518–519. Wilen, S. H.; Kremer, C. B. J. Chem. Educ. 1962, 39, 209–210. http://www.thalesnano.com/products/h-cube (accessed November 2012). The two articles we used to understand symbolic links or sym links were: “A brief tutorial on symbolic links” http://hints.macworld.com/ article.php?story=2001110610290643 and “Symbolic links made simple” http://www.macworld.com/article/1153437/symlinkservice.html (accessed November 2012). http://mestrelab.com/ (accessed November 2012). http://www.inmr.net/ (accessed November 2012). For digital copies of all of the handouts and the report given to the students, email the principal author at [email protected].

90 In NMR Spectroscopy in the Undergraduate Curriculum; Soulsby, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.

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