Molecular Properties of Caffeine Explored by NMR: A Benchtop NMR

Laboratory Experiment Cite This: J. Chem. Educ. XXXX, XXX, XXX−XXX

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Molecular Properties of Caffeine Explored by NMR: A Benchtop NMR Experiment for Undergraduate Physical-Chemistry Laboratories James E. Kent and Nicholle G. A. Bell* EaStCHEM, School of Chemistry, The University of Edinburgh, King’s Buildings, David Brewster Road, Edinburgh EH9 3FJ, United Kingdom

J. Chem. Educ. Downloaded from pubs.acs.org by WEBSTER UNIV on 02/21/19. For personal use only.

S Supporting Information *

ABSTRACT: NMR is a fundamental part of any undergraduate chemistry degree, and training students how to measure and interpret NMR spectra is essential for postgraduate research or chemistry-based careers. Traditionally, the undergraduate laboratory experience with NMR is restricted to sample submission for NMR analysis, which is typically conducted by a technician. However, instant access to NMR instrumentation is now possible because of the development of benchtop NMR spectrometers. These can be placed on the lab bench alongside other spectroscopic equipment. The simplicity of operation and variety of experiments available allows the development of new undergraduate experiments across organic-, inorganic-, and physical-chemistry laboratories. Here we describe a new experiment for undergraduate physical-chemistry laboratories that incorporates relaxation measurements to understand the behavior of caffeine in a variety of conditions. The experiment uses the T1 and T2 experiments available on most benchtop spectrometers, which can be processed using a variety of software packages. The experiment, performed in the third year of a 4 or 5 year Bachelor’s or Master’s degree, follows a five-lecture unit on NMR spectroscopy as part of a third undergraduate chemistry course. It can be conducted in pairs or groups of three in three 3 h laboratory sessions, but it can be tailored to reduce the required time. The experiment allows students to become proficient users of a new piece of equipment, incorporate taught theory, and handle data in different platforms and software packages. Allowing students hands-on access to NMR instrumentation gives them a complementary experience to their NMR lectures and offers a unique way to inspire a new generation of chemists. KEYWORDS: NMR Spectroscopy, Upper-Division Undergraduate, Physical Chemistry, Hands-On Learning/Manipulatives, Laboratory Equipment/Apparatus, Inquiry-Based/Discovery Learning

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INTRODUCTION NMR spectroscopy is one of the most important analytical techniques in the chemists’ tool box. The growing number of applications aided by the increasing ease of use of modern NMR spectrometers means the technique is now stepping out of chemistry laboratories into other fields, such as the food industry,1 environmental science,2 and materials science.3 NMR is now firmly embedded in any undergraduate chemistry degree; furthermore, it now features increasingly in high-school curricula, where pupils now learn about spin−spin coupling constants in addition to 1H chemical shifts in their final-year chemistry courses. However, the high running costs and limited availability of NMR time has traditionally restricted hands-on access to this technique to final-year undergraduate © XXXX American Chemical Society and Division of Chemical Education, Inc.

projects or postgraduate students. Considering that NMR is commonly used in academic research and R&D of numerous industries, this limited access means that many chemists are leaving their education with a “black-box” attitude toward NMR, with little knowledge on the practical aspect of measuring NMR spectra. However, the recent availability of more affordable benchtop NMR spectrometers paves the way for new teaching opportunities in undergraduate-degree programs. These low-field (45−80 MHz) instruments are simple to setup and intuitive to operate and can perform basic Received: August 4, 2018 Revised: February 4, 2019

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DOI: 10.1021/acs.jchemed.8b00633 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

I(τ ) = I0e−τ / T2

1D but also more advanced 2D homo- or heteronuclear experiments.4 Several NMR experiments for organic-chemistry, inorganic-chemistry, and biochemistry undergraduate laboratories have been published for use with benchtop spectrometers4−8 or are available as application notes from the benchtop-NMR manufacturers.9−11 There are many more examples of published organicchemistry undergraduate experiments originally developed for high-field instruments that can now be easily conducted on benchtop systems.12−15 However, in terms of physical and inorganic chemistry, there is only a handful of published NMR experiments,16−20 and to our knowledge, only a few of those papers can be classed as physical-chemistry experiments. This includes the use of a 45 MHz benchtop NMR spectrometer to determine the octanol−water partition coefficient.21 Two other benchtop-NMR-manufacturer application notes describe the measurement of the pKa of pyridine10 and the determination of the partition coefficients of some common solvents.9 Opportunities thus exist to expand the utilization of benchtop NMR in physical-chemistry undergraduate teaching. This paper introduces a new physical-chemistry experiment involving measurement of relaxation parameters of a small organic molecule in different environments and how these can report on various physical phenomena. Relaxation is a fundamental concept in NMR spectroscopy, as it affects the efficiency of NMR-spectrum collection and the validity of quantification and is fundamental to the understanding of magnetic-resonance imaging. This experiment provides an important opportunity for students to experience the attributes of modern scientific research: mastering a new piece of equipment, learning a new experiment, handling data on new platforms, processing data with a variety of software packages, and bringing everything together to understand the properties of molecules from the physical-chemistry point of view.

Some benchtop NMR spectrometers use a single spin echo instead of the CPMG sequence, which does not suppress Jevolution and therefore can only be used to measure the T2 values of singlets.

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EXPERIMENT OVERVIEW In this experiment, the NMR-relaxation parameters of caffeine are measured in different solutions to investigate the effects of the solvent and the addition of a relaxation agent or a compound known to interact with caffeine. Caffeine was chosen because it is a simple molecule whose signals present themselves as singlets at 60 MHz and also because it is a wellrecognized molecule by the general public as a result of its presence in soft drinks, tea, coffee, and foods like chocolate. It has been studied extensively by NMR with several studies focusing on caffeine’s complex formation or self-aggregation.24−28 Using a benchtop spectrometer, the experiment allows students to • become proficient operators of a benchtop NMR spectrometer; • understand the basic experimental parameters involved in the acquisition of 1D NMR spectra; • gain experience with processing spectra and data analysis using NMR software; • gain appreciation of the importance of spin relaxation, its measurement, and the interpretation of experimental T1 and T2 data; and • investigate the effects of viscosity, paramagnetic species, and molecular interactions on spin relaxation. In order to analyze the data obtained in this experiment, students have to learn the appropriate data-analysis tools provided by NMR software. We decided to use Mnova as this is encountered by students in their second-year organicchemistry undergraduate teaching laboratory for examining the NMR spectra of their synthetic products. The experiment is conducted by third-year chemistry students as part of their physical-chemistry-laboratory investigations in three 3 h sessions, which allows sufficient time to acquire data, process data, and start the experimental write-up. To help students become familiar with the benchtop NMR spectrometer before the laboratory session, a prelab video was created, and a link to this video is given in the handout for students in the Supporting Information (SI). This covers the basic operation of the spectrometer, the use of a sample preheater, and a few practical tips for operating basic laboratory equipment (e.g., microbalances).

Spin−Lattice (T1)- and Spin−Spin (T2)-Relaxation Times

Spin−lattice relaxation is a measure of how fast Boltzmann equilibrium is restored after the spins are taken out of equilibrium following a radio-frequency pulse, whereas spin− spin relaxation describes the loss of coherence or dephasing of the spins in the xy-plane. These two processes are characterized by the T1 and T2 relaxation times, respectively. The spin−lattice relaxation is measured by the inversionrecovery method,22 whereas the spin−spin relaxation can be measured by a spin-echo sequence or the Carr−Parcell− Meiboom−Gill (CPMG) method.23 Inversion recovery involves inverting proton-spin populations before allowing the spins to relax during a variable relaxation delay, τ. The amount of restored magnetization is captured by a final read pulse. If the delays are set appropriately, students should see NMR signals that start negative, go through zero intensity and gradually become positive. The relaxation-time constant, T1, can be extracted from the data using eq 1: I(τ ) = I0(1 − 2e−τ / T1)

(2)

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BENCHTOP NMR SPECTROMETERS IN THE TEACHING LABORATORY The experiment is performed in the teaching laboratory and provides instant access to a benchtop NMR spectrometer. Any benchtop NMR spectrometer can be used to conduct this experiment. The key factors to consider when using a benchtop NMR spectrometer are the following: • Position: the spectrometer should be placed on a stable benchtop away sources of temperature variance (e.g., doors or air-conditioning systems). • Homogeneity: spectrometers should be shimmed by the laboratory technician or demonstrator before each session to ensure good signal-line shapes.

(1)

where I(τ) is the integral intensity as a function the relaxation delay, τ, and I0 is the equilibrium integral intensity. The CPMG sequence starts by flipping the magnetization into the transverse plane by a 90° pulse, before applying a train of short (∼1 ms) spin echoes of increasing length, τ, which suppresses J-evolution. The signal decays exponentially as a function of τ according to eq 2: B




Figure 1. 1H NMR spectrum (60 MHz) of 70 mM caffeine, recorded with 16 scans and acquired on the NMReady 60e spectrometer.

Figure 2. Stack plot of 1D 1H 60 MHz spectra obtained in an inversion-recovery experiment of caffeine in D2O (left), where each individual spectrum represents a different τ delay. T1 fitting curves for the three methyl resonances of caffeine in D2O (right). Red: H14, yellow: H12, blue: H10.

• Sample preparation: sample volumes should be between 500 and 700 μL, and it is recommended that samples be prepared at higher concentrations than for high-field NMR spectrometers (70 mM in this experiment). Specific details regarding the benchtop spectrometer used for this experiment can be found in the SI.

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benchtop NMR spectrometers because of magnetic field inhomogeneity. Once a 1D 1H spectrum of caffeine in D2O is acquired, the students proceed to measuring the T1 and T2 spectra of the same sample. Both relaxation experiments are implemented on all benchtop NMR spectrometers. For example, the NMReady 60e requires only the maximum and minimum τ delays plus the number of points to be entered. This simple setup allows students to focus on tailoring these three parameters appropriately so that the exponential dependencies are sampled appropriately. The procedure is repeated for three new samples containing • caffeine (70 mM) dissolved in CDCl3 • a 1:1 solution of 70 mM caffeine and 70 mM chlorogenic acid dissolved in D2O • caffeine (70 mM) with 10 μL of 0.09 mM stock solution of a paramagnetic inorganic complex dissolved in D2O For the third, students are given the names of two possible complexes; they then decide which one is paramagnetic. Each group should make up a different solution and record the spectra. The data is then shared among groups at the end of each session. While the experiment is running, students are instructed to proceed with learning how to process and analyze data using Mnova or use the time to search the literature. Students use the first session to learn how to operate the benchtop spectrometer, and collect data from caffeine in both D2O and CDCl3. Session 2 is used to collect the rest of the

EXPERIMENTAL PROCEDURE

The students are given the experimental procedure in written form (see the handout for students in the SI), which contains the necessary instructions to begin their investigation. Before starting the experiment, students should watch the prelab video and complete the prelab investigation. Students, working in pairs or groups of three, begin the laboratory session by discussing the prelab questions as well as the risk assessment with the demonstrator. The experiment starts with sample preparation and acquisition of a 1D 1H NMR spectrum of 70 mM caffeine in the first solvent, D2O (Figure 1). Students assign the spectrum by a comparison with literature values of chemical shifts of caffeine, which they should have found as part of the prelab questions before conducting the experiment.29 The spectrum of caffeine features two true singlets from methyl protons H10 and H12 and two slightly broad singlets from methyl proton H14 and the CH proton H8, which are coupled by a small four-bond coupling constant (0.6 Hz). This coupling is not resolved in spectra acquired on C




data. Session 3 is designated for finishing data collection, if required, and completion of data analysis and the write-up. Please see the instructor notes in the SI for the full experimental details.

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each condition must be tabulated and compared. The differences between values obtained must be justified and explained with reference to literature data. Students are free to go into as much detail as they wish with regard to explaining NMR relaxation in the context of this experiment and their experimental observations.

HAZARDS

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Before students are allowed to start the experiment, they must read and understand the risk assessment provided. The quantities of the chemicals used in this experiment are small (∼4−8 mg of the compounds and 600 μL of solvent). D2O is harmful if ingested. CDCl3 is harmful to the eyes and skin and is toxic if inhaled. The paramagnetic complex is an irritant to the eyes and skin and has specific organ toxicity. CDCl3 and the paramagnetic complex must be handled in the fume hood while wearing gloves and, in the case of gadolinium, under the supervision of a demonstrator. Safety glasses and lab coats must be worn at all times while in the laboratory.

ASSESSMENT AND OBSERVATIONS The experiment has been successfully conducted by 36 students over four semesters. Most notable was the ability and efficiency of the students in learning how to operate the spectrometer given only the manual and a prelab video. All groups understood the basic parameters associated with NMR acquisition and were able to choose the appropriate parameters for the relaxation experiments. All students were able to recognize that the differences in the T1 and T2 values were caused by the different conditions, which drew upon the lecture material already given to them in their second- and third-year chemistry lectures (e.g., π-stacking interactions). The data collected by students varied in quality, reflecting a few technical issues with the spectrometer or sample preparation. It is crucial that students check the collected data on the same day. A source of poor-quality data were, for example, if the spectrometer was left in “standby mode” for too long, after which it requires reshimming (this may be particular to the NMReady 60e or the particular laboratory environment). This issue was addressed by making sure demonstrators kept a close eye on the condition of the spectrometer. A second issue was the CDCl3 sample volume. The high vapor pressure of this solvent causes dripping from air-cushioned pipets and therefore transfer of variable volumes of the solvent. This issue was addressed by instructing students to prewet the pipet tip. After initial trials, the experiment was modified. The first modification applicable to all benchtop NMR spectrometers was the removal of the H8-proton-relaxation-time determination. This was due to the longer relaxation time of this proton (T1 = 3 s in D2O) and its substantially lower signal intensity compared with those of the signals of the methyl groups. In order to sample the relaxation curves adequately, a different set of relaxation points would have to be chosen (and possibly more scans taken), which would at least double the experimental time. This was deemed too long, and the focus was set to the methyl-group protons. The second modification made depends on the benchtop spectrometer being used to conduct this experiment. The spectrometer used in our laboratory uses a single spin-echo experiment to sample the signal decay for the measurement of T2. This leads to Jmodulation of coupled protons (H8 and H14 in the case of caffeine), which distorts the integral values. The coupled methyl proton H14 was therefore excluded from the analysis. A common point of misunderstanding for students was why spectra need to be processed with a phase or baseline correction. To aid students’ understanding, demonstrators were given instructions on how to explain these concepts after the first spectrum was recorded (see the instructor notes in the SI). When the above issues were addressed the data obtained was of high quality and reproducibility. T1 and T2 values within 0.1 s were achieved. Comparing all reports submitted from the 36 students, the lowest and highest marks obtained were 61 and 91%, respectively, and the average mark was 72%. Considering the quality of the submitted reports, the main differences were in

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PROCESSING AND ANALYSIS OF RESULTS Most benchtop NMR spectrometers (NMReady, Spinsolve, and Picospin) produce DX or JDX files containing the T1 or T2 spectra stored in a pseudo-2D manner that will open in all major NMR software packages (e.g., Mnova and TopSpin). Note that Pulsar produces MNOVA files directly. The files are transferred from the spectrometer using a USB pen drive and simply dragged and dropped into the chosen software. The step-by-step instructions for processing the data in Mnova are given in the instructor notes in the SI. Each spectrum obtained has to be processed to ensure the peak phase and baseline are correct and that the quality of the data is high. An example of a processed, stacked plot of T1 spectra obtained for caffeine in D2O on the NMReady 60e is shown in Figure 2. Each individual spectrum represents a different τ delay. As τ increases, the peaks change from negative through zero to positive. Once processed, the integral intensity of each caffeine resonance can be fitted using the appropriate equation (eq 1 or 2 for T1 and T2, respectively). NMR software packages like Mnova allow easy determination of T1 and T2 values. It is important to note that the τ values may need to be inputted manually into NMR software like Mnova. In terms of the NMReady 60e, an additional CSV file is also created that contains the τ delays used to acquire each spectrum as well as the relaxation data interpreted by the benchtop NMR spectrometer. This data uses the integral regions chosen by the initial integration of the 1D spectrum. Note that the data can also be evaluated in Excel or Origin by plotting the intensities versus the delays and using regression analysis based on eq 1 or 2. An example of the T1 fitting curves obtained for the three methyl signals of caffeine in D2O is shown in Figure 2. Exemplar data obtained from the NMReady 60e NMR spectrometer can be found in the Supporting Information. After completion of the experiment, students are required to write up the experiment in a journal format. The required content for the report and marking scheme are found in both the instructor notes and the handout for students in the SI. Briefly, these reports should introduce the key terms and concepts behind T1 and T2 relaxation and the inversionrecovery and CPMG or spin-echo experiments. The procedure students take from processing the spectra to extracting relaxation values must be demonstrated, including the graphs and spectra. The T1 and T2 values of caffeine obtained under D




feedback on the experiment and S. Mooney, the physicalchemistry technician, for looking after the experiment and the equipment. J.K. would like to acknowledge M. Kosar for his input when running the original experiments.

the level of detail in explaining the phenomenon of relaxation and the rationalization of the observed values. Although some students cited size, viscosity, and the magnetic moments of electrons, others went as far as trying to explain relaxation trends by introducing the spectral-density function. In terms of presentation, the best reports demonstrated the data processing (stack plot of spectra) and analysis (fitting curves) on one condition and provided only the T1/T2 values for the other sample conditions. The data and analysis of the other conditions were provided in appendices. A common mistake by a number of students was to forget to quote all the NMR parameters used. When asked, all students reported that the experiment had improved their understanding of the workings of NMR and its relevance to the study of molecules and their interactions.

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CONCLUSIONS The investigation described in this paper offers a new experiment that uses a benchtop NMR spectrometer in a physical-chemistry lab. Benchtop NMR is a welcome addition to undergraduate laboratories as a way of introducing NMR to students. It can be used in all laboratories, organic, inorganic, and physical, for different purposes. The contribution of this experiment is that it allows students to learn key parameters that influence acquisition of all NMR experiments in an easy and straightforward manner and connects a rather esoteric concept of NMR relaxation with tangible molecular properties. Fitting of exponential functions is prevalent in the natural sciences, and the NMR-relaxation process is a good example of this phenomenon. As many students will use NMR during their final-year projects or on industrial placements, the skills learned demystify NMR and provide a good platform for expanding their knowledge in this area. This experiment epitomizes many of the attributes of everyday scientific research: mastering a new piece of equipment and handling the data in different platforms and software.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.8b00633. Handout for students (PDF, DOCX) Instructor notes (PDF, DOCX) Sample data obtained on a 60 MHz spectrometer (PDF) Details of the benchtop NMR spectrometer tested (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Nicholle G. A. Bell: 0000-0001-7887-2659 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS N.G.A.B. would like to acknowledge J. Hardie, R. Thakor, G. Coulter, M. Compton, B. Hoggan, E. Andrews, P. O’Kane, K. Bisset, and T. Callaghan who initially performed and provided E




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Molecular Properties of Caffeine Explored by NMR: A Benchtop NMR

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