Laboratory Experiment Cite This: J. Chem. Educ. XXXX, XXX, XXX-XXX
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qHNMR Analysis of Purity of Common Organic SolventsAn Undergraduate Quantitative Analysis Laboratory Experiment Peter T. Bell, W. Lance Whaley, Alyssa D. Tochterman, Karl S. Mueller, and Linda D. Schultz* Department of Chemistry, Geosciences, and Physics, Box T-0540, Tarleton State University, Stephenville, Texas 76402, United States S Supporting Information *
ABSTRACT: NMR spectroscopy is currently a premier technique for structural elucidation of organic molecules. Quantitative NMR (qNMR) methodology has developed more slowly but is now widely accepted, especially in the areas of natural product and medicinal chemistry. However, many undergraduate students are not routinely exposed to this important concept. This article describes a simple and practical lab experiment that has been successfully performed by students in a Quantitative Analysis class for several years and is based on a comparison of relative integration areas of species present in spectra of compound mixtures. In this experiment, 1H NMR spectroscopy is used to determine the purity of common organic solvents using dimethyl sulfoxide as an internal standard in D2O. Groups of students analyze unknown samples containing one of the following solvents: methanol, ethanol, 2-propanol, tetrahydrofuran, or acetone, to which water has been added as an impurity. Over a period of five years, 54 students analyzed samples ranging from 60% to 99% purity with an average error of 2.64%. This experiment fills a niche in the initial portion of a standard Quantitative Analysis lab sequence by differentiating between qualitative and quantitative analysis, providing exposure to equipment not usually encountered in an introductory analytical lab, and generating numerical data for students to analyze and evaluate. KEYWORDS: First-Year Undergraduate/General, Second-Year Undergraduate, Analytical Chemistry, Organic Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, NMR Spectroscopy, Quantitative Analysis, Instrumental Methods
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to qNMR8 detail the progress made in this area, and in 2014 absolute quantitative 1H NMR spectroscopy became listed in the Guidelines for Authors as an accepted method for the establishment of compound purity for papers published in the Journal of Medicinal Chemistry.9 Although several abbreviations describing qNMR techniques have been used in publications, qHNMR is suitable for specification of methods using 1H NMR spectroscopy.8 1 H NMR spectroscopy can be used to determine the relative amounts of components in mixtures by analyzing the relative integration values of proton signals of each compound, provided that the different compounds have sufficiently distinct NMR resonances and the absolute amount of one major (>1%) compound can be used as an internal standard.10,11 Some early applications of qHNMR, including several in the field of chemical education,12−19 utilized analysis of relative integration areas, and a classic experiment for undergraduate Instrumental Analysis involved the quantitative determination of a mixture of benzene and ethyl alcohol in CDCl3 by 1H NMR spectroscopy on the basis of relative integration areas.20 Some more recent qHNMR experiments for the teaching laboratory have utilized standard addition methods,21,22 and qHNMR has been incorporated into kinetics and mechanistic studies.23−26
INTRODUCTION Nuclear magnetic resonance (NMR) theory was proposed as long ago as 1925 by Wolfgang Pauli but was first demonstrated independently in 1946 by Bloch and Purcell, for which they shared the 1952 Nobel Prize. The first commercial NMR spectrometer was marketed in 1953, and it became a common analytical tool in the 1960s.1 Two indicators of the general acceptance of the technique are that contemporary introductory organic chemistry texts contain entire chapters devoted solely to NMR spectroscopy and that access to NMR instrumentation is a requirement for American Chemical Society undergraduate chemistry program certification. Small benchtop instruments currently available have even been transported to public schools to introduce high school students to the concepts of NMR spectroscopy.2 NMR spectroscopy has many characteristics of a good quantitative analytical method: it requires minimal sample, is rapid and nondestructive, yields information on both the identity and relative amount of each analyte present, and is now readily accessible to most universities. Historically, NMR spectroscopy has been considered almost exclusively a tool for structural elucidation and to be of limited use in quantitative applications. However, improvements in instrumentation have changed this image, and quantitative NMR (qNMR) had become a widely accepted technique by 2001, especially in the areas of natural product and medicinal chemistry.3,4 An excellent series of review articles5−7 and a Web site dedicated © XXXX American Chemical Society and Division of Chemical Education, Inc.
Received: May 22, 2017 Revised: September 19, 2017
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DOI: 10.1021/acs.jchemed.7b00343 J. Chem. Educ. XXXX, XXX, XXX−XXX
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Figure 1. Partial 1H NMR spectrum of ethanol and DMSO in D2O.
commercial samples, but they can also be prepared by the instructor. Student results should depend only on the care and precision of the student’s lab technique. The principles of qNMR make it an attractive candidate for inclusion as a laboratory experiment in a Quantitative Analysis course. Such an experiment fits well into the early portion of the curriculum, where the foundations of analytical chemistry are first introduced. A qHNMR experiment illustrates differences between qualitative and quantitative analysis and classical and instrumental methods and provides practice in solution preparation, pipetting, and dilution techniques. This experiment also generates numerical data that students can analyze and evaluate for precision while the basic techniques for classic gravimetric and volumetric analyses are still being covered in lecture. The students have already been introduced to the use of analytical balances and volumetric glassware during the lab check-in process, and complexity of sample preparation is minimal for this experiment. The use of D2O as an NMR solvent allows investigation of many common water-soluble components and eliminates hazards associated with qNMR procedures that utilize CDCl3 or other toxic compounds, and unknown sample preparation is fast and simple for the instructor. The experiment described herein also utilizes some volumetric equipment that is simple to use but may be new to some students, such as the syringe-type pipets. Finally, the experiment is fast, and individual students can obtain their raw data in only one lab period. Students are provided with a review of the fundamentals of NMR theory and instrumentation at least 1 week prior to performing the experiment. An essential consideration in this experiment is that speed is crucial. Since most of the compounds involved are volatile, all solutions should be prepared and analyzed within a time frame of less than 1 h to obtain optimal results. Therefore, unknown sample preparation time by the instructor, student sample preparation time, and instrument and operator availability must be carefully scheduled. Ideally, the student should work with the instrument operator, who explains the operational procedures and output to the student so that the NMR spectrometer is not just a “black box”.
However, the focus of the majority of these experiments has been on the relative concentrations of the components of mixtures, not the purity of an individual compound, which should be a topic of interest to undergraduate students. Although the above-referenced works bear testimony to the increasing utility and acceptance of qHNMR as an analytical technique, it is still not widely recognized in introductory analytical chemistry courses. Therefore, a need exists for a simple, practical laboratory experiment to introduce this concept to undergraduate chemistry students utilizing safe, less toxic (greener) chemicals with a focus on determination of purity. This experiment fits well into the early portion of a traditional introductory course by familiarizing students with the lab techniques of weighing on analytical balances and precisely pipetting very small volumes of solvents. The students also generate (and process) data from an instrument with which they are familiar, but they use these data as the basis of quantitative rather than qualitative analysis. This experiment can be done during that awkward period early in the course when basic material essential for performing later experiments is still being covered in lecture.
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EXPERIMENTAL OVERVIEW The goal of this project was to develop a simple laboratory experiment using 1H NMR spectroscopy to introduce principles of quantitative analysis to a group of undergraduate students with diverse levels of chemistry laboratory experience. Although Quantitative Analysis is considered to be an advanced-level course in a “typical” curriculum, individual student scheduling issues typically lead to a class mixture containing individuals with greatly varying degrees of previous chemistry experience at the authors’ institution. However, the majority of these students have already taken Organic Chemistry and thus have been exposed to NMR theory and spectral interpretation of structure. Additionally, this is the first course that most students encounter in which the balance of the grade is based on the accuracy of quantitative results. Therefore, the “unknowns” must have a value that is known to the instructor as a basis for the grade. Typically, these are B
DOI: 10.1021/acs.jchemed.7b00343 J. Chem. Educ. XXXX, XXX, XXX−XXX
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The basis of qHNMR is that the integration area of a peak is directly proportional to the number of nuclei responsible for that individual peak. Therefore, a pure sample of a specific compound is not required for calibration; it is only necessary that an identifiable resonance of the analyte does not overlap resonances of other sample constituents. The concentration of the analyte is then directly obtained by comparing the signal areas of its protons with those of a known concentration of an internal standard.1 The probability of overlapping resonances becomes greater with increasing sample complexity, but this should not be a factor in unknowns prepared by an instructor by dilution of relatively pure compounds to obtain student unknowns of known composition for analysis in a quantitative analysis experiment. Selection of an appropriate internal standard compound is essential to a qNMR experiment. The standard should ideally be inexpensive and readily available in a highly pure form, contain a small number of magnetically equivalent protons that do not resonate in an area of the spectrum that overlaps with analyte peaks, be stable and soluble in the NMR solvent of choice, have low volatility, and be nontoxic and inert.26 The internal standard selected for this experiment, dimethyl sulfoxide (DMSO) satisfies most of these requirements. It is inexpensive and readily available in high purity, has six magnetically equivalent protons that do not overlap with those of the compounds being analyzed in this experiment, is water-soluble, has low toxicity, and is nonreactive with the analytes. It is hygroscopic and volatile, but no more so than the compounds being analyzed. The relevant portion of an NMR spectrum of ethanol and DMSO is shown in Figure 1. It should be noted that although both DMSO and ethanol have six protons, only five of the ethanol protons are used for calculations because of the rapid ethanol hydroxyl proton exchange with D2O. The calculations are based on relative peak areas of the analyte and internal standard as described by Wallace:10
Laboratory Experiment
EXPERIMENTAL PROCEDURES
A student handout briefly describing basic NMR theory and instrumentation was prepared by the instructor using material from a current Instrumental Analysis text1 and given to the students 1 week prior to the experiment. The focus of the handout was not on spectral interpretation, because that information is presented in the Organic Chemistry courses. The laboratory portion of the Quantitative Analysis course on this campus consists of two three-hour lab periods per week. However, for this experiment the students were assigned specific lab times to arrive and begin their individual data acquisitions, which were expected to require approximately one hour per student. Students were scheduled at 30 minute intervals. Upon arrival, each student first prepared a solution containing approximately equal volumes of his or her “pure” analyte and DMSO. To minimize the relative weight changes caused by evaporation, sample volumes of about 10 mL of both analyte and DMSO were used. All of the mixtures were prepared by transferring these volumes into preweighed, ovendried, glass-stoppered 50 mL volumetric flasks, and the weights of each were determined by difference. All of the weights were determined to 0.1 mg on a Metler-Toledo AB 104-S analytical balance, and when the requisite amounts of analyte and DMSO had been combined, the flasks were immediately stoppered and swirled to mix. Appropriate sample and D2O volumes were transferred to glass NMR tubes using Eppendorf pipettes, and NMR spectra were obtained as quickly as possible. After the spectrum of the “pure” analyte with DMSO had been obtained, the student returned to the lab where he/she received an “unknown” analyte sample that had been freshly prepared by the instructor. This sample contained analyte and an unknown amount of deionized water. An NMR sample of the student unknown mixture was prepared in the same manner as for the pure analyte. Spectra obtained prior to 2013 were obtained on a Varian Gemini 200 MHz 1H FT-NMR spectrometer; later spectra were obtained on a Bruker Fourier 300 spectrometer. As previously noted, proper regulation of instrumental parameters markedly improves the accuracy of relative proton integration areas in a compound. Specific details are included in the student lab handout in the Supporting Information.5,30 All students doing this experiment in the same lab period analyzed the same “pure” analyte and compared their results. It should be noted that sample purity depends only upon the actual amount of analyte present in a known sample mass. All of the chemicals were purchased as 99.5% pure or better, and most were in new, unopened bottles. However, the values of some of these “pure” compounds were markedly less than 100%, as noted by Wells,27 because the analysis of purity had been obtained by other methods and in some cases the bottles had been previously opened and exposed to moisture. The students calculated the purities of both their “pure” analytes and their unknown solutions by means of the relationship between the integration areas of the analyte and the internal standard. Water was chosen to manipulate the analyte concentration because it is a common impurity in alcohols and is nontoxic. Then, since the unknowns had been prepared by dilution of the “pure” analyte samples, a correction factor was applied by dividing the experimental unknown value by the experimental percentage of the pure analyte to obtain a
na A N = a × s ns As Na
where na and ns are the molar amounts, Aa and As are the integral areas, and Na and Ns are the numbers of resonating protons in the analyte and internal standard compounds, respectively. Solutions for analysis are prepared by the students and contain known masses of the analyte of unknown concentration and the internal reference, so the molar amount of the internal standard is related to the molar amount of the analyte in the solution of unknown concentration. The molar amount of analyte is converted to the mass of analyte, which is divided by the mass of the analyte of unknown concentration analyzed to give the purity of the analyte as percent by mass. Although relative proton integration areas in a compound are not always perfect, they can be optimized by regulating the delay times between pulses as well as other instrumental parameters, and when total integration areas of different compounds are compared, good quantitative results at the percent level are obtained for mixtures.28,29 This accuracy range is typical of that obtained by students using commercial unknown samples in a classical undergraduate Quantitative Analysis course. C
DOI: 10.1021/acs.jchemed.7b00343 J. Chem. Educ. XXXX, XXX, XXX−XXX
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normalized percentage for comparison purposes. Realistic unknown values should be greater than 80%; therefore, only a few unknowns with purities below this value were analyzed, and none had a purity of less than 60%. Student performance was evaluated using the same standards as all other laboratory experiments in this course. Results were reported as a component of a formal lab report that discussed the basic theory of the chemistry and analytical principles of the procedure. Separate evaluations were made of the accuracy of the unknown results compared to the theoretical value, and the quality of the content and written communication of the lab reports was compared to standards expressed in rubrics that were included in the course syllabus and are included in the Supporting Information. As per university policy for all experimental procedures involving students, the primary author (course instructor) underwent IRB training, and an IRB approval form was submitted to the University Institutional Review Board for review in 2015. The application was approved as Exempt (under 45 CFR 46.101b1), and IRB protocols were followed throughout the duration of the experiment. However, this experiment had been in use since 2011, which preceded implementation of these procedures on our campus, and some of the data included in this article were obtained during prior years. All of the information from this period was carefully examined and subjected to the same standards regarding participant confidentiality as the more current results.
Figure 2. Correlation of normalized qHNMR-based results with actual mass percent of analyte for five common organic solvents in solvent− water mixtures.
recommended as a student unknown. Because of the time limitations in a structured laboratory class, the students in the class analyzed only one pure sample and one mixture, but the research students did analyze duplicate samples with good correlation. The primary sources of error were found to be weighing and/or pipetting errors by the students and errors caused by sample evaporation if excessive time elapsed between sample preparation and analysis. Student outcomes for the qNMR experiment as measured by grades on numerical results and lab reports were similar to those of the traditional experiments in the Quantitative Analysis class and are shown in Table 2 in the Supporting Information. qNMR was not a topic covered in the textbook, so it was not included among the exam topics for the course. Hence, student feedback about the experiment was primarily anecdotal. Those advanced students who had already been exposed to the NMR instrument found the quantitative application of the instrument enlightening, while those students who had been exposed only to the theory of the instrument were pleased to actually prepare samples and observe the operation of the instrument on an individual basis. Some of the students who had completed only General Chemistry were a bit overwhelmed by the instrument, but they were also able to successfully complete the experiment and write a satisfactory lab report. The main source of negative feedback came from schedule breakdowns when delays occurred and students had to wait for an extended period of time before their turn to obtain spectra. However, when the students received their unknown grades and were shown the graph of their class results (similar to Figure 2 above), complaints vanished and the students were impressed by the outcome of the experiment and recognized the potential of the method as a validation of purity.
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HAZARDS AND SAFETY Care must be taken in handling all chemicals. Methanol, ethanol, 2-propanol, tetrahydrofuran, dimethyl sulfoxide, and acetone are all flammable and toxic. Therefore, none of these solvents should be exposed to an open flame or ingested. Small amounts of excess ethanol and 2-propanol may be disposed of by flushing down a sink with adequate volumes of water. However, all of the samples containing other organic solvents should be collected and disposed of with proper precautions. Proper clothing, shoes, gloves, and goggles with splash protection are required, and caution must be exercised in handling glass NMR tubes to prevent injury.
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RESULTS AND DISCUSSION
The normalized results obtained by 54 undergraduate students in Quantitative Analysis classes during the period 2011−2015 are shown in Table 1 in the Supporting Information. (The instrument was not operational at the time the experiment was scheduled in 2016). Errors were expressed as the absolute value of the difference between the experimental and theoretical values and were random in nature, as can be seen by the plot of theoretical versus experimental values shown in Figure 2. The average error was 2.64%, with a standard deviation of 1.87%. Four results were eliminated by Q test at the 95% confidence level31 and were excluded from the calculation of the average value and Figure 2. All of the results reported were obtained by students enrolled in the Quantitative Analysis classes. However, in prior years undergraduate research students performed this procedure multiple times with all of the above analytes to validate the method and generally obtained more consistent results. N,NDimethylformamide (DMF) was also examined but yielded inconsistent results, possibly due to interactions between the DMF and water. 32 Therefore, this compound is not
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CONCLUSIONS Analysis of the purity of water-soluble solvents on the basis of relative 1H integration areas of a pure standard used to analyze a second relatively water-soluble solvent by 1H NMR spectroscopy with D2O solvent showed great promise as a laboratory experiment in an undergraduate Quantitative Analysis course. Optimization of instrumental parameters increased the accuracy, and the use of large sample sizes and rapid analysis after sample preparation minimized problems due to evaporation. The technique was rapid, and sample preparation time was minimal. Ethanol, methanol, 2-propanol, tetrahydrofuran, and acetone mixtures of varying concenD
DOI: 10.1021/acs.jchemed.7b00343 J. Chem. Educ. XXXX, XXX, XXX−XXX
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(11) Eberhart, S. T.; Hatzis, A.; Rothchild, R. Quantitative NMR assay for aspirin, phenacetin and caffeine mixtures with 1,3,5-trioxane as internal standard. J. Pharm. Biomed. Anal. 1986, 4, 147−154. (12) Kolthoff, I. M.; Elving, P. J. Treatise on Analytical Chemistry, Part I, Volume 4: Theory and Practice; John Wiley and Sons: New York, 1963. (13) Hollis, D. P. Quantitative analysis of aspirin, phenacetin, and caffeine mixtures by nuclear magnetic resonance spectroscopy. Anal. Chem. 1963, 35, 1682−1684. (14) Smith, W. B. Quantitative analysis using NMR. J. Chem. Educ. 1964, 41, 97−99. (15) Markow, P. G.; Cramer, J. A. An Analysis of a Commercial Furniture Refinisher: A Comprehensive Introductory NMR Experiment. J. Chem. Educ. 1983, 60 (12), 1078−1079. (16) Phillips, J. S.; Leary, J. J. A qualitative-quantitative proton-NMR experiment for the instrumental analysis laboratory. J. Chem. Educ. 1986, 63, 545−546. (17) Peterson, J. 1H NMR analysis of mixtures using internal standards: a quantitative experiment for the instrumental analysis laboratory. J. Chem. Educ. 1992, 69, 843−845. (18) Clarke, D. W. Acetone and ethyl acetate in commercial nail polish removers: a quantitative NMR experiment using an internal standard. J. Chem. Educ. 1997, 74, 1464−1465. (19) Doscotch, M. A.; Evans, J. P.; Munson, E. J. Fourier Transform Nuclear Magnetic Resonance Spectroscopy Experiment for Undergraduate and Graduate Students. J. Chem. Educ. 1998, 75, 1008−1013. (20) Sawyer, D. T.; Heineman, W. R.; Beebe, J. M. Chemistry Experiments for Instrumental Methods; John Wiley & Sons: New York, 1984; p 294. (21) Hoffmann, M. M.; Caccamis, J. T.; Heitz, M. P.; Schlecht, K. D. Quantitative analysis of nail polish remover using nuclear magnetic resonance spectroscopy revisited. J. Chem. Educ. 2008, 85, 1421−1423. (22) Rajabzadeh, M. Determination of unknown concentrations of sodium acetate using the method of standard addition and proton NMR: An experiment for the undergraduate analytical chemistry laboratory. J. Chem. Educ. 2012, 89, 1454−1457. (23) Clark, M. A.; Duns, G.; Golberg, G.; Karwowska, A.; Turgeon, A.; Turley, J. NMR Analysis of Product Mixtures in Electrophilic Aromatic Substitution. J. Chem. Educ. 1990, 67, 802. (24) Peterson, T. H.; Bryan, J. H.; Keevil, T. A. A Kinetic Study of the Isomerization of Eugenol. J. Chem. Educ. 1993, 70, A96−A98. (25) Friesen, J. B.; Schretzman, R. Dehydration of 2-Methyl-1cyclohexanol: New Findings from a Popular Undergraduate Laboratory Experiment. J. Chem. Educ. 2011, 88, 1141−1147. (26) Her, C.; Alonzo, A. P.; Vang, J. Y.; Torres, E.; Krishnan, V. V. Real-Time Enzyme Kinetics by Quantitative NMR Spectroscopy and Determination of the Michaelis−Menten Constant Using the Lambert-W Function. J. Chem. Educ. 2015, 92, 1943−1948. (27) Wells, R. J.; Cheung, J.; Hook, J. M. Dimethylsulfone as a Universal Standard for Analysis of Organics by QNMR. Accredit. Qual. Assur. 2004, 9, 450−456. (28) Pauli, G. F.; Jaki, B. U.; Lankin, D. C. A Routine Experimental Protocol for qHNMR Illustrated with Taxol. J. Nat. Prod. 2007, 70, 589−595. (29) Weizman, H. Why Are 1H NMR Integrations Not Perfect? An Inquiry-Based Exercise for Exploring the Relationship between Spin Dynamics and NMR Integration in the Organic Laboratory. J. Chem. Educ. 2008, 85 (2), 294−296. (30) Bharti, S. K.; Roy, R. Quantitative 1H NMR Spectroscopy. TrAC, Trends Anal. Chem. 2012, 35, 5−26. (31) Rorabacher, D. B. Statistical Treatment for Rejection of Deviant Values: Critical Values of Dixon’s “Q” Parameter and Related Subrange Ratios at the 95% Confidence Level. Anal. Chem. 1991, 63, 139−146. (32) Mishustin, M. I.; Kessler, Yu. M. Interactions Between Water and Dimethylformamide in the Liquid Phase. Zh. Strukt. Khim. 1974, 15, 205−209.
trations were analyzed using this technique by undergraduate students with good results. Dimethyl sulfoxide proved to be satisfactory as an internal standard. The goal of the experiment, to demonstrate to undergraduate Quantitative Analysis students the utility of qHNMR as a quantitative tool to assay the purities of some common organic solvents, was achieved. The experiment also worked well as an initial experiment in the laboratory sequence.
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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.7b00343. Student laboratory handout, notes for instructors, sample student results, and assessment rubrics for lab reports (PDF, DOC)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Linda D. Schultz: 0000-0001-6086-1484 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank the students in the Quantitative Analysis class of 2015 for participating in the experiment and Dr. Bernat Martinez for help with the abstract graphic. The financial assistance of The Robert A. Welch Foundation, Chemistry Departmental Grant AS-0012 is gratefully acknowledged.
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REFERENCES
(1) Skoog, D. A.; Holler, F. J.; Crouch, S. R. Principles of Instrumental Analysis, 7th ed.; Cengage Learning: Boston, 2018; p 453. (2) Bonjour, J. L.; Pitzer, J. M.; Frost, J. A. Introducing High School Students to NMR Spectroscopy through Percent Composition Determination Using Low-Field Spectrometers. J. Chem. Educ. 2015, 92, 529−533. (3) Maniara, G.; Rajamoorthi, K.; Rajan, S.; Stockton, G. W. Method Performance and Validation for Quantitative Analysis by 1H and 31P NMR Spectroscopy. Applications to Analytical Standards and Agricultural Chemicals. Anal. Chem. 1998, 70, 4921−4928. (4) Evilia, R. F. Quantitative NMR spectroscopy. Anal. Lett. 2001, 34, 2227−2236. (5) Pauli, G. F.; Jaki, B. U.; Lankin, D. C. Quantitative 1H NMR: development and potential of a method for natural products analysis. J. Nat. Prod. 2005, 68, 133−149. (6) Pauli, G. F.; Jaki, B. U.; Gödecke, T.; Lankin, D. C. Quantitative 1 H NMR: development and potential of a method for natural products analysis - An Update. J. Nat. Prod. 2012, 75, 834−851. (7) Pauli, G. F.; Chen, S.-N.; Simmler, C.; Lankin, D. C.; Gödecke, T.; Jaki, B. U.; Friesen, J. B.; McAlpine, J. B.; Napolitano, J. G. Importance of Purity Evaluation and the Potential of Quantitative 1H NMR as a Purity Assay. J. Med. Chem. 2014, 57, 9220−9231. (8) The Quantitative NMR Portal. http://qNMR.org (accessed August 2017). (9) Cushman, M.; Georg, G. I.; Holzgrabe, U.; Wang, S. Absolute Quantitative 1H NMR Spectroscopy for Compound Purity Determination. J. Med. Chem. 2014, 57, 9219. (10) Wallace, T. Quantitative analysis of a mixture by NMR spectroscopy. J. Chem. Educ. 1984, 61, 1074. E
DOI: 10.1021/acs.jchemed.7b00343 J. Chem. Educ. XXXX, XXX, XXX−XXX
