Laboratory Experiment pubs.acs.org/jchemeduc
A First Laboratory Utilizing NMR for Undergraduate Education: Characterization of Edible Fats and Oils by Quantitative 13C NMR Charles G. Fry,* Heike Hofstetter, and Matthew D. Bowman Department of Chemistry, University of WisconsinMadison, Madison Wisconsin 53706, United States S Supporting Information *
ABSTRACT: Quantitative 13C NMR provides a straightforward method of analyzing edible oils in undergraduate chemistry laboratories. 13C spectra are relatively easy to understand, and are much simpler to analyze and workup than corresponding 1H spectra. Average chain length, degree of saturation, and average polyunsaturated fatty acid content of common edible oils are obtained directly using integrals of the proper regions in the 13 C 1D spectra. These spectra segue to 2D 1H−13C HSQC experiments that provide an excellent path for students to verify assignments, in particular those associated with polyunsaturated fatty acids. Data acquisition and analysis can be completed in one to two lab sections, although spectrometer scheduling may be necessary for groups of students greater than six. 13C relaxation measurements of common oils are presented that enable accurate quantitative 13C data to be obtained in reasonable amounts of time (30−40 min per sample) on modern 400 MHz NMR spectrometers. KEYWORDS: First-Year Undergraduate/General, Second-Year Undergraduate, Analytical Chemistry, Organic Chemistry, Inquiry-Based/Discovery Learning, Consumer Chemistry, Fatty Acids, Instrumental Methods, NMR Spectroscopy, Quantitative Analysis
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We present a laboratory that provides 13C NMR-based quantitative measurements of average chain length, degree of saturation, and average polyunsaturated fatty acid3 content of common edible oils. This work was inspired by publications by Crowther,4 and Hartel and Moore.5 1H NMR takes a prominent place in both papers. Complementary techniques such as IR4 and extraction plus gravimetric measurements5 are included. However, for our nonchemistry majors’ course, the combined set of lectures and laboratories that would be required is too involved. We were still presented with difficulties when reducing the scope to just 1H NMR. The analysis of the methine glycerol protons from vinyl protons in most edible oils is formidable, due to spectral overlap of these different protons. Simple integration is insufficient to obtain accurate quantitative data, due to the extent of overlap. 13C NMR, on the other hand, provides spectra that are completely resolved in these regions. We therefore investigated the plausibility of performing quantitative 13 C NMR.6 An experimental protocol is presented that enables accurate measurements to be made on any modern NMR spectrometer. Experiment times are 30−40 min per sample on a 400 MHz spectrometer equipped with a standard broadband or 13C direct-observe probe.7 A carbon-optimized liquid-heliumcooled cryoprobe provides maximum performance, where 1 scan is sufficient to obtain high-quality data.8 Experiments were
MR spectroscopy is a premier tool for chemical characterization. Its instruction is therefore a crucial component of undergraduate organic laboratories. NMR provides a framework for the pedagogy of understanding chemical bonding and stereochemistry through three key characteristics of NMR spectra: chemical shift, integration, and coupling. While 1H NMR is the most common technique utilized in research, 13C NMR has been lauded as an entry point into spectroscopy in undergraduate courses as the lack of spin− spin splitting and the decreased possibility of coincidental overlap make interpreting the spectra more intuitive: one type of carbon gives one distinct chemical shift. Due to its relatively straightforward nature, 13C NMR has been incorporated into general chemistry courses and even college preparatory programs.1 The focus has been on the use of chemical shift as 13C chemical shift information is sufficient for many assignments. Improvements in modern 13C chemical shift predictions and empirical chemical shift calculations2 provide powerful tools to complement the data. Very important for these studies is the purely quantitative nature of NMR. As a pedagogical bridge to 1H NMR, 13C NMR can be made quantitative allowing the incorporation of the concept of integration. In general, the low natural abundance of 13C (1.1%) greatly inhibits the use of the nucleus at natural abundance for quantitative studies. For edible oils, however, sample concentrations can be made quite high (neat oils are feasible; we use oil:solvent at 50:50%), enabling good quantitation in 13C 1D spectra to be achieved in reasonable amounts of spectrometer time. © XXXX American Chemical Society and Division of Chemical Education, Inc.
Received: January 23, 2017 Revised: May 23, 2017
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DOI: 10.1021/acs.jchemed.7b00057 J. Chem. Educ. XXXX, XXX, XXX−XXX
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run their own sample. Assuming 30 min per sample for a run, data can readily be acquired in two overnight runs on a 400 MHz spectrometer with an autosampler. Access to a cryoprobe will further reduce the required spectrometer time. Setup of samples using automation software is easy even for novice NMR users after a short introduction. Alternatively, a TA or NMR staff can queue samples. This experiment has been run in our course since 2012. Data processing and analysis completes the assignment. Nonmajor students will receive processed data and will only perform the analysis. One optional extension, suitable for any level student, involves a second lab that combines the newly acquired skills from the first lab with an extraction lab. Students bring samples of other food items to investigate their fat content. Another optional extension, most suitable for chemistry majors and honors sections but requiring an additional second lab, has students acquiring a 1H−13C edited-HSQC spectrum of their edible oil (and optionally the other food item). Data acquisition can be carried out as described before. Students confirm a variety of 13C and 1H assignments utilizing the 2D spectrum combined with assignments aids.
carried out on a Bruker AVANCE 400 MHz spectrometer equipped with a BBFO+ probe or a Bruker AVANCE 500 MHz instrument with a DCH cryoprobe. Standard pulse sequences were used; implementation of these experiments on Varian/ Agilent and JEOL spectrometers is straightforward. The 1D 13C measurements can be complemented by 1 H−13C single-bond correlation 2D spectroscopy, editedHSQC. This optional segue to 2D NMR enables the students to confirm many aspects of the 13C assignments: (a) aliphatic vs vinyl vs carbonyl regions; (b) glycerol carbons; (c) polyunsaturated methylenes; and (d) cis- versus trans-vinyl carbons. 1 H assignments can be made using the HSQC spectra for students curious about other methods (e.g., as described in refs 4 and 5). Inclusion of HSQC transitions an NMR-based laboratory suitable for nonchemistry majors into one suitable for chemistry majors and honors students. Another optional addition to the laboratory is to have students perform the measurements on food items of their choosing: French fries, cake frosting, donuts, salad dressing, bacon grease, etc. Extraction is accomplished via a simple method. Data acquisition and analysis are identical to those performed on the edible oils. This addition further engages the students, allowing them to benefit from their curiosity, and can provide surprising and interesting results. One difficulty of first time NMR instruction is the amount of material to cover. The three main characteristics of proton NMR, chemical shift, integration, and coupling, can take three to four lectures to cover. In a laboratory course that meets once a week, this amounts to almost one-fourth of a semester. That is compounded if the students, e.g., nonchemistry majors, have no prior experience in organic chemistry. One of the key reasons to opt to teach 13C NMR before 1H NMR is that there is generally only a single item to pay attention to, the chemical shift. The chemical shift can be associated with basic structural information and compared to a chemical shift table. Integration is conceptually easier to understand than chemical shift. A drawback to 13C NMR is that when using conventional acquisition methods, a vast majority of the time spectra are not quantitative, and integration is therefore meaningless. On the other hand, proton decoupling is used to simplify spectra and improve sensitivity as a result of the nuclear Overhauser effect. Simple modifications described here make 13C NMR quantifiable, such that two of the three characteristics of NMR can be taught, namely, chemical shift and integration. These topics are mainly dependent on the concepts of symmetry and hybridization, which are covered in a previous general chemistry course. No prior formal organic chemistry lectures are necessary to start and interpret the results of the spectra. As an added bonus, a majority of our students that take the class are dietetics majors, and the idea of analyzing something they are familiar with piques their interest.
Sample Preparation
For edible oils, samples were prepared by mixing oils with chloroform-d (50:50%). Chloroform or chloroform-d may be used; deuterated solvents are not required. Instructions for acquiring data with proteo-solvent or no solvent (i.e., a no-D) setup is provided in the Supporting Information (SI). For other food items, chloroform or chloroform/methanol extraction, similar to standard saturated fat measurements, was carried out for isolation of fats.9 For chemistry majors and honors laboratories, we are considering adopting the more detailed extraction and gravimetric analysis of Hartel and Moore.5,10 Quantitative
13
C NMR
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Quantitative C spectra cannot be obtained with protocols used for obtaining standard 1D 13C NMR data, as this protondecoupled carbon, 13C{1H}, experiment generates nuclear Overhauser enhancements (NOEs) of the carbons from neighboring protons. Modifying the standard experiment to eliminate NOEs is not in itself sufficient. To obtain accurate quantitative results from 13 C data, four conditions must be met: 1. The proper pulse sequence must be used. 2. The repetition delay, combined with proper pulse angle, must be sufficiently long. 3. The data must be acquired with enough data points to accurately represent the shape of each 13C resonance line. 4. The data must be processed correctly.
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These simple modifications are described in section IV of the SI.
EXPERIMENTAL SECTION Students in our nonchemistry majors laboratory course (Chem 342) are introduced to 13C NMR during the class’s first lab period. For the first assignment they choose an edible oil out of a collection commonly available at local markets, e.g., flax, olive, avocado (see Table 2). Students prepare the NMR samples, and acquire quantitative 13C NMR data. Enrollment in our two lab sections generally is 30 students. All of them prepare and
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HAZARDS
Chloroform is known to be toxic. Acute effects include skin and eye irritation. Repeated or prolonged exposure might produce damage in target organs such as liver, kidneys, and heart. Chloroform is also a proven carcinogen in animals and potentially carcinogenic for humans. B
DOI: 10.1021/acs.jchemed.7b00057 J. Chem. Educ. XXXX, XXX, XXX−XXX
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RESULTS AND DISCUSSION
Primary Lab
Edible oils consist primarily of triglycerides, an example of which is shown in Figure 1. All edible oils contain a mixture of
Figure 3. 13C{1H} quantitative NMR spectrum of olive oil, 50% in CDCl3. Peak labels follow Figure 1. (See Figure S3 for an example showing integrals.)
Figure 1. Example triglyceride containing cis- and trans-vinyl moieties (d and e), as well as an ω-6 (Ω) polyunsaturated moiety. Other labels include carbonyl (a), glycerol (b, c), and aliphatic (f).
such compounds. Their assignments are therefore a complex task to perform with 1H 1D spectra. As indicated in Figure 2, the glycerol protons (b,c) overlap with vinyl protons (d) [olive oil contains no measurable trans-vinyl moieties (e)].
a∝
∫ [region 175 → 171 ppm]
(1)
b∝
∫ [region 70 → 60 ppm]
(2)
d+e∝ f∝
∫ [region 134 → 125 ppm]
∫ [region 40 → 10 ppm]
(3) (4)
where the left-hand side is defined in Table 1, and in Figure 1. We have determined via HSQC experiments that CC C*H2CC carbons resonate at 25.5 ppm.6,12 From this observation Ω∝
∫ [region 25.8 → 25.4 ppm]
(5)
In eqs 2−5, “correct” integrals must be performed on observable peaks in well-phased, baseline-corrected spectra. More details for proper workup of the data are provided in the SI.13 From these equations, the primary results are obtained as14 average chain length = [(d + e) + (b + c) + f ]/(b + c)
Figure 2. 1H 1D NMR spectrum of olive oil, 50% in CDCl3, acquired at 400 MHz; peak labels follow Figure 1.
(6)
average degree of unsaturation = (d + e)/[2 × (b + c)] (7)
average polyunsaturated moieties = Ω/(b + c)
This overlap is the primary issue that led us to the pursuit of quantitative 13C spectra. Vinyl carbon resonances are wellresolved from all other carbons, as shown by the group/ chemical type assignments shown for olive oil in Figure 3. The inset plot shows that many different types of vinyl carbons are also well-resolved, providing details about the mixture of components in the oil that are inaccessible, at least in a straightforward manner, by 1H NMR. In addition to NMR lectures in organic chemistry classes, students are provided with homework and discussion group material (section V in the SI) that will lead them to sufficient understanding of 13C NMR for its application in the determination of average chain length, degree of unsaturation, and average polyunsaturated fatty acids content. From this information, the following equations should then become clear:11
(8)
Typical values found are listed in Table 2. Optional Lab 1: Other Food Items
Students bring in one other food item that can include the following: French fries, crackers, cake icing, bacon, donuts, etc. Extraction is performed using CDCl3 (or CHCl3) or chloroform/methanol.9 Data acquisition and analysis are performed as for the edible oils. Results for all students are tabulated and discussed. Some examples are provided in Table S5. Students are asked whether the results match expectations: What might contribute to surprising results?15 How might the measurements be improved?16 Optional Lab 2: 1H−13C HSQC
Students set-up and run a standard 1H−13C edited-HSQC experiment on their edible oil. Details of the experimental setup C
DOI: 10.1021/acs.jchemed.7b00057 J. Chem. Educ. XXXX, XXX, XXX−XXX
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Table 1. Definitions of Terms Presented Here Expression
Description no. no. no. no. no.
a b+c d+e f Ω
of of of of of
Type of Carbon OC*O C*H2OCO and (CH2)2C*HOCO C*C aliphatic carbons (sp3) CCC*H2CC
carbonyl carbons aliphatic glycerol carbons vinyl (unsaturated) carbons saturated moieties polyunsaturated fatty acids3
Table 2. Quantitative 13C NMR Results from Fall 2014 Chem 342 Oil/Fat flax walnut grapeseed corn canola 05 canola 08 sesame shortening hazelnut sunflower 13 sunflower 01 avocado olive lard whipped butter coconut
Average Chain Length
Degree of Unsaturation
Average Polyunsaturated Fatty Acids
17.4 17.7 17.6 17.4 17.7 17.5 17.4 17.4 17.5 17.5 17.5 17.4 17.1 17.3 14.1
2.27 1.79 1.50 1.42 1.35 1.34 1.34 1.21 1.08 1.05 1.04 0.95 0.94 0.73 0.28
1.28 0.84 0.57 0.54 0.38 0.36 0.45 0.54 0.14 0.11 0.11 0.10 0.12 0.17 0.03
11.8
0.03
0.00
Figure 5. Fatty acids used to verify triglyceride chemical shift assignments, especially for polyunsaturated-3,6 CCCH2C C moieties.
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SUMMARY A laboratory suitable for nonchemistry majors has been presented that employs quantitative 13C NMR measurements for the characterization of triglycerides in edible oils. 13C NMR provides excellent pedagogy for learning how carbonyl, vinyl, glycerol, and aliphatic carbons can be identified by chemical shifts. Sample preparation is straightforward, and data workup can be done by the students or instructor. Students determine the average chain length, degree of unsaturation, and average polyunsaturated fatty acid content. An optional extension to the laboratory is presented that allows students to investigate fat content in other foods of their own choosing. A second optional extension uses 2D 1H−13C HSQC NMR to enable the students to clarify assignments, including proton, based on standard chemical shift tables. The students also empirically verify polyunsaturated 13C and 1H assignments based on data from standard fatty acids.
are provided in section IV of the SI. An example spectrum for olive oil is shown in Figure 4.
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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.7b00057. Information about sample preparation and experimental setup; student handout and homework questions, including instructions for data analysis; NMR 13C 1D and 1H−13C HSQC parameters; instructions for no-D NMR; 1H−13C HSQC spectra; and quantitative 13C NMR results from other foods (PDF, DOCX)
Figure 4. 1H−13C edited-HSQC spectrum of olive oil in CDCl3. Assignments are shown for vinyl (d), glycerol >CH (c), glycerol CH2 (b), and polyunsaturated CCC*H2CC moieties (see Figure 1).
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They then confirm a variety of 13C and 1H assignments utilizing the 2D spectrum combined with assignments aids. The assignment of the polyunsaturated crosspeak can be confirmed empirically by having students also acquire, or have distributed to them, NMR data of “standard” triglyceride components. We have used steric, oleic, linoleic, and α-linolenic acids for this purpose (Figure 5; data are provided in section V of the SI).
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Charles G. Fry: 0000-0002-9049-0781 D
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(13) For the present work, we find (b+c) to be more consistent with the other integrals during workup, and therefore use it rather than a in eqs 6−8. (14) An early goal of the laboratory was to measure % saturated fats, and compare that measurement to product labels. Hartel and Moore provide methods to do this,5 but we have not included these in our current experiments. We are considering doing so for laboratories directed toward chemistry majors and honors students. (15) An example is a high trans-fat content observed for a local (and favorite) donut shop, whereas a national brand name donut purchased from a convenience store showed no trans-fat. The measurement of trans-fat content involves HSQC assignments that go beyond the scope of this work (but will be detailed in ref 6); for the current laboratories, we therefore provide the assignments for students. (16) Primary examples would be to lead students to the publications of Crowther,4 and to those of Hartel and Moore.5
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The Bruker AVANCE 400 MHz NMR spectrometer was supported by the National Science Foundation under Grant NSF CHE-1048642. A generous gift from Paul J. Bender enabled the purchase of the Bruker AVANCE 500 MHz NMR spectrometer.
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REFERENCES
(1) (a) Chamberlain, P. H. Identification of an Alcohol with 13C NMR Spectroscopy. J. Chem. Educ. 2013, 90 (10), 1365−1367. (b) Pulliam, C. R.; Pfeiffer, W. F.; Thomas, A. C. Introducing NMR to a General Chemistry Audience: A Structural-Based Instrumental Laboratory Relating Lewis Structures, Molecular Models, and 13C NMR Data. J. Chem. Educ. 2015, 92 (8), 1378−1380. (c) McCaw, C. S.; Thompson, M. A. A New Approach to Chemistry Education at PreUniversity Level. Nat. Chem. 2009, 1, 95−96. (2) See, for example: (a) ACD/ChemSketch 2014, ACD/C+H NMR Predictors and DB; Advanced Chemistry Development, Inc.: Toronto, Cananda. http://www.acdlabs.com/ accessed May 2017. (b) Mnova V11.0 with NMRpredict plugin; Mestrelab Research S.L.: Santiago de Compostela, Spain. (http://www.mestrelab.com/ accessed May 2017). (c) nmrdb.org (http://www.nmrdb.org/13c/index.shtml?v= v2.34.1, accessed May 2017); NMRShiftDB (http://nmrshiftdb.nmr. uni-koeln.de/, accessed May 2017). (d) Brown, D. W. A Short Set of 13 C-NMR Correlation Tables. J. Chem. Educ. 1985, 62 (3), 209−210. (3) The phrase “polyunsaturated fatty acid” in this paper refers specifically to all CCCH2CC− chemical moieties in a triglyceride. The primary examples of these moieties in edible oils are ω-3 and ω-6 fatty acids. In plant based oils, these include the essential fatty acids, linoleic acid and α-linolenic acid. (4) Crowther, M. W. NMR and IR Spectroscopy for the Structural Characterization of Edible Fats and Oils. An Instrumental Analysis Laboratory. J. Chem. Educ. 2008, 85 (11), 1550−1554. (5) Hartel, A. M.; Moore, A. C. Extraction and 1H NMR Analysis of Fats from Convenience Foods: A Laboratory Experiment for Organic Chemistry. J. Chem. Educ. 2014, 91 (10), 1702−1705. (6) Hofstetter, H.; Zhu, L.; Bowman, M.; Fry, C. G. Paper in preparation. (7) When using noncryogenically cooled probes, a direct broadband or 13C optimized probe is recommended. Inverse probes, such as Bruker’s BBI, have lower 13C sensitivity, and the time per sample to obtain quantitative 13C spectra will be longer (perhaps by a factor of 4). (8) A Bruker AVANCE III 500 MHz spectrometer equipped with LHe-cooled DCH cryoprobe was particularly useful for obtaining T1(13C) values.6 Many inverse cryoprobes now have cold 13C preamps. These probes have very good 13C sensitivity (typically better than 400 MHz broadband probes), and will be usable for this lab. (9) Chloroform extraction: 1 mL of CDCl3 was added to 1 g of sample. Lipids were extracted by vortexing and centrifuging, after which the upper phase was pipetted off and used for NMR analysis. See the SI for more details. (10) The extraction and gravimetric measurements detailed in ref 5 enable measurement of total fat content, which then allows the students to obtain results that can be compared to product labels: e.g., grams of saturated fat. (11) Symbols and expressions are used as follows: a represents the chemical shift of carbonyl carbons (see Figure 1), whereas a represents the number of carbonyl carbons proportional to the integration of the region 175 → 171 ppm. Note that eqs 2−5 are correct relative to each other; they are not meant to represent absolute quantitation. (12) Students can determine this assignment themselves with the HSQC lab addition as described in the next section. E
DOI: 10.1021/acs.jchemed.7b00057 J. Chem. Educ. XXXX, XXX, XXX−XXX
