In the Laboratory
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Quantitative Determination of Methylcyclohexanone Mixtures Using 13C NMR Spectroscopy A Project for an Advanced Chemistry Laboratory Joseph W. LeFevre* and Augustine Silveira, Jr.† Chemistry Department, SUNY at Oswego, Oswego, NY 13126; *
[email protected] In order to teach the principles of 13C NMR spectral analysis to our chemistry majors, we have introduced a project involving the quantitative analysis of methylcyclohexanone mixtures. Project-oriented laboratories have been the backbone of our curriculum in our Advanced Chemistry Laboratory for many years (1). The project is suitable for a variety of advanced chemistry laboratories including analytical chemistry, instrumental analysis, organic chemistry, and NMR spectroscopy. Several analyses of methylcyclohexanones have appeared in this Journal. In the first, a qualitative identification of 2-, 3-, or 4-methylcyclohexanone was made using NMR spectroscopy (2). The second analysis utilized capillary gas chromatography (GC) to examine the stereochemistry of a commercial 2,6-dimethylcyclohexanone mixture (3). In the third analysis, capillary GC was also used to identify a mixture of dimethylcyclohexanones, which were formed via the kinetic and thermodynamic enolates of 2-methylcyclohexanone (4). Background The use of 13C NMR for quantitative purposes is not common owing to several inherent difficulties. First, 13C NMR suffers from poor sensitivity due to the low 1.1% natural abundance of this isotope. Broadband proton irradiation, which decouples the protons from their respective carbon atoms to produce singlets, addresses this problem by increasing the sensitivity via nuclear Overhauser enhancements (NOEs), but introduces a second more serious problem. The resulting NOEs are not uniform and produce peaks of varying intensity (5). Third, quaternary carbons with long spin-lattice relaxation times (T1’s) produce low peak intensities under standard broadband decoupling conditions. The last two problems can be overcome by a technique known as inverse-gated decoupling (6, 7 ) and by the addition of the paramagnetic relaxation reagent chromium(III) acetylacetonate [Cr(acac)3] (6 ). The project was performed in three laboratory periods lasting three hours each by pairs of students who analyzed mixtures containing varying proportions of 2-methylcyclohexanone, 1, 2,2-dimethylcyclohexanone, 2, trans-2,6-dimethylcyclohexanone, 3, and cis-2,6-dimethylcyclohexanone, 4. The structures of these compounds are shown below. The mixtures were analyzed using standard broadband decoupling and inverse-gated decoupling techniques, the latter being done both with and without Cr(acac)3. † Author to whom requests for spectra should be directed,
[email protected].
O
O
O
7 6
6 7
1
2
O 2
3
1
4
The students were exposed to a wide variety of current NMR techniques including COrrelated SpectroscopY (COSY), Distortionless Enhancement by Polarization Transfer (DEPT), HETeronuclear CORrelated spectroscopy (HETCOR), T1 determination by the inversion-recovery method (7), standard broadband decoupling, inverse-gated decoupling, and the addition of a paramagnetic relaxation reagent. The students compared the results obtained from the various decoupling methods and showed that inversegated decoupling in the presence of Cr(acac)3 was the best method in terms of accuracy and time. The percentages of each component were within 1% of the actual values for all of the mixtures that were analyzed using the inverse-gated decoupling method. Results and Discussion In order to illustrate the problems of unequal NOEs and long T1’s, students first studied a pure sample of 1 before analyzing their mixtures. They assigned the 13C spectrum using a combination of COSY, DEPT, and HETCOR spectroscopy. They acquired the 13C spectrum under standard broadband decoupling conditions and observed the wide variation in peak integrations between the carbonyl resonance and the other protonated carbons. They then acquired the data using inverse-gated decoupling conditions both with and without Cr(acac)3. In both cases, all peak integrations were essentially identical, but the Cr(acac)3 decreased the acquisition time from just over one hour to less than four minutes. Since inversegated decoupling requires a delay of five times the T1 of the slowest relaxing carbon between pulses, students determined the T1 of the carbonyl carbon of 1 both with and without Cr(acac)3. They saw how the relaxation reagent dramatically decreased the T1 from 25 seconds to less than one second. Before analyzing its mixture, each student pair had to assign a specific resonance in each of the four compounds. We chose the C-6 methylene carbon of 1 at 42.57 ppm, the C-7 methyl carbons of 2, both of which resonate at 25.68 ppm, the C-2 and C-6 methine carbons of 3, both of which resonate at 43.81 ppm, and the C-1 carbonyl carbon of 4 at 216.04 ppm. A different carbon type was chosen in each compound in order to exploit the full range of NOEs under standard broadband decoupling conditions, and to see these
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In the Laboratory
Table 1. Percentage Composition Data for Mixture 1 ExperDecoupling Method iment
Percentage Delay Acquisition between Time/min 1; C-6 (CH2a) 2; C-7 (CH3 ) 3; C-2, C-6 (CH) 4; C-1 (C=O) Pulses/s 42.57 ppm 25.68 ppm 43.81 ppm 216.04 ppm
1
Standard broadband
0.685
17
57.6
20.7
7.3
14.4
2
Inverse-gated
120 b
270
46.8
16.5
6.4
30.3
3
Inverse-gated + Cr(acac)3
46.3
17.1
6.0
30.6
4
Inverse-gated (student choice of peaks) c
45.6
17.2
6.3
31.0
45.5
17.0
6.3
31.1
5b 120 b
26.2 270
Actual Value d aRelative
to 49.00 ppm, which is the central line of the CD3OD solvent septet. are 5 × T1. cThe carbonyl resonances were used in this experiment. dBased upon carefully weighed mixture components. bValues
effects disappear using inverse-gated decoupling. In order to make the assignments, students were given three commercial samples, A, B and C. Sample A was pure 1, and the C-6 resonance was identified in their earlier work on 1. Sample B was 93.5% 2 and 6.5% 1.1 The large C-7 resonance due to the two methyl groups of 2 was easily identified by DEPT spectroscopy. Sample C was 16.6% 3 and 83.4% 4.2 The students were told that sample C was a mixture, but they were not told which was the major and which was the minor component. Instead, they were directed to reference 3 to find the answer. Once they knew that 3 was the minor component, the C-2 and C-6 methine resonance was easily identified by DEPT spectroscopy. The C-1 carbonyl of 4 was clearly the larger of the two carbonyl resonances of Sample C in the standard broadband decoupled spectrum and was assigned without difficulty. With the correct resonances assigned, each student pair was then given a mixture to analyze. Samples were run first under standard broadband decoupling conditions. The peaks of interest were integrated and the percent composition calculated. As is evident for mixture 1, the results were far from the actual values, which were based upon careful weighing of the mixture components. The results are shown in experiment 1, Table 1. Most noteworthy is the erroneously high value (57.6% vs 45.5% actual) for the C-6 methylene resonance of 1 due to its large NOE, and the erroneously low value (14.4% vs 31.1% actual) for the C-1 carbonyl carbon of 4 due to its lack of NOE and long T1. Next, the sample was run using inverse-gated decoupling conditions. Owing to the several-hour acquisition time that was required, the sample was run for the students outside of class. This was followed by the addition of Cr(acac)3 and inverse-gated decoupling. In both cases, the experimental values were very close to the actual values, as shown in experiments 2 and 3, Table 1. Finally, the students were asked to pick another set of resonances to determine the percentage composition. The inverse-gated decoupled spectrum acquired earlier was reanalyzed using the students’ choices. All of them chose the four carbonyl groups as the resonances of interest that were well resolved and easily assigned. The percentage composition using these new resonances also agreed closely with the actual values, as shown in experiment 4, Table 1. The students were
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not given the actual weights of the four compounds until all of their percentage composition calculations were completed. They then used the weights to calculate the actual percentage composition of the mixture. The first year that we ran the experiment, students were assigned the C-4 methylene resonance in all four compounds for determining the percentage composition. The data were acquired using both normal broadband decoupling and inversegated decoupling conditions. Because the NOEs and T1’s of the C-4 resonances of each compound were essentially identical, both the standard broadband and inverse-gated decoupling conditions gave results that were very close to the actual values. The second year, we ran the experiment using a different carbon type in each of the four molecules, as described above. We found this second method to be pedagogically more satisfying since students could see directly the problems that arise from differing NOEs and T1’s when using normal broadband decoupling and how these problems are eliminated using inverse-gated decoupling both with and without Cr(acac)3. The project was well received by the students and illustrated for them the power of 13C NMR as a quantitative analysis tool using inverse-gated decoupling. The materials are readily available from Aldrich Chemical Company, Inc. The project can be shortened by deleting the 2-methylcyclohexanone studies and the T1 analyses, and simplified by omitting 2,2-dimethylcyclohexanone from the mixtures. Copies of all relevant spectra are available from the corresponding author upon request. Acknowledgments We thank the students in Che 334L, Advanced Chemistry Laboratory, for their diligent work on this project. We gratefully acknowledge the National Science Foundation’s Instrumentation and Laboratory Improvement program (ILI), which provided partial funding (Grant #228-0388A) for the purchase of a Varian UNITYINOVA 300-MHz NMR spectrometer and for its continued support of project-oriented laboratories at SUNY Oswego. We thank the State University of New York at Oswego, which provided matching funds for the purchase of the spectrometer and other financial support. Finally, we thank Linda LeFevre for typing the manuscript.
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Supplemental Material
Supplemental material for this article is available in this issue of JCE Online. Detailed student procedures, questions, and instructor’s notes are provided. The instructor’s notes contain a schedule of the experiments, 13C NMR acquisition parameters, T1 determinations, percentage composition calculations, data on additional mixtures, answers to questions, mixture preparation procedures, and a reagent list. Notes 1. Aldrich reports a ratio of 92% 2 and 8% 1. These numbers differ slightly from our values. We used 1H NMR spectroscopy to quantitate the mixture by integrating the appropriate methyl signal for each compound. Average integration values of three spectra were used in the calculations. A single 90° pulse was used to generate the spectra. 2. Reference 3 reports a ratio of 19% 3 and 81% 4 using capillary GC as the quantitation method. These numbers differ
slightly from our values. The samples were analyzed by 1H NMR as described in note 1.
Literature Cited 1. LeFevre, J. W. J. Chem. Educ. 1998, 75, 1287. Silveira, A. Jr.; Evans, J. M. J. Chem. Educ. 1995, 72, 374. LeFevre, J. W. J. Chem. Educ. 1990, 67, A278. Silveira, A. Jr.; Orlando, S. C. J. Chem. Educ. 1988, 65, 630. Silveira, A. Jr.; Koehler, J. A.; Beadel, E. F. Jr.; Monroe, P. A. J. Chem. Educ. 1984, 61, 264. Silveira, A. Jr.; Bretherick, H. D.; Negishi, E. J. Chem. Educ. 1979, 56, 560. Silveira, A. Jr.; Satra, S. K. J. Org. Chem. 1979, 44, 873. Silveira, A. Jr. J. Chem. Educ. 1978, 55, 57. 2. Gurst, J. E. J. Chem. Educ. 1992, 69, 774. 3. Garner, C. M. J. Chem. Educ. 1993, 70, A310. 4. Silveira, A. Jr.; Knopp, M.; Kim, J. J. Chem. Educ. 1998, 75, 78. 5. Gunther, H. NMR Spectroscopy; Wiley: New York, 1980. 6. Schmedake, T. A.; Welch, L. E. J. Chem. Educ. 1996, 73, 1045. 7. Breitmaier, E.; Voelter, W. Carbon-13 NMR Spectroscopy, 3rd ed.; VCH: New York, 1987.
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