Lipid Technology
February 2009, Vol. 21, No. 2
1
DOI 10.1002/lite.200900004
Analysis
Using proton nuclear magnetic resonance as a
rapid response research tool for methyl ester
characterization in biodiesel
Marc ter Horst, Stephanie Urbin, Rachel Burton, and Christina MacMillan
Marc ter Horst is an NMR Spectroscopist and NMR Facility co-Director, Stephanie Urbin is a graduate student, and Christina MacMillan
is an undergraduate student in the Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599, USA; tel.: 919-8435802; fax: 919-962-2388; e-mail: terhorst@email.unc.edu
Rachel Burton is the Laboratory Manager at Piedmont Biofuels, Pittsboro, NC 27312, USA; tel.: 919-321-8260; fax: 919-542-0886;
e-mail: rachel@biofuels.coop
Summary
Reliable and rapid analysis remains a high priority for quality control in biodiesel production. Quantifying biodiesel with alternative
analytical tools such as proton nuclear magnetic resonance (1H NMR) can provide total methyl esters distributions without significant
sample pretreatment. Using unique spectra of individual methyl esters, we investigate the feasibility of using 1H NMR spectroscopy to
identify and quantify relative and absolute concentrations of methyl esters in a biodiesel.
Introduction
1
Biodiesel is composed of a mixture of monoalkyl esters of longchain fatty acids of either vegetable oils or animal fats. Gas chromatography (GC) analysis is used to determine the distribution
of methyl esters. For each sample, standards and derivatization
are required to resolve the various saturated and unsaturated
methyl esters in a biodiesel mixture. Additional techniques such
as mass spectrometry (MS) and proton nuclear magnetic resonance (1H NMR) provide complementary information with
different demands on sample preparation and allowances for
sample variability. MS provides detailed molecular weight information and requires only a very small amount of sample. NMR
data is sensitive to unique molecular environments which yield
unique spectra for different molecules. Although MS is a more
sensitive technique, NMR can provide detailed molecular information once a spectrum is acquired with a sufficiently high signal-to-noise ratio. For most samples generated in the biodiesel
industry, sample quantity is not an issue and NMR can be
applied to biodiesel and biodiesel mixtures. NMR has been used
to monitor the transesterification reaction used in the production of biodiesel, see for example (1), and to monitor the oxidation of methyl esters in biodiesel (2). Previous work by Diehl and
Randel has shown the ability of NMR to quantify blends of biodiesel and petroleum diesel (3). Careful analysis with NMR can
also determine relative amounts of identified components
within a mixture such as biodiesel. Knothe and Kenar have
shown integrals of resonances in 1H spectra can be used to determine the relative amounts of fatty acids in vegetable oils and
methyl ester mixtures when the source of the oil feedstock is
known (4). We deconstruct 1H NMR spectra of biodiesel using
spectra of individual methyl esters. Using characteristic 1H spectra of various methyl esters, we attempt to determine the relative and absolute concentrations of methyl esters in a biodiesel
sample through the fitting of pure methyl ester spectra to a spectrum of biodiesel. The uniqueness of this work is the analysis of
the biodiesel spectrum using all features of the NMR spectra.
Proton NMR provides a good probe for biodiesel since 1H is the
most naturally abundant and most sensitive NMR active isotope.
Relatively narrow line widths of a few Hertz are obtained for 1H
spectra so that magnetically unique nuclei are resolved at many
field strengths. Fig. 1 displays 1H spectra of individual methyl
esters and a soy-based biodiesel sample. Methyl esters displayed
are: methyl palmitate (16:0), methyl stearate (18:0), methyl oleate (18:1), methyl linoleate (18:2) and methyl linolenate (18:3).
The peaks at 5.35 ppm, 2.8 ppm and 2.1 ppm are related to the
1
H located at or near the double bond(s) within the unsaturated
methyl esters, 18:1, 18:2, and 18:3. The sharp peak at 3.7 ppm is
due to the ester methyl located next to the carbonyl carbon and
the triplets around 0.9 ppm are from the terminal alkyl methyl
in each of the methyl esters. The methylene alpha to the ester
group is at 2.3 ppm and the beta group is at 1.6 ppm. The
remaining CH2 group protons have similar resonance frequencies and overlap in the range of 1.2–1.4 ppm. The total intensity
in this region is the sum of the individual contributions from
the remaining CH2 groups in the molecule.
For the biodiesel spectrum, the integral of this region is proportional to the total number of these CH2 protons in each of the
methyl esters. The unique chemical shifts of unsaturated methyl
esters can be used to determine the saturated component of the
mixture by first identifying and then subtracting the contribution of the unsaturated methyl esters. The saturated methyl
esters are either analyzed collectively or identified assuming the
feedstock is known so that the variety of methyl esters is known.
The intensity profile of the overlapping CH2 peaks, however,
does vary between the individual saturated methyl esters. The
l1.25 ppm peak in 18:0 has different intensity and shape than
the same peak in 16:0 at the same concentration. Normalized to
the terminal methyl, 18:0 will have an integral of the CH2 region
two units larger than that of 16:0 and 4 more than that of 14:0.
At an external field corresponding to a 500 MHz resonance frequency for proton, the exact location of maximum and the
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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
H NMR analysis
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February 2009, Vol. 21, No. 2
Lipid Technology
Figure 1. Proton NMR spectra of soy-based biodiesel and individual methyl esters. See text for details.
Figure 2. Proton NMR spectra of saturated methyl esters,
methyl myristate (14:0), methyl palmitate (16:0) and methyl stearate (18:0), scaled to the terminal alkyl methyl triplet.
shape of the CH2 region show differences between these saturated methyl esters, see Fig. 2.
Commercially available methyl esters were used as purchased.
All samples were dissolved in deutrated chloroform with 1% v/v
TMS. A standard (1,3,5 (tristrifluromethyl)benzene) was used to
quantify the exact concentration of the methyl esters in the
NMR tube. All spectra were acquired on a Bruker DRX 500 at
25oC. The acquisition parameters were selected to provide sufficient time for the complete relaxation of the methyl ester resonances based on previously determined relaxation times in biodiesel. As a result, each 8 transient spectrum was acquired in less
than one minute.
NMR spectra of the individual methyl esters were iteratively
scaled to match the biodiesel spectrum using the Chenomix
NMR Suite (5). The quantification standard was used to determine accurate concentrations of the methyl esters based on the
fits. The process involved fitted the 2.1 ppm region of the unsaturated methyl ester spectra starting with 18:3 due to its limited
overlap with the resonances in 18:2 and 18:1 in this region. The
residual was fit with the 18:2 spectra and the 18:1 spectrum was
fit to the final remainder. At each step the complete contribution of all protons in each methyl ester was subtracted from the
entire biodiesel spectrum, so that after all unsaturated methyl
ester basis spectra were used only the saturated component in
the biodiesel spectrum remained. Using the region between
1.2 ppm and 1.4 ppm, the 18:0 and 16:0 spectra were fit to the
Lipid Technology
February 2009, Vol. 21, No. 2
Table 1. Percent composition of individual methyl esters in a soy-based
biodiesel sample.
Soy-based
biodiesel*
Run 1
Run 2
Run 3
16:0
18:0
18:1
18:2
18:3
6–10%
2–5%
20–30%
50–60%
5–10%
13
9
12
6
8
7
20
22
21
53
54
52
8
7
8
* Building a successful biodiesel business, J. Von Gerpen et al.,
2006.
remaining saturated component of the biodiesel spectrum. The
residuals from these fits were not improved with the added fitting of the 14:0 spectrum and the 14:0 spectrum was not used in
the analysis. The results for three runs using the same spectra
are summarized in Table 1. Consistently, the unsaturated
methyl esters are well resolved, varying by 1% and within the
accepted range for soy-based biodiesel. The saturates were more
uncertain.
Conclusion
The 1H NMR spectra of the individual methyl esters in this study
have differences that lead to a unique analysis of biodiesel spectra. The analysis works well with unique resonances from protons near carbon-carbon double bonds in the unsaturated
methyl esters. For the saturated methyl esters, the analysis can
vary from run to run. This uncertainty will be a major factor for
biodiesel derived from more diverse feedstocks such as animal
fat, yellow grease and trap grease. Better results might be
obtained in different solvents, at elevated temperatures or using
neat samples. Work is underway to consider additional
approaches including the use of carbon spectra (6).
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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Acknowledgements
This work was made possible by funding from NSF Center for Enabling
New Technologies through Catalysis (CENTC) and the Department of
Chemistry at UNC-CH for supporting an outreach effort to involve
undergraduate and high school students. Tom O'Connell in the UNC
Metabolomics Laboratory provided assistance in using the Chenomix
NMR Suite.
References
[1] Morgenstern, M., Cline, J., Meyer, S., and Cataldo, S.
(2006) Determination of the Kinteics of Biodiesel Production Using Proton Nuclear Magnetic Resonance Spectroscopy (1H NMR), Energy & Fuels, 20, 1350 – 1353.
[2] Knothe, G. (2006) Analysis of oxidized biodiesel by 1HNMR and effect of contact area with air, Eur. J. Lipid Sci.
Technol., 108, 493 – 500.
[3] Diehl, B. and Randel, G. (2007) Analysis of biodiesel, diesel
and gasoline by NMR Spectroscopy: a quick and robust
alternative to NIR and GC, Lipid Technol., 19, 258 – 260.
[4] Knothe, G. and Kenar, J.A. (2004) Determination of fatty
acid profile by 1H NMR spectroscopy, Eur. J. Lipid Sci. Technol., 106, 88 – 96.
[5] Weljie, A. M., Newton, J., Mercier, P.M., Carlson, E., and
Slupsky, C.M. (2006) Targeted Profiling: Quantitative
Analysis of 1H-NMR Metabolomics Data, Anal. Chem., 78,
4430 – 4442.
[6] Bowden, M., Rieth, A., and ter Horst, M.A., The Effects of
Cold Flow Additives on Soy-based Biodiesel as Determined
by NMR Spectroscopy, poster presented at the Southeast
Regional Meeting of the American Chemical Society (SERMACS), Greenville, SC, October 24 – 27, 2007.
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