1350

Energy & Fuels 2006, 20, 1350-1353

Determination of the Kinetics of Biodiesel Production Using Proton

Nuclear Magnetic Resonance Spectroscopy (1H NMR)
Mark Morgenstern,* Jessica Cline, Sally Meyer, and Simon Cataldo
Chemistry Department, Colorado College, Colorado Springs, Colorado 80903
ReceiVed NoVember 14, 2005. ReVised Manuscript ReceiVed May 26, 2006

Methyl esters were produced at several temperatures (10, 35, and 45 C) by transesterification batch reactions
of soybean oil with methanol utilizing KOH and NaOH catalysts. The reactions were monitored by aliquot
removal and subsequent proton nuclear magnetic resonance spectroscopy (1H NMR) analysis. 1H NMR analysis
allowed for the calculation of the average degree of fatty acid unsaturation (DU ) 1.52) in oil and methyl
ester. 1H NMR analysis also provided initial rates of methyl ester formation and an activation energy of 27.2
kJ/mol. The time-dependent concentration data revealed substantial reaction progress toward equilibrium after
only 120 s at a reduced temperature of 10 C. Understanding the resonance shifts in the 1H NMR spectra of
starting materials and products allows for quantitation of reaction progress that is in good agreement with
results obtained using other analytical methods.

Introduction
Rudolf Diesel first envisioned his diesel engine invention as
a powerful engine that could operate on agricultural products
such as vegetable oils to counter the dependency on petroleum
fuels that was prevalent as early as 1905.1 His engine was a
great success, but his desire to use agricultural-derived fuels
was not realized because of the inexpensive petroleum production of diesel fuels and the need to chemically modify plant
oils prior to use in the diesel engine. However, global events
and rising petroleum costs have recently revitalized interest in
Rudolf Diesels dream. The diesel engine can indeed be run on
straight vegetable oil, but many problems result ranging from
starting failure and engine wear to polymerization of the
lubricating oils.2 Most of the problems associated with the use
of straight vegetable oil can be avoided by chemical modification
through transesterification of the vegetable oil triglyceride
molecules to smaller alkyl esters of the fatty acids commonly
referred to as biodiesel.2 This transesterification is normally
accomplished using methanol as the alcohol and either NaOH
or KOH as a base catalyst (see Figure 1). Reviews of biodiesel
production have been published, and the transesterification
reaction has primarily been monitored using gas chromatography
(GC).3,15
The use of proton nuclear magnetic resonance spectroscopy
(1H NMR) has recently been employed to monitor the kinetics
and product distributions in transesterification reactions (alcoholysis) between vegetable oils and alcohols.4-6 The use of 1H
NMR is convenient and fast when monitoring a reaction,
because a small aliquot can be extracted from the batch reaction
at any given time and the 1H NMR spectrum analysis provides
* To whom correspondence should be addressed. E-mail: mmorgenstern@
coloradocollege.edu.
(1) Krawzcyk, T. Inform 1996, 7, 801-815.
(2) Harwood, H. J. J. Am. Oil Chem. Soc. 1984, 61, 315-324.
(3) Ma, F.; Hanna, M. A. Bioresour. Technol. 1999, 70, 1-15.
(4) Suppes, G. J.; Bockwinkel, K.; Lucas, S.; Botts, J. B.; Mason, M.
H.; Heppert, J. A. J. Am. Oil Chem. Soc. 2001, 78, 139-145.
(5) Knothe, G. J. Am. Oil Chem. Soc. 2000, 77, 489-493.
(6) Gelbard, G.; Bres, O.; Vargas, R. M.; Vielfavre, F.; Schuchardt, U.
F. J. Am. Oil Chem. Soc. 1995, 72, 1239-1241.

Figure 1. General reaction for the transesterifaction of a triglyceride

using an alcohol.

quantitative information pertaining to the chemical species

present in the reaction. In this process, concentrations of reactant
oil and product methyl ester can be determined at early times
during a batch reaction and the initial rates of the reaction can
be obtained. The rate of oil conversion to methyl ester has been
reported using aliquot sampling and 1H NMR analysis.5 The
work presented in this paper expands the previous study in three
significant ways: (i) a detailed interpretation of the 1H NMR
spectrum of unsaturated vegetable oil is presented; (ii) initial
rates and activation energy for the reaction are obtained; and
(iii) the transesterification (alcoholysis) reaction is studied using
a stoichiometric 3:1 molar ratio of alcohol/soybean oil as
opposed to a 6:1 or larger ratio used in most previous
studies.3-5,7,8 Recent work has found that the optimum molar
ratio is approximately 3.5:1 and increased quantities of methanol
over this ratio result in separation problems during subsequent
steps in the process.9
In previous 1H NMR studies of vegetable oil alcoholysis
reactions, quantitation is achieved by comparing the integration
of the resonance signal from the glyceridic protons in the oil to
the methyl ester protons in the product to determine the extent
of the reaction at each sampling time.5 Additionally, the olefinic
proton signals in the product are compared to the methyl ester
signal in the product to determine the average degree of
unsaturation in the fatty acid chains.4 However, previous work
(7) Darnoko, D.; Cheryan, M. J. Am. Oil Chem. Soc. 2000, 77, 12631267.
(8) Freedman, B.; Butterfield, R. O.; Pryde, E. H. J. Am. Oil Chem. Soc.
1986, 63, 1375-1380.
(9) Dorado, M. P.; Ballesteros, E.; Mittlebach, M.; Lopez, F. J. Energy
Fuels 2004, 18, 1457-1462.

10.1021/ef0503764 CCC: $33.50 2006 American Chemical Society

Published on Web 07/04/2006

Kinetics of Biodiesel Production Using 1H NMR

incorrectly assigned the glyceridic proton signal at 4.2 ppm as

a five-proton signal, while it is actually only a four-proton
dimethylene signal, with the remaining methine proton showing
up further downfield as part of the olefinic proton signal at 5.25
ppm.5 The shift of the glyceridic methine proton can easily be
established by obtaining the 1H NMR spectrum of a saturated
triglyceride, such as tributyrin or trimyristin, where there are
no olefin protons and the methine glyceridic proton clearly
shows up as a multiplet downfield from the four methylene
glyceridic protons. Therefore, when using the 1H NMR integration values in unsaturated oils, the olefinic proton integrations
must be corrected for the underlying glyceridic methine proton
and the glyceridic methylene resonance should be treated as a
four-proton signal.
The time interval used in aliquot removal from a vegetable
oil alcoholysis reaction using methanol must be rapid when the
reaction is performed at room temperature or higher. Over 50%
conversion to the product within the initial 5 min has been
reported for the reaction of soybean oil with methanol at 45 C
using the KOH catalyst.5 This fast reaction would require several
aliquots to be removed and analyzed within the first minute to
obtain the initial rates for the disappearance of soybean oil and
the appearance of the methyl ester product.
In the present work, the kinetics of a 3:1 stoichiometric ratio
of methanol/oil is studied in contrast to previous papers that
utilize a 6:1 ratio of methanol/oil. It has been reported that
performing the transesterification as a two-stage reaction results
in a more complete reaction.16 In the two-stage transesterification
process, the first stage is allowed to approach equilibrium using
a 3:1 stoichiometric ratio of methanol/oil before the glycerol is
removed. The second stage consists of the addition of an
additional equivalent of methanol and stirring to drive the
equilibrium further to products and therefore a second separation
of the glycerol byproduct. Therefore, this work is partially
initiated to determine the time required for the first stage to
approach equilibrium when a 3:1 stoichiometric ratio of reactants
is used. 1H NMR can be used to monitor the extent of the
reaction as an alternative to GC.
Experimental Section
The progress of the transesterification reaction between soybean
oil and methanol was monitored by the extraction of aliquots from
a batch reaction at given time intervals. The extent of the reaction
was determined by 1H NMR analysis of the aliquots. Batch reactions
were carried out at different temperatures and with different
catalysts.
Batch Reactions. The batch reactions were performed with a
stoichiometric molar ratio (3:1) of methanol/soybean oil. A 200
mL two-necked round-bottom flask was charged with 2.50 mmol
of catalyst (0.100 g of NaOH or 0.140 g of KOH) and 9.40 mL
(0.232 mol) of methanol. A magnetic stir bar was added, and with
continuous stirring on a magnetic stir plate, the catalyst was
completely dissolved in about 15 min. Once the catalyst was in
solution, 80 mL of soybean oil (0.077 mol based on an average
molar mass of 895.9 g/mol from percent composition data) was
added and the rate of stirring increased to approximate a homogeneous system. The batch reactions were performed in an ice bath
(10 C) and in an oil bath (35 and 46 C) to obtain the temperaturedependent data for the reaction. The oil and alcohol/catalyst
solutions were allowed to equilibrate at the desired temperature
prior to mixing and initiation of the reaction. A reflux condenser
was employed at the higher temperatures to prevent the alcohol
from evaporating.
Aliquot Removal. Aliquots (1.0 mL) were removed from the
batch reaction using an autopipet with disposable tips. The
maximum rate of aliquot extraction was one sample every 10-30
s using this method. The aliquots were immediately transferred into

Energy & Fuels, Vol. 20, No. 4, 2006 1351

Table 1. Reported Percent Fatty Acid Composition of Soybean Oil
and Corresponding Degree of Unsaturation per Fatty Acid
(FA)10,11,12
fatty acid identity

average ( SD

myristic (14:0)
palmitic (16:0)
stearic (18:0)
oleic (18:1)
linoleic (18:2)
linolenic (18:3)
average degree of unsaturation per FA

0.2 ( 0.1
10.7 ( 1.0
3.0 ( 0.9
25.0 ( 3.5
53.4 ( 2.3
7.3 ( 1.2
1.54 ( 0.02

a test tube containing 2.5 L of 12 M HCl (0.030 mmol) and placed

on ice to quench any further alcoholysis reaction in the test tube.
Acetone (0.100 mL, 1.35 mmol) was added to each aliquot to serve
as an internal standard for the 1H NMR analysis of concentrations
present in a sample. Several 1H NMR analyzes were performed
with known concentrations of oil and methyl ester, and back
calculations using the acetone standard showed a consistent 10%
error. Individual spectra of each aliquot were obtained using a
Varian 200 MHz NMR and CDCl3 (99.8%) as the solvent.
Reagents. Soybean oil, King Soopers brand, was purchased in
1 gallon jugs from the local King Soopers grocery store (Colorado
Springs, CO). Methanol (99.9% purity), KOH (88.1% purity),
NaOH (98.0% purity), and concentrated (12 M) HCl were all
certified ACS-grade and purchased from Fischer Scientific (Fairlawn, NJ). Acetone (99.8% purity) was certified ACS-grade and
purchased from the J. T. Baker Chemical Company (Phillipsburg,
NJ). The CDCl3 [99.8% D with 0.03% (v/v) tetramethylsilane
(TMS)] used as the NMR solvent was purchased from Aldrich
Chemical Co. (Milwaukee, WI). All reagents were used without
further purification.

Results and Discussion

The general reaction for the transesterification of a triglyceride
with an alcohol (alcoholysis) is shown in Figure 1. Note that
this reaction is reversible and every triglyceride molecule reacts
with 3 equiv of the alcohol to produce glycerol and three fatty
acid ester molecules. In base-catalyzed reactions, the catalyst
is typically NaOH or KOH. The general fatty acid chains
denoted as R1, R2, and R3 vary in identity and percent
composition depending upon the triglyceride source (see Table
1 for soybean oil composition). Plant oils generally contain a
high percent of unsaturated fatty acid chains, where R1, R2, and
R3 would be oleic, linoleic, and linolenic acid chains. The high
percent of unsaturated fatty acid chains results in plant-derived
triglycerides having a liquid physical state (oil) at moderate
temperatures. The alcohols (ROH) used in the transesterification
of triglycerides are normally methanol or ethanol, with methanol
being the most common because of the low cost, low reaction
temperatures, fast reaction times, and higher quality methyl ester
product.9,14
Typical 1H NMR spectra of soybean oil and methyl ester
product resulting from transesterification with methanol are
presented in Figures 2 and 3. The corresponding 1H resonance
assignments are presented in Figure 4. It is interesting to note
that the presence of polyunsaturated fatty acids can be readily
detected by the appearance of a resonance at 2.72 ppm from
the methylene group between two double bonds of a linoleic,
linolenic, or higher polyunsaturated fatty acid chain. The
splitting pattern for the protons in the glyceride moiety is an
ABX pattern because of the chemical-shift inequivalence of the
glyceride methylene protons at 4.21 ppm, and the measured
coupling constants are Jab ) 12 Hz, Jax ) 6 Hz, and Jbx ) 4
Hz for the system represented by R1OCHaHbC(OR2)HxCHaHbOR3.13 In this system, there is a plane that runs along the
carbon-carbon bond and the Ha protons are on the same side
of this plane as the -OR2 group, while the Hb protons are on

1352 Energy & Fuels, Vol. 20, No. 4, 2006

Morgenstern et al.

Figure 2. Typical 1H NMR spectrum of soybean oil (CDCl3, Varian 200 MHz NMR).

Figure 3. Fatty acid methyl ester product from soybean oil alcoholysis with methanol.

Figure 4. Useful 1H NMR assignments in the CDCl3 solvent for a

hypothetical triglyceride of soybean oil, containing the three most
prominent fatty acid chains as examples (the assignments are downfieldshifts relative to TMS).

the side of the plane with the Hx proton. Therefore, the Ha

protons (4.30 ppm) show up as the downfield doublet of
doublets, and the Hb protons (4.15 ppm) are a doublet of
doublets further upfield (see the inset of Figure 2). The large
coupling constant, Jab ) 12 Hz, is assigned to the geminal coupling interaction between the nonequivalent methylene protons
of both methylene groups. The long fatty acid chains R1, R2,
and R3 are very similar or even identical in the effect that they
have on the environment of the glyceride protons. If the groups
were very different, the resulting pattern at 4.21 ppm would be

much more complex because the four glyceride methylene

protons would all have different chemical shifts. The complex
patterns observed in this region for mono- and diglycerides have
been observed and serve as an example.5 The major differences
between the 1H NMR spectra of the starting soybean oil (Figure
2) and the resulting fatty acid methyl ester (Figure 3) are the
disappearance of the glyceride protons at 4.21 ppm (4H) and
the appearance of the methyl ester protons at 3.67 ppm (3H).
The average degree of unsaturation (DU) for the fatty acid
methyl ester product can be calculated on the basis of a
comparison of 1H NMR integration values for the methyl group
(3.67 ppm) and the olefin protons (5.35 ppm) in the methyl
ester product. The average DU can also be obtained from the
starting oil by a comparison of the integration value for the
methylene group adjacent to the carbonyl group (2.31 ppm) to
that of the olefin protons (adjusted for the underlying methine
proton). The DU value from soybean oil obtained in this manner
was 1.52, which corresponds with the DU values calculated
using percent composition data in Table 1 and also agrees with
a DU value of 1.487 reported in previous research using 1H

Kinetics of Biodiesel Production Using 1H NMR

Figure 5. Kinetic plot of the KOH-catalyzed methanolysis of soybean

oil at 10 C.

NMR analysis of fatty acid methyl esters synthesized from

soybean oil.4 Similar 1H NMR analysis of fatty acid methyl
esters synthesized from olive oil gave a DU value of 1.06, which
also corresponds to the value of 0.94 calculated from percent
composition data.10-16 These calculations and comparisons serve
to provide confidence in the functional group integration values
of the vegetable oil and the methyl ester product to calculate
concentrations during kinetic runs.
The kinetics of the base-catalyzed transesterification reaction
between methanol and soybean oil in a 3:1 molar ratio was
studied by 1H NMR at temperatures of 10, 35, and 45 C. An
example kinetic plot for the reaction at 10 C using the KOH
catalyst is presented in Figure 5. It is obvious from the kinetic
plot that the reaction has significantly approached equilibrium
within 120 s even at a reduced temperature of 10 C. Therefore,
in a two-stage process, the first stage can begin separation after
a very short stirring period of only a few minutes. Furthermore,
the kinetic plot shows that the methyl ester product has a much
faster rate of formation than the rate of total glyceride depletion.
This has been observed in previous studies and is expected in
this work because the concentration of glycerides is not effected
until all three fatty acid chains are cleaved and glycerol is
produced.8 Only with the production of glycerol is the 1H NMR
signal for the glyceride methylene protons diminished because
the mono- and diglyceride intermediates still contribute to the
proton integration at 4.25 ppm and are noticeable as complex
patterns in addition to the triglyceride pattern discussed earlier.
(10) Wade, L. G., Jr. Organic Chemistry, 2nd ed.; Prentice Hall: New
Jersey, 1987; p 1150.
(11) Goering, C. E.; Schwab, A. W.; Daugherty, M. J.; Pryde, E. H.;
Heakin, A. J. Trans. Am. Soc. Agric. Eng. 1982, 25, 1472-1483.
(12) Kincs, F. R. J. Am. Oil Chem. Soc. 1985, 62, 815-818.
(13) Silverstein, R. M.; Bassler, G.C.; Morrill, T. C. Spectroscopic
Identification of Organic Compounds, 4th ed.; John Wiley and Sons: New
York, 1981; pp 192-212.
(14) Tickell, J. From the Fryer to the Fuel Tank: The Complete Guide
to Using Vegetable Oil as an AlternatiVe Fuel, 3rd ed.; Tickell Energy
Consultants, 2000.
(15) Khan, A. K. Research into Biodiesel Kinetics and Catalyst DeVelopment; University of Queensland: Brisbane, Queensland, Australia, 2002.
(16) Encinar, J. M.; Gonzalez, J. F.; Rodriguez-Reinares, A. Ind. Eng.
Chem. Res. 2005, 44, 5491-5499.

Energy & Fuels, Vol. 20, No. 4, 2006 1353

Figure 6. Arrhenius plot of data presented in Table 2 to determine

the activation energy for methanolysis of soybean oil using the NaOH
catalyst.
Table 2. Temperature Dependence of Initial Rates of Methyl Ester
Formation Obtained by 1H NMR Analysis
temperature (C)

catalyst

methyl ester initial rate (M/s)

10
35
45
10

NaOH
NaOH
NaOH
KOH

6.4 10-3
1.6 10-2
2.3 10-2
2.7 10-3

The glycerol product is not observed in the 1H NMR analysis

because of insolubility in the system. Analogous kinetic data
using NaOH as the catalyst provided the initial rates for methyl
ester formation at several temperatures and are presented in
Table 2. Figure 6 shows the Arrhenius plot (of the initial rates
presented in Table 2) used to obtain the activation energy from
the temperature dependence of the initial rates for the methanolysis of soybean oil using the NaOH catalyst. An activation
energy (Ea) value of 27.2 kJ/mol was obtained from the slope
of the plot according to the equation: ln(rate) ) -Ea/RT + C.
In this equation, R is the gas constant (8.31 J mol-1 K-1), T is
the absolute temperature (in Kelvin), and C is a constant. The
plot provided an excellent linear fit (R2 ) 0.9999), and a
calculated Ea of 27.2 kJ/mol is in agreement with the lower
values reported from experiments using a 6:1 molar ratio
(methanol/oil) and GC analysis.3,7,8 This activation energy would
relate to the conversion of diglyceride to monoglyceride as the
rate-determining step in the proposed multistep mechanism.8
However, more work with this 1H NMR method is necessary
to obtain rate data and an Ea for each step in the proposed
mechanism for base-catalyzed methanolysis of soybean oil.
Although the 1H NMR spectra of starting oils and product
methyl esters are very similar, it is possible to differentiate
resonance signals to the extent that quantitation of the reaction
progress can be achieved. The results obtained using 1H NMR
analyses agree with the results reported from the more traditional
GC analysis of the soybean oil transesterification reaction.
Acknowledgment. J. C. would like to thank the Colorado
College Otis and Margaret Barnes Trust for financial support of
undergraduate research.
EF0503764