Articles

FT-Raman Spectroscopy Quantification of Biodiesel in a Progressive

Soybean Oil Transesterification Reaction and Its Correlation with
1
H NMR Spectroscopy Methods
Grace Ferreira Ghesti,* Julio Lemos de Macedo, Ines Sabioni Resck, Jose Alves Dias, and
S lvia Claudia Loureiro Dias*
Laboratorio de Catalise, Instituto de Qu mica, UniVersidade de Bras lia, caixa postal 4478,
Bras lia-DF, 70904-970, Brazil
ReceiVed December 29, 2006. ReVised Manuscript ReceiVed May 29, 2007
Biodiesel fuel (fatty acid esters) has become more and more attractive due to its environmental benefits.
Transesterification is the most common and important method for making biodiesel from vegetable oils or
animal fats. Several studies have focused on the development and improvement of analytical methods for
monitoring biodiesel production and determining the fuel quality. Analytical procedures reported in the literature
include chromatographic methods (e.g., gas chromatography, high-performance liquid chromatography, gel
permeation chromatography, etc.) and spectroscopic methods [e.g.,
1
H and
13
C NMR, near infrared, Fourier
transform infrared spectroscopy, and recently, Fourier transform (FT)-Raman]. The study presented in this
paper expands our previous research, in which FT-Raman spectroscopy combined with partial least squares
(PLS) multivariate analysis was successfully applied to the quantification of soybean oil/ethyl ester mixtures.
The FT-Raman/PLS methods developed by our group were used to monitor and quantify a transesterification
reaction process involving soybean oil and ethanol to produce fatty acid ethyl esters (biodiesel) over 22 h
catalyzed by a heterogeneous Lewis acid catalyst. The results were successfully correlated with two
1
H NMR
spectroscopic methods reported in the literature and a new
1
H NMR method proposed in this work that can be
easily extended to other vegetable oils. The correlation coefficients (R
2
) obtained from the linear fit between
FT-Raman measurements and the above
1
H NMR methods were 0.9974, 0.9847, and 0.9972, respectively.
1. Introduction
Biodiesel is defined by the American Society for Testing and
Materials (ASTM) as a fuel comprised of monoalkyl esters of
long-chain fatty acids derived from vegetable oils or animal
fats meeting the requirements of ASTM D 6751.
1,2
Biodiesel
has distinct advantages when compared to petroleum-derived
diesel fuel (petrodiesel). It is derived from renewable resources;
is biodegradable; is nontoxic; has low emission profiles, a higher
flash point, and excellent lubricity; and can be used either pure
or blended with petrodiesel fuel.
1,3,4
The use of vegetable oils as fuel has been known since the
Paris Exposition in 1900.
4
However, due to its higher molecular
mass and kinematic viscosity, its direct use in diesel engines
resulted in several operational problems (e.g., poor atomization,
carbon deposits due to incomplete combustion, oil ring sticking,
lubricating problems, etc.).
1,4,5
To solve these problems, four
possible solutions were investigated in literature: transesteri-
* Corresponding authors. Phone: 55-(61)-3307-2162. Fax: 55-(61)-3368-
6901. E-mail: scdias@unb.br (S.C.L.D.) and grace@unb.br (G.F.G.).
(1) Ma, F.; Hanna, M. A. Bioresour. Technol. 1999, 70, 1-15.
(2) ASTM D 6751-03a. Annu. Book ASTM Stand. 2005, 05.04, 609-
614.
(3) Knothe, G. J. Am. Oil Chem. Soc. 1999, 76, 795-800.
(4) The Biodiesel Handbook; Knothe, G., Gerpen, J. V., Krahl, J., Eds.;
American Oil Chemists Society Press: Champaign, IL, 2005.
(5) Meher, L. C.; Sagar, D. V.; Naik, S. N. Renew. Sustain. Energy ReV.
2006, 10, 248-268.
VOLUME 21, NUMBER 5 SEPTEMBER/OCTOBER 2007
Copyright 2007 American Chemical Society
10.1021/ef060657r CCC: $37.00 2007 American Chemical Society
Published on Web 07/17/2007
fication (also called alcoholysis), pyrolysis, dilution with con-
ventional petroleum-derived diesel fuel, and microemulsifi-
cation.
1,4-12
The transesterification reaction is the only method that leads
to products defined as biodiesel (i.e., alkyl esters) and is by far
the most usual method to make biodiesel.
1,4
In transesterification,
the triacylglyceride (TAG) molecules found in animal fats or
vegetable oils (e.g., soybean,
12-14
peanut,
12
rapeseed,
1
etc.)
reacted with an alcohol (e.g., methanol,
12
ethanol,
12-14
etc.) in
the presence of a catalyst (e.g., NaOH,
12-14
H
2
SO
4
,
12
lipase,
11
etc.) to form esters and glycerol (Figure 1).
1,3-5,10,12
In addition
to the advantages previously mentioned, biodiesel fuel produced
by transesterification also has similar properties to petroleum-
derived diesel fuel (e.g., cetane number, viscosity, molecular
mass, density, etc.),
15-18
and no diesel engine modifications are
required.
19
In Brazil, ethanol is a less expensive alcohol than methanol
due to its high production volume from biomass sugarcane. In
this sense, the biodiesel produced from ethanol (fatty acid ethyl
esters or FAEE) can be seen as a truly renewable fuel. In 2003,
the Brazil Federal Government started its own biodiesel pro-
grams (PROBIODIESEL and PNPB), which intend to stimulate
the scientific and technologic development of biodiesel and to
implement the sustainable production and use of biodiesel,
respectively, through the gradual increase of renewable resources
in the Brazilian energetic matrix.
20
The development and
optimization methods for biodiesel production in Brazil and in
other countries have motivated a great number of publications
and patents.
21
The analytical procedures reported in the literature
for the determination of fuel quality and the monitoring of
biodiesel production
22
include chromatographic methods [e.g.,
gas chromatography (GC),
23
high-performance liquid chroma-
tography,
24
gel permeation chromatography (GPC),
22,25
etc.] and
spectroscopic methods [e.g., nuclear magnetic resonance
(NMR),
14,26,27
near-infrared (NIR),
3,27
Fourier transform infrared
spectroscopy (FTIR),
13,25
and recently, FT-Raman spectros-
copy
28
].
Spectroscopic techniques are fast, easily adapted in routine
process analysis, and allow nondestructive measurements of the
samples
22,29
versus time-consuming chromatographic methods.
NMR spectroscopy has become one of the most powerful
techniques to investigate and identify the structure of chemical
compounds and dynamics of molecular systems in almost all
branches of chemistry.
30,31
Although NMR spectroscopy does
not present the detection limit accuracy for the quantification
of minor components as chromatographic methods do, NMR is
a suitable method to monitor a chemical reaction, since small
amounts are required to obtain a quantitative spectrum with
significant information related to the substances of interest in
the reaction media.
32
Alternatively, the employment of vibrational spectroscopic
techniques (NIR, FTIR, and Raman) in quality-control monitor-
ing has been growing quickly due to several qualities (e.g., fast
measurements, easy handling, accuracy, reliability, possibility
of on-line monitoring with fiber-optic probes, etc.).
22,33
For
example, NIR spectroscopy is being used to analyze free fatty
acid contents in oils,
34,35
and Raman spectroscopy has been
widely used in the pharmaceutical
36
and polymer industries.
37
Uni- and multivariate regression analyses have been widely used
to develop calibration models based on vibrational spectroscopic
data.
38-40
Actually, all reports in the literature until now
describing vibrational spectroscopy methods to monitor and
quantify biodiesel fuel production were based on regression
analysis.
3,13,27
Recently, our group reported the advantages of FT-Raman
spectroscopy to quantify the concentration of ethyl esters in
(6) Schwab, A. W.; Bagby, M. O.; Freedman, B. Fuel 1987, 66, 1372-
1378.
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9, 363-378.
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(20) Programa Nacional de Produc ao e Uso de Biodiesel Home Page.
http://www.biodiesel.gov.br (accessed May 2007).
(21) Pinto, A. C.; Guarieiro, L. L. N.; Rezende, M. J. C.; Ribeiro, N.
M.; Torres, E. A.; Lopes, W. A.; Pereira, P. A. P.; Andrade, J. B. J. Braz.
Chem. Soc. 2005, 16, 1313-1330.
(22) Knothe, G. Trans. ASAE 2001, 44, 193-200.
(23) Plank, C.; Lorbeer, E. J. Chromatogr., A 1995, 697, 461-468.
(24) Holcapek, M.; Jandera, P.; Fischer, J.; Prokes, B. J. Chromatogr.,
A. 1999, 858, 13-31.
(25) Dube, M. A.; Zheng, S.; McLean, D. D.; Kates, M. J. Am. Oil Chem.
Soc. 2004, 81, 599-603.
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F. J. Am. Oil Chem. Soc. 1995, 72, 1239-1241.
(27) Knothe, G. J. Am. Oil Chem. Soc. 2000, 77, 489-493.
(28) Ghesti, G. F.; Macedo, J. L.; Braga, V. S.; Souza, A. T. C. P.;
Parente, V. C. I.; Figueredo, E. S.; Resck, I. S.; Dias, J. A.; Dias, S. C. L.
J. Am. Oil Chem. Soc. 2006, 83, 597-601.
(29) Drago, R. S. Physical Methods for Chemists, 2nd ed.; Saunders
College Publishing: New York, 1992; pp 162-192.
(30) Silverstein, R. M.; Bassler, G. C.; Morril, T. C. Spectrometric
Identification of Organic Compounds, 7th ed.; John Wiley & Sons: New
York, 2005; pp 165-202.
(31) Engelhardt, G.; Michel, D. High-Resolution Solid-State NMR of
Silicates and Zeolites; John Wiley & Sons: New York, 1987; p 1.
(32) Morgenstern, M.; Cline, J.; Meyer, S.; Cataldo, S. Energy Fuels
2006, 20, 1350-1353.
(33) Cooper, J. B.; Wise, K. L.; Jensen, B. J. Anal. Chem. 1997, 69,
1973-1978.
(34) Sato, T. Biosci. Biotechnol. Biochem. 2002, 66, 2543-2548.
(35) Zhang, H.-Z.; Lee, T.-C. J. Agric. Food Chem. 1997, 45, 3515-
3521.
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G. TrAC, Trends Anal. Chem. 2002, 21, 869-877.
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15, 18-25.
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1995, 67, 4096-4100.
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87, 477-481.
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1821.
Figure 1. Scheme of a generic transesterification reaction between
one triacylglyceride (TAG) molecule and three ethanol (EtOH)
molecules (stoichiometric relation) to form three molecules of ethyl
esters (FAEE or biodiesel) and one glycerol (GLY) molecule.
2476 Energy & Fuels, Vol. 21, No. 5, 2007 Ghesti et al.
known standard mixtures containing soybean oil.
28
The present
paper extends that work, and FT-Raman/partial-least-squares
(PLS) calibration models were used to monitor and quantify a
progressive transesterification reaction with soybean oil and
ethanol. The results obtained were correlated with three
1
H NMR
spectroscopic methods, two of them already reported by other
authors
14,41
and a new approach reported here.
2. Experimental Section
2.1. Materials. Commercial refined soybean oil (Soya), NaOH
(Vetec, 99%), NaCl (Vetec, 99.5%), and concentrated HCl (Vetec,
38%) were used as received. Ethanol (Vetec, 99.8%) was dried
over 3A molecular sieves (Aldrich) for at least 24 h before the
experiments, and MgSO
4
7H
2
O (Vetec, 98.0%) was dried at 300
C for 4 h.
2.2. Preparation of the Heterogeneous Catalyst. Cerium tris-
dodecylsulfate was used as a heterogeneous Lewis acid catalyst
for the transesterification reaction. The synthesis, characterization,
and modification have been described elsewhere.
42,43
For all reaction
procedures, the catalyst was activated in a muffle furnace at 100
C for 4 h.
2.3. Preparation of Biodiesel and Soybean Oil/Biodiesel
Standard Mixtures. Ethyl esters were prepared by transesterifi-
cation according to conditions suggested in the literature
12
for base-
catalyzed reactions. The reaction was performed in a 50 mL glass
round-bottom flask containing 20.00 g of soybean oil, 6.34 g of
anhydrous ethanol (1:6 oil-to-alcohol mole ratio), and 0.20 g of
NaOH (1% w/w of oil). The system was kept stirring at 80 C
under reflux for 90 min. Then, the product was cooled to room
temperature, washed several times with consecutive aqueous
solutions of HCl (0.5 wt %) and NaCl (5 wt %), and dried over
anhydrous magnesium sulfate and residual alcohol was removed
in a rotary evaporator at 70 C. Molecular weights of biodiesel
and soybean oil were calculated using the composition of fatty acids
obtained from the literature.
44
The quality of the product was
verified by NMR (section 2.6) and GC (GC-17A Shimadzu
chromatograph with a flame ionization detector and polydimeth-
ylsiloxane column, CBPI PONA-M50-042). Since no significant
contaminants were observed, the biodiesel was treated as 100%
ethyl ester. Six soybean oil/ethyl ester samples (0:100, 20:80, 40:
60, 60:40, 80:20, and 100:0%) were prepared by weighing the
feedstock (soybean oil) and the ethyl ester standards produced
(biodiesel).
2.4. Transesterification Reaction. The heterogeneous transes-
terifications were carried out in 50 mL glass round-bottom flasks
kept under stirring at 100 C under reflux conditions. A total of
six reactions (2, 6, 10, 14, 18, and 22 h) were made under identical
conditions in order to reproduce a continuous 22 h reaction. For
each run, 10.00 g of soybean oil, 15.85 g of anhydrous ethanol
(1:30 oil-to-alcohol mole ratio), and 1.00 g of the catalyst (10%
w/w of oil) were used. After the reaction, the system was cooled
to room temperature, centrifuged to remove the catalyst, washed
three times with a 5 wt % NaCl solution, and dried over anhydrous
magnesium sulfate; residual alcohol was removed in a rotary
evaporator at 70 C; and then the system was kept in a refrigerator
for FT-Raman and NMR analyses. The reaction conditions used
above were determined to be ideal for this catalyst according to
Ghesti.
45
2.5. FT-Raman/PLS Analysis. FT-Raman spectra were recorded
on a Bruker FRA 106/S module attached to a Bruker Equinox 55
spectrometer using a 1 cm quartz cuvette with a mirrored surface
toward the scattering direction (128 scans and 4 cm
-1
resolution).
The laser excitation (Nd:YAG) and laser power were 1064 nm and
250 mW, respectively, and the signal was detected by a liquid N
2
cooled Ge detector. All spectra were recorded at room temperature.
The complete procedure of the method developed and theoretical
explanations were described in a previous publication.
28
For
multivariate analysis, PLS-1 (OPUS-NT Quant software, from
Bruker) methods were used, also as described previously.
28
2.6.
1
H NMR Measurements. NMR experiments were per-
formed at 7.05 T using a Varian Mercury Plus NMR spectrometer
equipped with 5 mm Varian probes (ATB and SW) using CDCl
3
as solvent.
1
H (300 MHz) spectra were recorded with a pulse
duration of 45, a recycle delay of 1.36 s and 16 scans. The spectra
were referenced to tetramethylsilane ( ) 0.0 ppm).
13
C (75.46
MHz) spectra were recorded with a pulse duration of 45 , a recycle
delay of 0.28 s and 300 scans. The spectra were referenced to CDCl
3
( ) 77.0 ppm). The two-dimensional experiment heteronuclear
multiple quantum correlation (HMQC) was obtained with the field
gradient mode, and the attached proton test (APT) pulse sequence
was used to distinguish
13
C NMR multiplicities.
3. Results and Discussion
3.1.
1
H NMR Methods. The first reports involving biodiesel
synthesis by transesterification and
1
H NMR analysis were
primarily focused on yield determination of progressive metha-
nolysis
26,27
or ethanolysis
14,41
reactions (i.e., transesterification
of a vegetable oil or animal fat with methanol or ethanol,
respectively). In an article by Knothe,
27
the rate of oil conversion
to fatty acid methyl ester (FAME) was also studied by removing
aliquots through the reaction and analyzing by
1
H NMR. This
latter work was expanded by recent papers,
32,46
where NMR
spectroscopy was used to elucidate aspects related to the kinetics
and the mechanism of biodiesel production. The transesterifi-
cation process to produce biodiesel fuel from TAGs follows a
stepwise mechanism of consecutive reversible reactions: (1)
conversion of TAG into diacylglyceride (DAG) and FAME, (2)
conversion of DAG into monoacylglyceride (MAG) and FAME,
and (3) conversion of MAG into glycerol (GLY) and FAME.
47
Morgenstern and co-workers
32
calculated, from initial rates of
FAME formation, an activation energy of 27.2 kJ mol
-1
for
the rate-determining step (DAG to MAG) in the multistep
mechanism proposed by Freedman and co-workers.
47
It has been
shown
48
that
13
C NMR can provide valuable information about
the acyl positional distribution of TAG (triacylglyceride) in
vegetable oils. Jin and co-workers,
46
by using
1
H and
13
C NMR
to identify the positional isomers of DAGs and MAGs formed
during the transesterification reaction, reported that the metha-
nolysis pathway occurs preferentially through sn-1,3-DAGs to
sn-1-MAG intermediates. These results showed that NMR
spectroscopy can be successfully applied in all steps of biodiesel
production.
(41) Silva, C. L. M. Obtenc ao de E steres Et licos a Partir da Transes-
terificac ao do O leo de Andiroba com Etanol. M.S. Thesis, University of
Campinas, Campinas, SP, Brazil, 2005.
(42) Ghesti, G. F.; Macedo, J. L.; Dias, J. A.; Dias, S. C. L. Green Lewis
Acid Catalysts for Biodiesel Production by Transesterification. Brazilian
Patent Appl. 325, 2007.
(43) Ghesti, G. F.; Macedo, J. L.; Parente, V. C. I.; Dias, J. A.; Dias, S.
C. L. To be submitted for publication, 2007.
(44) Gunstone, F. D. Fatty Acid and Lipid Chemistry; Aspen Publishers,
Inc.: Gaithersburg, MD, 1999; p 76.
(45) Ghesti, G. F. Estudo de Catalisadores para Obtenc ao de Biodiesel
por Transesterificac ao e Determinac ao do Rendimento por Espectroscopia
Raman. M.S. Thesis, University of Bras lia, Bras lia, DF, Brazil, 2006.
(46) Jin, F.; Kawasaki, K.; Kishida, H.; Tohji, K.; Moriya, T.; Enomoto,
H. Fuel 2007, 86, 1201-1207.
(47) Freedman, B.; Butterfield, R. O.; Pryde, E. H. J. Am. Oil Chem.
Soc. 1986, 63, 1375-1380.
(48) Mannina, L.; Luchinat, C.; Emanuele, M. C.; Segre, A. Chem. Phys.
Lipids 1999, 103, 47-55.
FT-Raman Spectroscopy Quantification of Biodiesel Energy & Fuels, Vol. 21, No. 5, 2007 2477
As described in the literature,
49
the transesterification reaction
catalyzed by Lewis acid sites occurs through the coordination
of acyl groups of the triacylglyceride molecule to lowest
unoccupied molecular orbitals of catalytic active centers. This
coordination increases acyl group polarization and forms a
carbocation that undergoes alcohol nucleophilic attack. The
tetrahedral intermediary so formed eliminates the diacylglyceride
molecule and produces an ester.
Quantification of biodiesel by NMR can be made by simple
equations,
26,27,41
by building an analytical curve
14
or by using
an internal standard (e.g., acetone).
32
Ethyl ester quantification
by
1
H NMR spectroscopy is more complex than methyl ester
quantification due to a superimposition of the glyceryl meth-
ylenic hydrogens in soybean oil and the -OCH
2
from ethyl
ester in biodiesel (see Figures 1-3). Figure 3 shows the
1
H
NMR spectra of pure soybean oil (doublet of doublets), pure
ethyl esters (quartet), and a 40:60 (m/m) soybean oil/ethyl ester
mixture from 4.00 to 4.40 ppm, where it illustrates the
difficulties created from signals overlapping caused by partial
conversion. To overcome this problem, two methods have
already been proposed by Neto and co-workers,
14
who prepared
an analytical curve, and Silva,
41
who used an equation. In the
next paragraphs, we will describe the use of both methods, and
a third alternative is suggested in this work.
To clarify the methods to be described, the following notation
was used (see Figure 2 and 3 for further clarification): (i) I
TAG
) integration of glyceryl methylenic hydrogens at 4.25-4.35
ppm; (ii) I
TAG+EE
) integration of glyceryl methylenic hydro-
gens and -OCH
2
of ethoxy hydrogens superimposed at 4.10-
4.20 ppm; and (iii) I
RCH2
) integration of R-acyl methylenic
hydrogens in soybean oil and ethyl esters at 2.20-2.40 ppm.
The analytical curve was built by plotting known concen-
trations of soybean oil/ethyl ester mixtures (see experi-
mental section 2.3) versus I
TAG
/I
TAG+EE
ratios. Values of 0 and
1 were attributed to pure ethyl esters and pure soybean oil in
I
TAG
/I
TAG+EE
, respectively. The correlation coefficient (R
2
) so
obtained was 0.9867.
The equation proposed by Silva
41
and presented below (eq
1) was applied to the same standard mixtures of soybean oil
and ethyl ester prepared (see experimental section 2.3) to
determine soybean oil conversion into biodiesel (C
EE
):
A plot of predicted values versus real values for eq 1 showed
an R
2
) 0.9977. However, in our
1
H NMR spectral analyses,
45
it was observed that intermediary compounds formed during
transesterification
46
persisted in biodiesel fuel after purification
steps, which regularly causes the signals from -OH groups in
mono- and diacylglyceride species to overlap with RCH
2
signals
at 2.20-2.40 ppm (Figure 4).
50
Their presence was also
observed at 3.50-3.80 ppm.
50
To use the integrated values in
eq 1, all spectra were treated by deconvolution and subtraction
steps to ensure reliable results. To avoid these time-consuming
procedures, a new equation is proposed in this work (eq 2),
where R-acyl methylenic hydrogens (RCH
2
) are not taken into
account:
As one can see, it is obvious that the above equation can be
simplified to %C
EE
) 100[(I
TAG+EE
- I
TAG
)/(I
TAG+EE
+ 2I
TAG
)].
However, to facilitate the following explanations, it was left in
the original form. The numbers 4 and 6 in eq 2 are related to
four glyceryl methylenic hydrogens present in TAG molecules
(49) Parshall, G. W.; Ittel, S. D. Homogeneous Catalysis, 2nd ed.;
Wiley: New York, 1992; pp 270-275.
Figure 2. Full
1
H NMR spectra from (a) pure soybean oil and (b)
pure ethyl ester.
Figure 3.
1
H NMR spectra at 4.00-4.40 ppm region from (a) pure
soybean oil, (b) 40:60 (m/m) soybean oil/ethyl esters, and (c) pure ethyl
ester.
Figure 4.
1
H NMR spectra at 2.20-2.40 ppm region from (a) pure
ethyl ester and (b) product from transesterification reaction after 2 h
(* indicates NMR signals from intermediate species).
%C
EE
) 100
(
(I
TAG+EE
- I
TAG
)
I
RCH
2
)
(1)
%C
EE
) 100
(
4(I
TAG+EE
- I
TAG
)
4(I
TAG+EE
- I
TAG
) + 6(2I
TAG
)
)
(2)
2478 Energy & Fuels, Vol. 21, No. 5, 2007 Ghesti et al.
and to six hydrogens formed in three ethyl ester products (see
Figure 1), respectively. The number of glyceridic hydrogens
assigned at 4.35-4.10 ppm was obtained from the litera-
ture,
32,46,48,50
and also from our own 2D heteronuclear correlation
in the HMQC experiment (not shown), where four glyceridic
hydrogens at 4.35-4.10 ppm were correlated with the CH
2
O-
carbon signal at 62.0 ppm and one glyceridic hydrogen at 5.20-
5.33 ppm was correlated with the CHO- carbon signal at 68.8
ppm. The APT experiment (not shown) and computational
simulation analysis were used to discriminate and correctly
assign carbon ressonances. The difference I
TAG+EE
- I
TAG
(i.e.,
integration from the superimposition between glyceryl meth-
ylenic hydrogens of TAG molecules and the -OCH
2
of ethoxy
hydrogens from ethyl ester molecules at 4.10-4.20 ppm minus
the integration of glyceryl methylenic hydrogens at 4.25-4.35
ppm) in eq 2 is associated with ethyl ester formation. To simplify
eq 2, the areas from glyceryl methylenic groups for soybean
oil at 4.25-4.35 and 4.10-4.20 ppm were assumed to be equal
(see Figure 3), that is, 2I
TAG
. Although this is not exactly true,
because the ratio between the two areas is 0.98 instead of 1.00,
the values obtained from eq 2 on standard soybean oil/ethyl
ester mixtures, considering hydrogen areas to be equivalent or
not, were very similar (see Table 1). Indeed, a plot of biodiesel
concentration predicted by eq 2 versus the true concentration,
using both calculations in Table 1, resulted in the same
correlation coefficient (R
2
) 0.9983).
3.2. FT-Raman/PLS Methods. Knothes work,
3
the first
report linking vegetable oil transesterification and vibrational
spectroscopic analysis, described biodiesel quantification and
fuel quality assessment by NIR spectroscopy using a fiber-optic
probe. The major spectral differences between soybean oil and
their respective methyl esters were distinguished at 4425-4430
and 6005 cm
-1
, where it was observed that methyl esters
displayed bands while soybean oil triacylglyceride molecules
exhibited only shoulders. These regions were analyzed for
biodiesel and contaminant quantifications (glycerol and metha-
nol) in the spectra obtained from soybean oil/methyl ester and
contaminant/methyl ester mixtures by PLS calibration models
with excellent results for both spectral regions. In a second NIR
paper,
27
Knothe successfully applied the calibration models built
in the first paper to a transesterification reaction in progress
and correlated, with good agreement, with
1
H NMR spectros-
copy. Although FTIR spectra of vegetable oil and biodiesel are
almost identical, Zagonel and co-workers
13
developed an
analytical procedure, based on CdO stretching vibration
displacement from 1746 cm
-1
(TAGs) to 1735 cm
-1
(ethyl
esters), to monitor soybean oil ethanolysis by FTIR multivariate
analysis models. The results obtained in a progressive transes-
terification reaction by the calibration model, built from FTIR
spectra of standard mixtures of triolein and ethyl esters analyzed
by principal component analysis and PLS regression, were
correlated with GPC analyses showing only small differences
(R
2
) 0.9837).
Raman spectra of soybean oil and their corresponding ethyl
esters showed several differences: (i) a peak at 2932 cm
-1
(
CH2
), whereas the soybean oil spectrum displays only a
shoulder; (ii)
CdO
displacement from 1748 (soybean oil) to
1739 cm
-1
(ethyl esters); and (iii) bands at 861 (assigned as

R-CdO
and
C-C
) and 372 cm
-1
(assigned as
CO-O-C
).
28
Raman spectra present well-defined bands and less congestion
than FTIR and NIR spectra, and as a consequence, there is a
smaller possibility of band overlapping, making quantitative
measurements simpler.
51,52
As mentioned earlier, in our previous work,
28
FT-Raman/
PLS methods showed good prediction capabilities when applied
to known samples treated as unknown. A data set of 63 spectra
from 21 standard soybean oil/ethyl ester mixtures (triplicate
measurements) was used, and from the numerous correlations
and regression methods tested for analysis, FT-Raman/PLS
methods at the CH stretching region (3100-2740 cm
-1
, mainly
the peak at 2932 cm
-1
) could be distinguished from the others
due to their predicted capabilities: (i) R
2
) 0.9994 for the model
using 21 samples averaged from 63 measurements as a data set
(model 4, notation used in ref 28) and (ii) R
2
) 0.9993 for the
model using all 63 measurements as a data set (model 5). A
detailed description of both methods was given elsewhere,
28
and
no further discussion will be mentioned here. The two models
were applied to the heterogeneous transesterification reaction,
and similar results were obtained. A plot of model 4 predicted
values versus model 5 predicted values results in an R
2
)
0.9976. However, when compared to the predicted values found
by
1
H NMR methods, model 4 was slightly better then model
5. Although multivariate analysis generally requires a large
number of samples to build reliable calibration models (at least
20 samples for each component), the articles of Knothe
3,27
using
NIR/PLS, and Zagonel et al.
13
using FTIR/PLS, were success-
fully applied to progressive transesterification reactions with
smaller data sets in their models. In our case, the effects of
increased data sets on FT-Raman/PLS models led to increased
predicted capabilities and linearity (see Table 2).
3.3. Correlation Between FT-Raman/PLS and
1
H NMR
Methods. In preceding articles dealing with spectroscopic
measurements of biodiesel content in transesterification reac-
(50) Jie, M. S. F. L. K.; Lam, C. C. Chem. Phys. Lipids 1995, 77, 155-
171.
(51) Baranska, H.; budzinska, A.; Terpinski, J. Laser Raman Spectros-
copy: Analytical Applications; Ellis Horwood Limited: Chichester, U.K.,
1987; pp 151-168.
(52) Skoog, D. A.; Holler, F. J.; Nieman, T. A. Principles of Instrumental
Analysis; Saunders College Publishing: Philadelphia, PA, 1997; pp 429-
443.
Table 1. Biodiesel True Concentration Values and Those Predicted
by Eq 2 with and without Assumption of Six Standard Soybean
Oil/Ethyl Esters Mixtures
true CEE
values (wt %)
predicted CEE
values (wt %)
without assumption
a
predicted CEE
values (wt %)
with assumption
b
0.00 0.00 0.63
20.45 20.64 21.19
40.39 42.46 42.90
59.85 60.62 60.94
79.62 76.80 77.00
100.00 99.40 99.41
a
CEE values calculated from eq 2 without assuming that glyceryl
methylenic hydrogens areas at 4.25-4.35 and 4.10-4.20 ppm are identical.
b
CEE values calculated from eq 2 assuming that glyceryl methylenic
hydrogens areas at 4.25-4.35 and 4.10-4.20 ppm are identical.
Table 2. Predicted CEE Values by Raman/PLS Models with
Increased Data Set for Standard Soybean Oil/Ethyl Ester Mixtures
of Known Composition Treated as Unknown
predicted CEE values (wt %)
true CEE
values (wt %)
a
6 samples
data set size
(R
2
) 0.9990)
11 samples
data set size
(R
2
) 0.9991)
21 samples
data set size
(R
2
) 0.9997)
0.00 3.73 3.71 0.52
a
20.01 25.91 25.72 21.88
a
49.82 51.23 51.34 50.28
a
79.69 80.52 80.42 80.64
a
100.00 99.64 99.18 99.31
a
Values obtained from ref 28.
FT-Raman Spectroscopy Quantification of Biodiesel Energy & Fuels, Vol. 21, No. 5, 2007 2479
tions, the results obtained were correlated with other techniques
to validate their values. Knothe
27
correlated NIR spectroscopy
and two
1
H NMR equations with good agreement. Neto and
co-workers
14
correlated their
1
H NMR analytical curves with
viscosity measurements, obtaining a linear correlation coefficient
of 0.9981. Zagonel and co-workers
13
used GPC to validate their
FTIR/PLS methods, obtaining an R
2
) 0.9837 between both
techniques. Figure 5 displays FT-Raman/PLS (model 4) and
1
H NMR methods applied to a progressive heterogeneous
transesterification reaction. The results obtained from all four
methods (FT-Raman/PLS and three
1
H NMR methods) showed
good correlation (Figure 5) with each other, indicating their
mutual validation. Indeed, the linear fit between FT-Raman
measurements and all NMR methods tested (i.e., analytical
curve, eq 1, and eq 2) showed R
2
values of 0.9974, 0.9847, and
0.9972, respectively (Figure 6). The difference observed between
biodiesel yield values from eq 1 and the other methods after 2
h of reaction is associated with integration difficulties produced
by signal superimposition from glyceridic intermediates (MAGs
and DAGs) and R-acyl CH
2
.
Although both methods presented in this work do not attend
the detection limits required by ASTM to determine low levels
of contaminants in biodiesel fuel as chromatographic methods
do, spectroscopic techniques allows a faster analysis of the
reaction by easily identifying the formation of peaks or bands
related to ethyl ester products. These methods, when coupled
to multivariate analysis, can be applied to fast reversible
reactions, such as transesterification,
3,13,14,26,27,32,46
to evaluate
a new catalyst potentiality to produce biodiesel. In conclusion,
FT-Raman/PLS models are an attractive alternative to rapidly
monitor biodiesel synthesis due to their inherent qualities and
their reduced costs when compared with other techniques,
including NMR. However,
1
H NMR methods, especially when
involving simple equations, are more easily adapted to other
vegetable oils methanolysis or ethanolysis reactions. Both
procedures described here were successfully correlated with each
other and validate by reported methods, evidencing and increas-
ing their potential for biodiesel synthesis and quality monitoring.
Acknowledgment. Acknowledgement is given to CNPq, CAPES/
PQI, FINATEC, Finep-CTPetro, Finep-CTInfra 970/01, UnB-IQ,
UnB-IG, and FAPDF/SCDT/CNPq.
EF060657R
Figure 5. Biodiesel yield from a heterogeneous transesterification
reaction determined by four methods:
1
H NMR analytical curve, eq 1,
eq 2, and FT-Raman/PLS model.
Figure 6. Plot from FT-Raman/PLS model predicted reaction values
vs
1
H NMR analytical curve, eq 1, and eq 2 predicted reaction values
(R
2
) 0.9974, 0.9847, and 0.9972, respectively).
2480 Energy & Fuels, Vol. 21, No. 5, 2007 Ghesti et al.