Energy & Fuels 2009, 23, 2273–2277 2273

Estimation of Free Fatty Acid Content in Oils, Fats, and Biodiesel

by 1H NMR Spectroscopy
Jitendra K. Satyarthi, D. Srinivas,* and Paul Ratnasamy*
National Chemical Laboratory, Pune 411 008, India

ReceiVed NoVember 24, 2008. ReVised Manuscript ReceiVed January 29, 2009

A novel 1H NMR method for the quantification of free fatty acid (FFA) content in vegetable oils, animal
fats, and biodiesel is reported. Nonedible oils and animal fats, which are increasingly being explored as cheaper,
renewable feed stocks for biodiesel production by transesterification with methanol, contain a significant amount
of FFA along with other acidic impurities. The 1H NMR spectroscopic method is found to be more accurate
than the conventional titrimetric analysis for the estimation of FFA content especially in those cases where
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acidic entities other than the FFA are also present in the feedstock. The titrimetric methods provide a gross
acid value which corresponds to that of FFA and other acidic impurities. Our NMR method provides the FFA
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content exclusively. In the case of refined edible oils (wherein the other acidic impurities are negligible), the
results obtained from the 1H NMR method are comparable with those from the titrimetic analysis.

1. Introduction
Biodiesel is gaining importance as an alternative fuel due to
its biodegradability, nontoxicity, renewability, and carbon
neutrality.1,2 It is prepared from vegetable oils by transesteri-
fication with methanol. Due to the “food verses fuel” problem,
use of edible oil as a fuel source may not be a viable, long-
term solution. Nonedible oils such as jatropha, pongamia, as
well as animal fat, tallow, etc., can replace vegetable oils as
fuel sources. These nonedible oils usually contain free fatty acids
(FFAs), phospholipids, sterols, water, odorants, and other
impurities.1,2 The FFA content of these feed stocks is an
important quality parameter.
Conventionally, the FFA content is determined by acid-base
titration methods.3,4 It is dependent on a visual end point which
is difficult to observe particularly in the cases of nonedible oils
which are dark-colored. To avoid the limitations of the
conventional titration method, various instrumental methods
such as potentiometry,5 pH metry,6 chromatography, especially
GC and HPLC,7-10 colorimetric ultramicro method,11 FTIR,12-14
* To whom correspondence should be addressed. Telephone: +91 20
2590 2018. Fax: +91 20 2590 2633. E-mail: d.srinivas@ncl.res.in (D.S.);
p.ratnasamy@ncl.res.in (P.R.).
(1) (a) Pinto, A. C.; Guarieiro, L. L. N.; Rezende, M. J. C.; Ribeiro,
N. M.; Torres, E. A.; Lopes, W. A.; Pereira, P. A. P.; de Andrade, J. B. J.
Braz. Chem. Soc. 2005, 16, 1313–1330. (b) Schuchardt, U.; Sercheli, R.;
Vargas, R. M. J. Braz. Chem. Soc. 1998, 9, 199–210.
(2) Di Serio, M.; Tesser, R.; Pengmei, L.; Santacesaria, E. Energy Fuels
2007, 22, 207–217.
(3) Li, S.-G.; Zhang, H.; Xue, W.-T. Eur. J. Lipid Sci. Technol. 2007,
109, 1088–1094.
(4) Che Man, Y. B.; Setiowaty, G. Food Chem. 1999, 66, 109–114.
(5) Lykken, L.; Porter, P.; Ruliffson, H.; Tuemmler, F. Anal. Chem.
1944, 16, 219–234.
(6) (a) Tur’yan, Y. I.; Berezin, O. Y.; Kuselman, I.; Shenhar, A. J. Am.
Oil Chem. Soc. 1996, 73, 295–301. (b) Kuselman, I.; Turýan, Y. I.; Burenko,
T.; Goldfeld, I.; Anisimov, B. Talanta 1999, 49, 629–637.
(7) Ackman, R. G. Food Sci. Technol. N.Y. 1992, 53, 47–63.
(8) (a) Ballesteros, E.; Gallego, M.; Valeareel, M. Anal. Chim. Acta 1993,
282, 581–588. (b) Ballesteros, E.; Gallego, M.; Valeareel, M. Anal. Chem.
1994, 66, 628–634. (c) Procida, G.; Ceccon, L. Anal. Chim. Acta 2006,
561, 103–106.
(9) Fuse, T.; Kusu, F.; Takamura, K. J. Chromatogr., A 1997, 764, 177–
182.
near IR,15,16 and 31P NMR17 have been reported for the
determination of FFA content in vegetable oils. A drawback of
some of these methods is that a chemical modification of the
sample is often necessary for analysis.
The NMR method can be used for quantitative analysis based
on the fact that the amplitude of a proton nuclear magnetic
resonance (1H NMR) signal is proportional to the number of
hydrogen nuclei contained in the molecule.18,19 Although GC
and HPLC are more sensitive techniques than NMR, the latter
is a more rapid and easy method to use than the former. The
area of the NMR peak depends mainly on the number of protons
and not on their response factor. Each and every component
FFA in the sample has a different response factor which needs
to be predetermined and used in the quantitative determination
by the chromatographic techniques. Such laborious processes
can be eliminated in the case of NMR detection. Hence, NMR
is more suited for quantification compared to chromatographic
methods such as GC and HPLC.
In the present work, we have, for the first time, applied 1H
NMR spectroscopy for the quantitative determination of FFA
(10) (a) Lam, S.; Grushka, E. J. Chromatogr. 1978, 158, 207–214. (b)
Komers, K.; Stloukal, R.; Machek, J.; Skopal, F.; Komersova, A. Fett/
Lipid 1998, 100, 507–512.
(11) (a) Novak, M. J. Lipid Res. 1965, 6, 431–433. (b) GyQrik, M.;
Ajtony, Z.; Dǒka, O.; Alebic-Juretić, A.; Dicanic, D.; Koudijs, A. Instrum.
Sci. Technol. 2006, 34, 119–128.
(12) Saad, B.; Ling, C. W.; Jab, M. S.; Lim, B. P.; Mohamad Ali, A. S.;
Wai, W. T.; Saleh, M. I. Food Chem. 2007, 102, 1407–1414.
(13) Al-Alawi, A.; van de Voort, F. R.; Sedman, J.; Ghetler, A. JALA
2006, 11, 23–29.
(14) Sherazi, S. T. H.; Mahesar, S. A.; Bhanger, M. I.; van de Voort,
F. R.; Sedman, J. J. Agric. Food Chem. 2007, 55, 4928–4932.
(15) Moschner, C. R.; Biskupek-Korell, B. Eur. J. Lipid Sci. Technol.
2006, 108, 606–613.
(16) Gerde, J. A.; Hardy, C. L.; Hurburgh Jr, C. R.; White, P. J. J. Am.
Oil Chem. Soc. 2007, 84, 519–522.
(17) Dayrit, F. M.; Buenafe, O. E. M.; Chainani, E. T.; de Vera, I. M. S.
J. Agric. Food Chem. 2008, 56, 5765–5769.
(18) Gelbard, G.; Brès, O.; Vargas, R. M.; Vielfaure, F.; Schuchardt,
U. F. J. Am. Oil Chem. Soc. 1995, 72, 1239–1241.
(19) (a) Knothe, G. Trans. ASAE 2001, 44 (2), 193–200. (b) The
Biodiesel Handbook; Knothe, G., Krahl, J., Van Gerpen, J., Eds.; AOCS
Press: Champaign, IL, 2005.

10.1021/ef801011v CCC: $40.75  2009 American Chemical Society

Published on Web 03/09/2009
2274 Energy & Fuels, Vol. 23, 2009
Figure 1. 1H NMR spectra and assignment of peaks: (a) soybean oil,
(b) fatty acid methyl ester (biodiesel), and (c) oleic acid.
in vegetable oils, animal fats, and biodiesel. We have used the
R-CH2 of FFA and ester (triglyceride of vegetable oil or methyl
ester of biodiesel) to estimate the FFA content. Application of
this method to monitor the esterification of FFA has also been
demonstrated. Esterification and transesterification reactions of
vegetable oils and animal fats were conducted using solid,
hydrophobic, double metal cyanide Fe-Zn catalysts. In the past
we had reported24 that these catalysts are highly efficient for
converting a range of edible and nonedible oils to biodiesel.
2. Experimental Section
All the chemicals were of analytical reagent grade and were used
as such without any further purification. Oleic acid was procured
from Loba Chemie, India. Sodium hydroxide, oxalic acid, phenol-
phthalein indicator (1% solution), and solvents were obtained from
Merck, India. All the vegetable oils (soybean oilscommercial
refined oil, karanjascrude oil, chicken fatscrude) were procured
from the local market and standard biodiesel from a commercial
vendor. Deionised Milli-pore water was used to prepare solutions
for titration. Chloroform-d (99.8% atom D with 0.1% v/v TMS)
from Aldrich was used as a solvent for 1H NMR sample preparation.
Satyarthi et al.
Scheme 1. Representative Structures of Vegetable Oil, FFA, and
Biodiesel
For the calibration of the 1H NMR method and for the
quantification of FFA in oils or biodiesel, standard solutions (oleic
acid in soybean oils0.5, 0.7, 1, 5, 10, 20, 30, and 40 wt %sand
oleic acid in biodiesels0.5, 0.7, 1, 2, 5, 10, 15, and 20 wt %) were
prepared and their compositions determined by both 1H NMR
method and the conventional titration method by titrating a sample
with 0.1 N NaOH using phenolphthalein solution (1%) as the
indicator. FFA content (1H NMR and titration) was determined
twice for each sample, and the average values are reported. For 1H
NMR, 20-25 mg of the sample was dissolved in 0.6 mL of CDCl3
and the spectrum was recorded at 298 K on a Bruker AV 200 MHz
spectrometer; 30 scans for each sample were taken during the
measurement. A standard 5 mm quadronuclei (1H, 31P, 13C, and
15N) probe (QNP) was used. An acquisition time of 3.9 s, relaxation
delay of 1 s, flip angle of 30°, and sweep width of 4.139 kHz were
employed in the spectral measurements. Calibration curves were
plotted, and correlations among the titrimetric and 1H NMR data
were established. The FFA content of nonedible oils was determined
by 1H NMR and compared with the titration method. The choice
of FFA content in standard samples preparation was made based
on its proportion (<40 wt %) generally present in nonedible oils
like karanja, chicken fat, and acid palm oil. To remove phospho-
lipids, chicken fat, for example, was treated with water at 60 - 80
°C for half an hour. Water was then separated, and the oil was
dried over anhydrous Na2SO4. The treated chicken fat oil was
subjected to further analysis.
Esterification of oleic acid with methanol was carried out as
reported by us earlier24 at 190 °C in a batch reactor using an oleic
acid to methanol molar ratio of 1:5 and a heterogeneous
catalyst24shydrophobic, double metal cyanide Fe-Zn (3 wt %).
At the end of the reaction, the catalyst was separated by filtration
and methanol was removed from the reaction mixture using a
rotavapor. The sample was then analyzed.
3. Results and Discussion
Application of NMR (both 13C and 1H) had been reported
for the fatty acid profiling of biodiesel (or a vegetable oil or
fat),20 the quantification of fatty compounds in oxidized
biodiesel,21,22 and the analysis of biodiesel blended diesel.23 We
(20) Knothe, G.; Kenar, J. A. Eur. J. Lipid Sci. Technol. 2004, 106,
88–96.
(21) Knothe, G. Eur. J. Lipid Sci. Technol. 2006, 108, 493–500.
(22) Hämäläinen, T. I.; Kamal-Eldin, A. Analysis of Lipid Oxidation
Products by NMR Spectroscopy. In Analysis of Lipid Oxidation; Kamal-
Eldin, A., Pokorny’, J., Eds.; AOCS Press: Champaign, IL, 2005; pp 70-
126.
Estimation of FFA Content by 1H NMR Energy & Fuels, Vol. 23, 2009 2275

Table 1. Assignment of 1H NMR Peaks of FFA, Vegetable Oil, and Biodiesel

compound/chemical shift, δ (ppm)
proton(s) functional group oleic acid soybean oil biodiesel
CH3-C terminal methyl group 0.88 0.80-1.01 0.8-1.0
-(CH2)n- backbone CH2 1.14-1.43 1.20-1.41 1.22-1.42
-CH2CH2COOH β-methylene proton 1.54-1.69 1.53-1.70 1.55-1.69
)CH-CH2- R-methylene group to one double bond 1.92-2.11 1.94-2.11 1.93-2.10
-CH2COOH R-methylene group to acid 2.34 - -
-CH2COOR R-methylene group to ester - 2.31 2.31
)CH-CH2-CH) R-methylene group to two double bonds - 2.76 2.77
-COOCH3 methyl group of ester - - 3.67
-CH2OCOR methylene group (C1and C3) of glyceride - 4.09-4.34 -
-CHOCOR methine proton at C2 of glyceride - 5.25 -
-CH)CH- olefinic protons 5.3-5.4 5.28-5.43 5.27-5.41

Figure 2. 1H NMR spectrum in R-CH2 region: (a) soybean oil and oleic acid (FFA) and (b) mixture of oleic acid and its methyl ester.

Figure 3. Correlation/calibration plots of FFA content in (a) soybean-oleic acid (standard deviation ) 0.12 (titration) and 0.44 (1H NMR)) and (b)
standard biodiesel-oleic acid (standard deviation ) 0.29 (titration) and 0.18 (1H NMR)).

Table 2. FFA Content (wt %) in the Synthetic Standard

report here, for the first time, its application for the quantitative
Mixture of Oleic Acid-Soybean Oil and Oleic Acid-Biodiesel
determination of FFA in vegetable oils, animal fats, and Determined by 1H NMR and Titration Methods
biodiesel. Scheme 1 describes the typical chemical structures,
oleic acid-soybean oil oleic acid-biodiesel
and Figure 1 depicts the 1H NMR spectra of vegetable oil, FFA,
and fatty acid methyl ester (biodiesel). Assignments of the 1H standard standard
(actual (actual
NMR peaks are listed in Table 1. Quantification of the FFA concentration) 1H NMR titration concentration) 1H NMR titration
content in vegetable oil and biodiesel by 1H NMR is based on
0.5 0.4 0.6 0.5 0.5 0.6
the fact that R-CH2 peaks of fatty acids appear at δ values higher 0.7 0.7 0.8 0.7 0.6 0.8
than those of the methyl (biodiesel) or glyceryl (vegetable oil) 1.0 0.9 1.0 1.0 0.9 1.2
esters. The difference in chemical shift (between the acid and 5.0 5.9 4.9 2.0 2.2 2.8
10.0 10.5 9.9 5.2 4.9 5.5
ester) is due to the greater deshielding effect of the carboxylic 20.0 19.8 19.9 10.0 9.7 10.6
group compared to the ester group. Due to this shift, one of the 30.0 29.3 29.9 15.2 15.2 15.9
peaks of the triplet of FFA (at 2.38 ppm in Figure 2a) shifts 40.0 39.5 40.2 20.0 19.5 20.5
out of the R-CH2 region of the ester, and the other two peaks (2.34 and 2.30 ppm, respectively) are merged with those due
to the ester at 2.35 and 2.31 ppm, respectively. In other words,
(23) (a) Diel, B.; Randel, G. Lipid Technol. 2007, 19, 258–260. (b) a sample containing FFA and ester (vegetable oil or biodiesel)
Monteiro, M. R.; Ambrozin, A. R. P.; Lião, L. M.; Ferreira, A. G. Fuel, shows a quartetlike spectral pattern in the R-CH2 region of the
2009, 88, 691–696. 1H NMR spectrum (Figure 2b) with the intensity of the peaks
(24) Sreeprasanth, P. S.; Srivastava, R.; Srinivas, D.; Ratnasamy, P. Appl.
Catal. A 2006, 314, 148–159. depending on the content of FFA in esters. The unmerged peak
2276 Energy & Fuels, Vol. 23, 2009
Figure 4. 1H NMR spectra in the R-CH2 region: reaction of oleic acid with methanol as a function of time.
Table 3. Evaluation of FFA Content by Titration and 1H NMR:
Esterification of Oleic Acid with Methanola
oleic acid conversion (%)
time (h) titration 1H
0.5 4.3
1 44.3
2 89.5
4 96.2
6 96.0
8 96.1
a Reaction conditions: oleic acid ) 10 g; oleic acid:methanol molar
ratio ) 1:5; reaction temperature ) 190 oC.
Table 4. FFA Content of Karanja Oil, Chicken Fat, and
Biodiesel Derived from Them
FFA content (wt %)
oil/fat/biodiesel titration
karanja oil 5.3
biodiesel from karanja oil 0.79
chicken fat 11.7
biodiesel from chicken fat 0.53
water-treated chicken fat 10.4
biodiesel from water-treated chicken fat 0.52
of the FFA triplet can be used to determine the FFA content in
vegetable oil, animal fat, and biodiesel (Figure 2b). The area
(AFFA) of the unmerged peak of the FFA triplet (appearing
around 2.38 ppm, out of the ester triplet) can be determined by
integration of the spectral region 2.37-2.41 ppm. The triplet
appears with an intensity ratio of 1:2:1. The total area corre-
sponding to the R-CH2 groups of the FFA, will thus, be four
times the area of the single unmerged FFA peak around 2.38
ppm. The total area corresponding to R-CH2 of both FFA and
ester can be determined by integrating the spectral region
2.20-2.41 ppm. The concentration of FFA (wt %) in oil or
biodiesel is thus
% of FFA )
4 × area of unmerged peak of R-CH2 of FFA
× 100
total area of R-CH2 of both FFA and ester
Satyarthi et al.
Alternatively, the peaks due to the ester and acid can be
deconvoluted, and the FFA content can be calculated using the
following equation
NMR
4.6 area of triplet of R-CH2 of FFA
% of FFA ) × 100
46.2 total area of R-CH2 of both FFA and ester
88.4
94.1
94.0 For the validation of this method, calibration curves for the
94.2 standard solutions of oleic acid (as the representative FFA) and
soybean oil (as a vegetable oil) were constructed based on both
the 1H NMR and titrimetric methods (Figure 3a; Table 2). A
very good correlation is seen.
Biodiesel can also contain FFA due the hydrolysis of the fatty
acid methyl esters by moisture or incomplete conversion of FFA-
containing, nonedible oils. We prepared standard mixtures
1H NMR containing 0.5-20% of oleic acid in commercial biodiesel, and
4.5 the FFA content was determined by both titration and 1H NMR
0 methods (Figure 3b). Again there is a good correlation between
10.4
0
the 1H NMR and titrimetic methods. This method of quantifica-
9.6 tion is valid irrespective of the type of FFA present in oils and
0 biodiesel. All the FFAs show R-CH2 triplet peaks in the 1H
NMR spectrum at 2.30, 2.34, and 2.38 ppm. It may, however,
be noted that 1H NMR has an intrinsic error of approximately
1%.
During the esterification of oleic acid with methanol, the
decrease of the oleic acid content in the reaction mixture was
followed, as a function of time, by both the 1H NMR and
titrimetric methods (Figure 4). At the start of the reaction, 1H
NMR spectrum in the R-CH2 region (2.3-2.4 ppm) showed a
triplet pattern corresponding to oleic acid. As the reaction
progressed, a quartetlike pattern developed. The intensity of the
triplet signals due to oleic acid decreased and that of the methyl
ester increased with the reaction time (Figure 4). Finally, at the
end of 8 h, the peaks corresponding to oleic acid had almost
disappeared, and only a triplet corresponding to that of the
methyl ester (biodiesel) was detected. The FFA content in the
reaction mixture at different time intervals determined by 1H
Estimation of FFA Content by 1H NMR
NMR spectroscopy correlated well with that determined by the
titrimetric method (Table 3).
Nonedible oils and fats often contain significant amounts of
phospholipids and other acidic impurities along with the FFAs.
While the titrimetric method determines the total acid value in
oils and fats, 1H NMR detects the FFA content exclusively. The
content of FFA in karanja oil (Table 4) estimated by titration
(5.3%) is indeed higher than that determined by the 1H NMR
(4.5%) technique. The FFA content in chicken fat oil (by both
the methods) had decreased after the water treatment (Table
4). The values estimated by the titrimetric method are still higher
than those determined by 1H NMR. After transesterification of
those oils into biodiesel, the titration method still shows the
presence of some acidity while no FFA is detected by 1H NMR.
These data, therefore, confirm that 1H NMR spectroscopy
determines the FFA in nonedible oils, fats, and biodiesel more
accurately than the titrimetric method. Control experiments
revealed that 1H NMR can detect FFA as low as 0.5 wt %.
Energy & Fuels, Vol. 23, 2009 2277
FFA content is an important quality parameter which has to be
determined before oil can be used for any purpose. FFA
determination is not only important for the biodiesel industry,
but a quick and reliable method is always desirable in the food
industry also.
4. Conclusions
A simple and fast method for the estimation of free fatty acids
in vegetable oil, animal fat, and biodiesel using proton NMR
spectroscopy is reported for the first time. This method is
nondestructive and requires only a small amount of the sample.
It can also be used as an in situ tool to monitor the esterification
reaction of FFA to biodiesel.
Acknowledgment. J.K.S. acknowledges the Council of Scientific
and Industrial Research (CSIR), New Delhi, for the award of
research fellowship.
EF801011V