Article

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H Nuclear Magnetic Resonance (NMR) Determination of the Iodine
Value in Biodiesel Produced from Algal and Vegetable Oils
Ravindra Kumar,* Veena Bansal, M. B. Patel, and A. S. Sarpal
Research and Development (R&D) Center, Indian Oil Corporation (IOC), Limited, Sector 13, Faridabad 121007, Haryana, India

ABSTRACT: A method for the determination of the iodine value in biodiesel produced from algal and vegetable oils has been
developed by nuclear magnetic resonance (NMR) spectroscopy. The determination of the iodine value is based on the average
molecular weight of the fatty acid methyl esters and the number of double bond, which are calculated from 1H NMR spectra. The
total methyl ester content (% ME) was estimated using the integral value of the OCH3 group peak at 3.67 ppm and the α CH2 at
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2.2 ppm. The results obtained from the 1H NMR method show good correlation (r2 = 0.9974) with the traditional American Oil
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Chemists’ Society (AOCS) method. The 1H NMR method does not depend upon the nature of biodiesel feedstock. It is
applicable to all methyl biodiesel samples obtained from different raw materials, including algal oils. The results show that a
higher iodine value indicates less oxidation stability, but there is not a linear correlation between the iodine value and oxidation
stability of biodiesel.

1. INTRODUCTION AOCS method (titration-based) for the determination of IV is

Biodiesel is defined as monoalkyl esters of vegetable oils or
animal fats. It is suitable as a replacement or complement for
conventional diesel fuel (petrodiesel) in a compression-ignition
(diesel) engine.1 The most common forms of biodiesel are
methyl esters of vegetable oils. The use of methyl esters offers
the advantage that the high viscosity of vegetable oils is reduced
by an order of magnitude to a value close to that of petrodiesel.
The produced biodiesel must meet a series of physical and
chemical specification parameters. The iodine value (IV) is one
of them, which indicates the degree of unsaturation of the fuel,
expressing its trend to oxidize. In industrial applications,
oxidation stability is an issue not only for biodiesel but also for
lubricants.2 It is well-known that the rate of autoxidation of
unsaturated fatty compounds depends upon the number and
position of the double bonds.3 The algal oil contains a high
amount of polyunsaturated fatty acids (PUFAs) compared to
seed oils; therefore, the oxidation stability of algal-derived
biodiesel is less.
In European Standard EN-14214, the IV has been included,
usually being set to a maximum of 120. The limitation of the IV
in biodiesel is currently one of the most discussed topics within
the different worldwide biodiesel specifications. The higher the
IV, the more extensive the unsaturation degree, meaning a
higher number of double bonds in the biodiesel. The iodine
number is a measure for the number of double bonds in a
sample. It specifies the amount of iodine in grams that is
consumed by 100 g of the biodiesel sample under the given
conditions. The determination of the iodine number by redox
titration with a solution of sodium thiosulfate is described by
the American Oil Chemists’ Society (AOCS). Some other
methods are also reported in the literature4−8 for estimation of
IV in vegetable oils and animal fats.
Nuclear magnetic resonance (NMR) has become one of the
most promising methods to determine IV of vegetable oils and
animal fats.9−11 There is no single method that has been
reported for the estimation of IV that can apply for all types of
biodiesel and especially for algal biodiesel. The traditional
time-consuming and not particularly effective for the analysis of
a large number of samples. To overcome the above problems,
an attempt has been made to develop a quick method for the
estimation of IV for methylic biodiesel, which is also applicable
for algal biodiesel. The developed method was applied to
biodiesel samples obtained from several vegetable oil seeds and
algal oil to test its applicability.
2. EXPERIMENTAL SECTION
2.1. Samples. Biodiesel samples were obtained from extracted
vegetable oil through the transesterification reaction using methanol as
the alcohol source. Algae samples (Scenedesmus, Chlorella, and
Spirulina) were provided by the Department of Agricultural and
Biological Engineering, University of Illinois at Urbana−Champaign
(UIUC), Urbana, IL, to the R&D Center of IOC, Ltd.
2.2. Extraction of Oil and Biodiesel Synthesis. Oils were
extracted from the different seeds by Soxhlet extraction using
petroleum ether (bp 60−80 °C) at 80 °C for 20 h.12 The oil
extracted seeds were again subjected to Soxhlet extraction under
identical conditions for an extra 5 h to ensure complete oil extraction.
The sample recovered after the extraction was monitored by thin-layer
chromatography (TLC) using a mixed solvent system of petroleum
ether (85 mL), diethyl ether (13.5 mL), and glacial acetic acid (1.5
mL).
Algal oil was extracted from the solid algal sample by the Bligh and
Dyer method.13 The extracted oils from seeds and algae were
converted into biodiesel through the transesterification reaction using
methanol as the alcohol source.14
2.3. NMR Spectroscopic Studies. All of the 1H NMR spectra
were recorded on a Bruker ACP-300 MHz NMR spectrometer. The
concentration of the sample used was 5−10% (w/w) in CDCl3 for 1H
NMR, containing tetramethylsilane (TMS) as an internal reference per
the following experimental conditions: spectral width, 3600 Hz (0.0−
12.0 ppm); spectral size, 16 K; digital resolution, 0.2197 ppm/point;
90° pulse, 18 μs; relaxation delay, 10 s; and number of scans, 64.
Received: June 14, 2012
Revised: September 22, 2012
Published: October 2, 2012

© 2012 American Chemical Society 7005 dx.doi.org/10.1021/ef300991n | Energy Fuels 2012, 26, 7005−7008
Energy & Fuels
3. RESULTS AND DISCUSSION
The 1H NMR spectrum of Jatropha methyl biodiesel is
presented in Figure 1, and this spectrum shows all of the
Figure 1. 1H NMR spectrum of Jatropha biodiesel.
characteristic signals of methyl biodiesel. A direct measure of
the unsaturation degree of the samples is obtained from the 1H
NMR spectra by integrating the olefinic hydrogen signals
between δ 5.40 and 5.26 ppm (L).
The OCH3 protons of the methoxy groups are observed at
the chemical shift of 3.67 ppm (R). Signals in the range of δ
0.5−2.8 ppm are due to the other methylenic hydrogens of the
fatty acid ester chain (M).
The calculation of the methyl ester yield (% ME) was carried
out, dividing the integral value of the methoxy OCH3 group at
3.67 ppm after the subtraction of one proton integral value of
the 1H NMR spectra by the integral area of the α CH2 at 2.3
ppm and multiplying the result by 100.
3.1. Total Percentage of Methyl Esters (% ME).
% ME = (R − r )/E × 100
where r is the area per hydrogen and E is the integral area of α
CH2 at 2.3 ppm.
3.2. Estimation of the IV. From the 1H NMR spectra, the
integral values at the chemical shift ranges designed as M, R,
and L (see Figure 1) are obtained. Therefore, it is possible to
calculate the average molecular weight (MW) of the fatty acid
methyl esters that constitute the biodiesel samples, as
demonstrated as follows: (1) First, an average molecular
formula that represents the methyl esters present in the
biodiesel samples will be used to calculate the average MW
according to Reda et al., except for the methyl ester identified
and estimated from the 1H NMR spectrum in the present
study, against the ethyl ester in Reda et al.10
MW = CH3(OCO)−(CH 2)q −(CHCH)x −CH3 (1)
The present study used the number of double bonds against the
area per hydrogen. The total number of double bonds (DBs) is
calculated by the olefinic hydrogen number (O).
number of double bonds (DBs) = O/2 (2)
(2) Calculation of the IV: The IV of the biodiesel samples were
calculated by means of eq 3.
IV = 253.808/DBs × MW × 100 (3)
As examples, the method was applied to a Jatropha biodiesel,
who’s 1H NMR spectrum is shown in Figure 1, and Scenedesmus
(algae), shown in Figure 2.
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Article
Figure 2. 1H NMR spectrum of algal methyl biodiesel (Scenedesmus).
3.3. Jatropha Biodiesel. (1) Calculation of M: For the
calculation of M, the values of the integrals within the chemical
shift range of 2.8 and 0.5 ppm of Figure 1 are summed.
Therefore, the value of M for the biodiesel Jatropha sample
studied is M = 2.945 + 10.279 + 15.081 + 12.486 + 99.162 +
16.438. M = 156.391. (2) From Figure 1, it is also possible to
obtain the value of L directly from the integrated area at δ 5.2
ppm: L = 10.555. (3) The calculation of r is then
r = 15/3, r=5
(4) The calculation of the total number of protons (T) results
in
T = (L + M + R )/r
T = (156.391 + 10.555 + 15.0)/5.0 = 36.39
(5) The value of O for the sample studied is calculated as
equation O = L/r.
O = 10.555/5.0 = 2.111
number of DBs=2.111/2 = 1.06
(6) The average MW is calculated by equation MW = 32 +
7(T) + 6(O).
MW = 32 + 7(36.39) + 6(2.111)
MW = 299.396
(7) Finally, it is possible to calculate the IV applying eq 3.
IV = 253.808/299.396 × 1.06 × 100
IV = 89.9
The IV obtained by the traditional AOCS method for the
Jatropha biodiesel is 91.0, which show good agreement with the
developed method.
3.4. Algal Biodiesel (Scenedesmus). The 1H NMR
method was applied to calculate the IV on three different
algal biodiesel samples (Scenedesmus, Chlorella, and Spirulina).
The 1H NMR spectrum of algal methyl biodiesel (Scenedesmus)
is shown in Figure 2. (1) Calculation of M: For the calculation
of M, the values of the integrals within the chemical shift range
of 3.5 and 0.5 ppm of Figure 2 are summed. Therefore, the
value of M for the algal biodiesel (Scenedesmus) sample studied
is M = 6.315 + 177.37, M = 183.685. (2) From Figure 2, it is
also possible to obtain the value of L directly from the
integrated area at δ 5.2−5.8 ppm: L = 15.802. (3) The
calculation of r is then
r = 15/3, r=5
dx.doi.org/10.1021/ef300991n | Energy Fuels 2012, 26, 7005−7008
Energy & Fuels Article

(4) The calculation of the total number of protons (T) results

in
T = (L + M + R )/r
T = (183.685 + 15.802 + 15.0)/5.0 = 42.897
(5) The value of O for the sample studied is calculated as
equation O = L/r.
O = 15.802/5.0 = 3.16
number of DBs=3.16/2 = 1.58
(6) The average MW is calculated by equation MW = 32 +
7(T) + 6(O).
MW = 32 + 7(42.897) + 6(3.16)
MW = 351.24
Figure 3. Correlation between the traditional AOCS method and the
(7) Finally, it is possible to calculate the IV applying eq 3. 1
H NMR method for the determination of IV.
IV = 253.808/351.24 × 1.58 × 100
3.5. Oxidation Stability versus IV of Biodiesel. To
IV = 114.2
compare the chemical stability of different biodiesel fuels, it is
The IV obtained by the traditional AOCS method for the algal desirable to have a measurement for the stability of the fuel
biodiesel (Scenedesmus) is 115.4, which shows good agreement against oxidation. Currently, the most common method for
with the value determined from 1H NMR. doing this and the method specified in many of the biodiesel
Other results presented in Table 1 indicate that the 1H NMR fuel specifications is called the iodine number or IV. The IV is
method employed for the determination of the IV is applicable not determined by measuring the stability of the fuel, rather it is
determined by measuring the number of double bonds in the
Table 1. Comparison of IV by the Traditional AOCS mixture of fatty acid chains in the fuel by introducing iodine
Method and the 1H NMR Method into 100 g of the sample under test and measuring how many
grams of that iodine are absorbed. Iodine absorption occurs at
IV by the IV by % ME by
biodiesel samples traditional method 1
H NMR 1
H NMR double bond positions; thus, a higher IV indicates a higher
Jatropha 1 91.0 89.9 97.3
quantity of double bonds in the sample. The IV does not
soybean 1 118.6 120 98.2
necessarily make the best measurement for stability because it
soybean 2 125.0 132 98.1
does not take into account the positions of the double bonds
sunflower 132.0 137.4 97.6
available for oxidation. Other measurements of stability are
biorema (round-robin- 114.0 111.6 97.4
available, such as “oil stability index (OSI)”, “allylic position
tested sample) equivalent (APE)”, and “bis-allylic position equivalent
coconut 10.0 11.4 98.8 (BAPE)”, which take into account both the number and
Jatropha 2 91.4 89.1 97.1 position of double bonds in the fatty acid chains.15 These
palm 49.0 50.6 97.6 measurements appear more suitable for assessing oxidative
Scenedesmus 115.4 114.2 96.8 stability of biodiesel. Because oxidative stability depends more
Chlorella 106.4 109 97.1 upon the nature of double bonds in a molecule and less upon
Spirulina 121.0 125 97.4 the MW, then the IV, which depends upon MW and the
methyl ester of linolenic 260.4 260.4 100.0 number but not nature of double bonds, cannot be a predictor
acid of oxidative stability.
The determination of the oxidation stability for the biodiesels
used in this study is performed following EN 14112,
for biodiesel samples obtained from different raw materials, determination of oxidation stability, with a 743 Rancimat
including algal biodiesel, covering the IV range from 10 to 260. (Metrohm, Inc.) instrument.16 A total of 3 g of biodiesel in a
The yield of the reaction for each biodiesel sample obtained test tube is heated to 110 °C and connected to an air bubbler
from the 1H NMR spectra is also shown in Table 1 (% ME). until the measurement of conductivity versus time in a water
The results shown in Table 1 indicate that the iodine numbers vessel attached to the sample test tube reaches the inflection
obtained by the traditional AOCS method correlate well with point. The result is performed in duplicate and expressed as an
the iodine numbers obtained by 1H NMR. The yields of the average in units of time (h). The higher the value, the more
transesterification reaction were considered satisfactory because stable the biodiesel is toward oxidation by air.
no values lower than 96% were obtained, demonstrating that In this study, IV estimated by 1H NMR is correlated to the
the process proposed for the preparation of the biodiesel oxidation stability of some biodiesel. The results shown in
samples was efficient. Figure 3 shows a good linear correlation Table 2 indicate that coconut biodiesel has a maximum
(r2 = 0.9974) between the IVs obtained from the traditional oxidation stability (35.1 h), in which IV is minimum (11.4), and
AOCS method and the 1H NMR method. sunflower shows a minimum oxidation stability (0.8 h), in
This fact indicates that the 1H NMR method is a reliable which IV is maximum (137.4). In the case of Jatropha and
alternative for the determination of the IV for vegetable and soybean, there is a large difference between their IVs but their
algal oil biodiesel. oxidation stability is close to each other. Thus, a higher IV
7007 dx.doi.org/10.1021/ef300991n | Energy Fuels 2012, 26, 7005−7008
Energy & Fuels Article

Table 2. IV versus Oxidation Stability of Methyl Biodiesel (2) Weller, D. E.; Perez, J. M. A study of the effect of chemical
structure on friction and wear: Part 2Vegetable oils and esters. Lubr.
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2 soybean 1 120 2.2 1998; p 19.
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Cd 1d-92, iodine value of fats and oils, cyclohexane−acetic acid
method. In Official Methods and Recommended Practices of the American
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Oil Chemists’ Society, 5th ed.; Firestone, D., Ed.; AOCS: Champaign,
IL, 1998.
indicates less oxidation stability (in Figure 4), but there is no (5) American Oil Chemists’ Society (AOCS). AOCS Recommended
linear correlation between IV and oxidation stability. Practice Cd 1b-87, iodine value of fats and oils, cyclohexane method.
In Official Methods and Recommended Practices of the American Oil
Chemists’ Society, 5th ed.; Firestone, D., Ed.; AOCS: Champaign, IL,
1998.
(6) American Oil Chemists’ Society (AOCS). AOCS Official Method
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Firestone, D., Ed.; AOCS: Champaign, IL, 1998.
(7) American Oil Chemists’ Society (AOCS). AOCS Official Method
Tg 1a-64, iodine value of fatty acids. In Official Methods and
Recommended Practices of the American Oil Chemists’ Society, 5th ed.;
Firestone, D., Ed.; AOCS: Champaign, IL, 1998.
(8) American Oil Chemists’ Society (AOCS). AOCS Official Method
Cd 3-25, saponification value. In Official Methods and Recommended
Practices of the American Oil Chemists’ Society, 5th ed.; Firestone, D.,
Ed.; AOCS: Champaign, IL, 1998.
(9) Sarpal, A. S.; Kapur, G. S.; Mukherjee, S.; Jayaparkash, K. C.; Jain,
Figure 4. Relation between IV and oxidation stability of biodiesel. S. K. Determination of iodine value of lubricating oils by nuclear
magnetic resonance (NMR) spectroscopy. Lubr. Eng. 1994, 51, 209−
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(10) Reda, S. Y.; Costa, B.; Freitas, R. J. S. Determination of iodine
value in ethylic biodiesel samples by 1H NMR. Appl. Magn. Reson.
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MW of the fatty acid methyl esters and the yield of methyl by 1H NMR spectroscopy. Eur. J. Lipid Sci. Technol. 2004, 106, 88−96.
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■
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AUTHOR INFORMATION
(16) BSI. EN 14112:2003, Fat and Oil Derivatives. Fatty Acid Methyl
Corresponding Author Esters (FAME). Determination of Oxidation Stability (Accelerated
*E-mail: kumarr88@indianoil.in. Oxidation Test); BSI: London, U.K., 2003.
Notes
The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS
The authors are thankful to the management of the R&D
Center, IOC, Ltd., Faridabad, India, for allowing the present
work to be published. The authors acknowledge the Depart-
ment of Agricultural and Biological Engineering, UIUC,
Urbana, IL, for providing the algae samples and Dr. Dheer
Singh for synthesis of biodiesel from algal and vegetable oils.

■ REFERENCES
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vegetable oils and their derivatives as alternative diesel fuels. In Fuels
and Chemicals from Biomass; American Chemical Society (ACS):
Washington, D.C., 1997; ACS Symposium Series, Vol. 666, Chapter
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7008 dx.doi.org/10.1021/ef300991n | Energy Fuels 2012, 26, 7005−7008