Thermal Formation of trans Fatty Acids in Romanian Vegetable

Oils Monitored by GC-MS and FT-IR Techniques

MIHAELA MIHALACHE1, AURELIA BRATU1, ANAMARIA HANGANU2*, NICOLETA-AURELIA CHIRA1, MARIA MAGANU2,
MARIA-CRISTINA TODASC1, SORIN ROSCA1
1
University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Costin D. Nenitzescu Organic
Chemistry Department, 1-7 Polizu Str., 011061, Bucharest, Romania
2
Romanian Academy, Organic Chemistry Center Costin D. Nenitzescu 202B Splaiul Independentei, 060023, Bucharest, Romania

Four different types of Romanian vegetable oils were heated in order to elucidate trans fatty acids accumulation
during thermal processing of oils in frequent domestic activities. The refined vegetable oils selected for this
study were sunflower, corn, soybean and linseed, heated at three different temperatures 180, 220 and 250oC
for 33 h. The amount of trans fatty acids formed during heating processes was determined using infrared
spectroscopy (IR), based on trans specific bonds absorption at 966 cm-1 (while the absorption of the cis
bonds appears at 724 cm-1). Based on the measurement of the absorption band at 966 cm-1, a calibration
curve was obtained. The results were compared with those obtained by gas chromatography-mass
spectrometry technique (GC-MS) used as reference method to quantify the trans fats. The results obtained
demonstrated that trans fatty acids formation in vegetable oils during heating, is closely related to process
temperature and time and also that trans fatty acids can only be formed under severe conditions.
Keywords: trans fatty acids, heated oils, Romanian vegetable oils, FT-IR, GC-MS

Trans fatty acids (TFAs) are unsaturated fatty acids that

contain one or more isolated (non-conjugated) carboncarbon double bonds in a trans configuration [1,2].
Naturally all unsaturated fatty acids in vegetable oils are
in cis configuration, but during partial hydrogenation or as a
result of manufacturing processes (thermal refining,
deodorization and bleaching) some cis-fatty acids may be
isomerized to the trans configuration. Other process that
can also lead to the formation of trans fatty acids are
thermal processing of oils in frequent domestic activities
(food-frying operations) [1-4].
Recent clinical and experimental studies reveal that
there is a close correlation between high level of TFAs
intake from the daily diet and the risk of cardiovascular
disease, by changing the plasma lipoprotein profile [1-4].
TFAs produce an increase in low-density lipoproteins (LDL),
so called bad cholesterol and a decrease of high-density
lipoproteins (HDL), so called good cholesterol [3,5].
It is suggested that fats for human daily consumption to
contain less than 2% (w/w) of the sum of all fatty acids
(total fat) as trans [5]. Today it is widely recommended to
break the trans fats group out of the total fat listing and the
amount of trans fat to be added to the amount of saturated
fat [5,6].
Gas chromatography (GC) and infrared spectroscopy
(IR) are the official methods used for the determination of
trans fatty acids in edible oils and fats [5,7].
American Oil Chemists Society developed using GC
method a total of 21 fatty acids: 15 cis fatty acids, 1 trans
oleic acid, 3 trans linoleic acid and 2 trans linolenic acid
[8].
Fourier transform (FT) infrared (IR) spectroscopy is a
simple and rapid technique for determination of trans fatty
acids with isolated trans-double bonds, applied directly on
the oil sample without any pretreatment, in comparison
with gas chromatography-mass spectrometry (GC-MS)
method that requires sample derivatisation of fatty acids
to fatty acid methyl esters (FAMEs) [3].

In FT-IR spectroscopy isolated trans fatty acids are

quantified based on the measurement of trans peak area
in the specific region from 990-945 cm-1 representing the
C-H out of plane deformation absorption (in trans
configuration). Using GC method, lower trans fatty acids
levels can be measured and also offers both identification
and quantification of individual trans fatty acids [1, 9, 10].
The purpose of this research is to analyze the
accumulation of trans fatty acids in four types of Romanian
vegetable oils during thermal processing. The four different
types of oils included sunflower, corn, soybean and linseed
oils, heated continuously at three different temperatures
180, 220 and 250oC for 33 h without any replenishment.
Experimental part
The commercially available vegetable oils used in the
thermal processes (sunflower, corn, soybean and linseed
oils) were purchased from different local Romanian
markets and stored at 4oC until use.
Heated oil samples were produced by heating the oils
without frying any food at three different temperatures 180,
220, 250oC for 33 h without any replenishment with fresh
oil.
Vegetable oils were heated in a commercially available
electrical deep fat fryer. The temperature of the oil was
monitored by a thermo recorder. Every day 500 mL of oil
was heated up for 1 h to reach the heating temperature
(180, 220 or 250oC) and then other 6 h continuously (until
33 h). Oil samples were collected at every 3 h. In total, 11
samples were collected for each oil at one temperature.
The oil samples collected from the thermal experiments
were derivatised in order to obtain fatty acids methyl esters.
Fatty acid methyl esters (FAMEs) were prepared by
transesterification of oils with methanol, using BF3-MeOH
complex as catalyst, according to the standard method
[11].
The standard mixture of 37 fatty acids methyl esters
(Supelco 37 Component FAME Mix) used for the gaschromatographic analyses was purchased from Supelco.

* email: ana.hanganu@cco.ro, tel : 0213167900

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REV. CHIM. (Bucharest) 63 No. 10 2012

Another standard mixture of fatty acids methyl esters

certified for its fatty acid profile used for the gaschromatographic analyses was BCR-162R, soya-maize oil
blend.
The gas-chromatograms of the fatty acid methyl esters
mixtures were recorded on an Agilent Technologies model
7890A instrument with mass detection Agilent
Technologies model 5975 C VL MSD with Triple Axis
Detector and auto-sampler Agilent. Separation into
components was made on a capillary column especially
designed for the FAMEs analysis (Supelco SPTM 2560,
characteristics: 100 m length, 0.25 mm inner diameter,
0.2 m film thickness). The injection solutions were
prepared in CH2Cl2 of HPLC purity grade.
Fatty acids identification was made by comparing each
peak retention time with those of two standard mixtures
of fatty acid methyl esters (SupelcoTM 37 Component FAME
Mix and BCR-162R). In the standard mixtures the exact
concentration of each component is known. Both standard
mixtures and each of the fatty acid methyl esters of the
analyzed oils were chromatographically separated under
the same conditions, using the same temperature program
(oven initial temperature 140oC to final temperature 240
o
C, heating rate 4oC/min.), injection volume 1L, split rate
100:1, carrier gas He according to the Supelco
specifications. The calibration of the signals was made by
taking into account the concentration of each component
of the standard mixture, correlated with the detector
response.
FT-IR spectra were recorded on a Bruker Vertex 70
spectrometer, with horizontal device for attenuated

reflectance and diamond crystal, on a spectral window

ranging from 4000 to 400 cm-1, at a spectral resolution of
2cm -1. Spectra were recorded without any sample
preparation and were processed with OPUS 5.5 software
(Bruker). A series of trans calibration standard mixtures
(used as primary standard) were prepared weighing
accurately to the nearest 0.0001 g (0.5-x) g of methyl oleate
and x g of methyl elaidate into a vial, in order to prepare a
series of trans fat standards of known concentrations. These
standard solutions were (1:1 w/w) diluted with ciclohexane [7]. The FT-IR spectra of the trans fat standard
solutions were recorded.
Results and discussions
IR spectroscopy
The infrared spectroscopy and gas-chromatography
have been widely used to determine the trans content in
edible oils and fats. The simplest method is the direct
analysis of oils by Fourier transform infrared (FT-IR)
spectroscopy.
Typical FT-IR spectra of oils are presented in figure 1.
By using specific trans absorption band at 966 cm-1,
several integration methods were performed using OPUS
5.5 software for a series of trans calibration standard
mixtures between fixed limits 990-945 cm -1. Several
calibration equations were obtained depending on the
specific area integration procedure [12], as described
below. A series of linear calibration equations were obtained
with variable correlation coefficients, as is shown in
table 1.

Fig.1 FT-IR spectra of oils

containing trans fatty acids
obtained in the spectral
region between 1000 and
930 cm-1, centered at 966 cm-1

Table 1
CORRELATIONS COEFFICIENTS OBTAINED FOR
DIFFERENT INTEGRATION METHODS

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Fig.2. Calibration curve and correlation

coefficient obtained with the bases of peak
areas between 990-945 cm-1

Table 2
CALCULATED PERCENTS OF TOTAL TRANS
ISOMERS FOR THE CONTROLLED HEATED
OIL SAMPLES USING IR SPECTRA

It can be remarked from table 1 that procedure B of

integration in the IR spectrum has the best linear correlation
coefficient, therefore the calibration curve obtained was
used to determinate the trans concentration in heated oil
samples by linear regression.
The calibration curve obtained correlates the areas of
the absorbance peak with corresponding methyl elaidate
concentrations (fig. 2).
Exploiting the specific trans absorption band from the
IR spectra of heated oils by linear regression, the calibration
equation y=0.0975x + 0.014 was used to calculate the
percent of trans isomers from the samples, using the
appropriate software.
The results are reported in table 2.
As it can be observed from table 2 there is an increase
in total trans isomers in all thermal experiments as the
heating time increases. The highest amount of trans fatty
acids was accumulated in the first thermal experiment
(250 oC), followed by 220 oC experiment, while 180 oC
experiment has the smallest quantity of trans fats farmed.
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Gas-chromatography
Another method used for identification and
quantification of trans fatty acids was gas-chromatography,
technique that allows to identify individual fatty acids. Using
this method we could notice when trans isomers begin to
form in thermally processed samples.
During the heating processes carried out, the following
trans isomers are identified:
- 9-cis 18:1 (oleic acid) generates 9-trans 18:1 (elaidic
acid);
- 9-cis, 12-cis 18:2 (linoleic acid) generates 9-trans, 12trans 18:2 (linolelaidic acid), 9-cis, 12-trans 18:2 and 9trans 12-cis,18:2.
Table 3 provides an overview on the individual trans fatty
acids composition detected in heated oils samples at three
different temperatures studied.
Elaidic acid (9-t C18:1) appeared after 18 h of severe
thermal degradation of oils (250oC) increasing with the
heating time. Considerable amounts of trans isomers
derived from linoleic acid are also formed in all edible oils.

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REV. CHIM. (Bucharest) 63 No. 10 2012

Table 3
TRANS FATTY ACID COMPOSITION OF HEATED OILS AT 250OC

Fig.3. Plot of trans values obtained from GC and FT-IR for

sunflower oil and corn oil

As it can be noticed drastic temperatures conditions

produced trans fatty acids.
In order to verify the similarity of the information provided
from the two methods used for TFAs quantification we
realized a series of calibration curves with good linear
correlation coefficients. An example of this type of
calibration curves for sunflower oil and corn oil, are given
in figure 3.
Figure 3 reveals that FT-IR and GC techniques give
comparable results. By plotting the results obtained from
GC and FT-IR a satisfactory calibration was achieved. It is
very clear that FT-IR results are in agreement with GC
results for sunflower and corn oil in which the major fatty
acids are linoleic acid and oleic acid, responsible for the
trans fatty acids formed during heating. In case of soybean
and linseed oils responsible for trans fatty acids formation
is linolenic acid also.
REV. CHIM. (Bucharest) 63 No. 10 2012

According to literature specifications, during heating

processes, linolenic acid (9-cis, 12-cis, 15-cis C18:3)
generates trans isomers like [13]:
- 9-trans, 12-cis, 15-cis C18:3;
- 9-cis, 12-trans, 15-cis C18:3;
- 9-cis, 12-cis, 15-trans C18:3;
- 9-trans, 12-trans, 15-cis C18:3;
- 9-trans, 12-cis, 15-trans C18:3;
- 9-cis, 12-trans, 15-trans C18:3;
- 9-trans, 12-trans, 15-trans C18:3.
The standard mixtures previously presented used for
trans fatty acids identification did not allowed us to identify
trans isomers derived from linolenic acid during heating,
so it is very clear that the results obtained using FT-IR
method (total amount of TFAs) differ from the results
obtained using GC techniques. The difference between the
results of the two methods represents linolenic acid trans
isomers not identified by GC.

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Conclusions
Drastic temperature conditions applied to edible oils
induce changes in fatty acids composition generating trans
isomers. No trans fatty acids were formed in sunflower,
corn and linseed oils during heating at 180oC. Trans fatty
acids accumulation in heated oils is closely related to
process temperature and heating time.
In conclusion, using edible oils in frequent domestic
activities (food-frying operations) at lower temperatures,
major degradation processes do not occur.
Both modern physical methods used, give similar results,
but both have advantages and disadvantages:
- FT-IR is a simple and rapid method applied directly on
oil samples, but the results obtained are reported as total
trans fats;
- GC is a more laborious method (oil samples require
derivatisation), but provides information about individual
trans fatty acids.
Further research is necessary to study the formation of
trans fatty acids in vegetable oils during heating by
exploiting the specific absorption band in the FT-IR spectra.
In a previous paper it was studied the authentication of
vegetable oils by [1] H-NMR [14].
Acknowledgments: The work has been funded by the Sectoral
Operational Programme Human Resources Development 2007-2013
of the Romanian Ministry of Labour, Family and Social Protection
through the Financial Agreement POSDRU/88/1.5/S/60203.

References
1. SHERAZI, S., KANDHRO, A., ARAIN, S., Food Chem., 114, 2009,
p. 323.
2. TSUZUKI, W., MATSUOKA, A., USHIDA, K., Food Chem., 123, 2010,
p. 976.
32.CHO, I.K., KIM, S., KHURANA, H.K., LI, Q.X., JUN, S., Food Chem.,
125, 2011, p. 1121.
4. BANSAL, G., ZHOU, W., TAN, T.W., NEO, F.L., LO, H.L., Food Chem.,
116, 2009, p. 535.
5. PRIEGO-CAPOTE, F., RUIZ-JIMNEZ, J., LUQUE DE CASTRO, M.D.,
Food Chem., 100, 2007, p. 859.
6. NAZ, S., SIDDIQI, R., SHEIKH, H., SAYEED, S.A., Food Res. Int., 38,
2005, p. 127.
7. PRIEGO-CAPOTE, F., RUIZ-JIMNEZ, J., GARCA-OLMO, J., Luque
de Castro, M.D., Anal. Chim. Acta, 517, 2004, p. 13-20.
8. LIU, W.H., INBARAJ, B.S., CHEN, B.H., Food Chem., 104, 2007,
p. 1740.
9. MAHESAR, S.A., KANDHRO, A.A., CERRETANI, L., BENDINI, A.,
SHERAZI, S.T.H., BHANGER, M.I., Food Chem., 123, 2010, p. 1289.
10. KANDHRO, A.A., SHERAZI, S.T.H., MAHESAR, S.A., BHANGER, M.I.,
TALPUR, M.Y., RAUF, A., Food Chem., 109, 2008, p. 207.
11. LI, Y., WATKINS, B. A., Current Protocols in Food Analytical
Chemistry, Ed. John Wiley and Sons Inc., New York, 2001, p. D1.2.1D1.2.15
12.TODASC, M.C., CHIRA, N, DELEANU, C, ROSCA, S., UPB Sci. Bull.,
Series B, 69(4), 2007, p.3.
13. DIJKSTRA, A. J., HAMILTON, R. J., HAMM, W., Trans fatty acids,
Ed. Blackwell, 2008, p. 31
14. MIHALACHE, M., BRATU, A., HANGANU, A., CHIRA, N.-A., TODASCA,
M.C., ROSCA, S., Rev. Chim. (Bucharest), 63, no. 9, 2012, p. 877
Manuscript received: 2.05.2012

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