Thermal degradation and isomerisation kinetics of triolein studied by infrared spectrometry and GC-MS combined with chemometrics

Article abstract of DOI:10.1016/j.chemphyslip.2008.12.002

Triolein, a triglyceride containing oleic acid as the only acid moiety in the glyceride molecules has been isothermally treated at 280, 300, and 325 °C in glass vials under nitrogen atmosphere. The products formed during the thermal treatment at each temperature have been analysed both by infrared spectrometry and GC-MS. The GC-MS analysis was performed after derivatisation of the fatty acids into their methyl esters (FAMEs). Chemometric tools were used in determining the concentrations of the main products namely triolein and trieaidin in the thermally treated mixtures. The concentration profiles of the trielaidin formed during thermal treatment at the above three temperatures were used in determining activation energy for the cis-trans isomerisation of triolein. The combined analysis reveals that the thermal treatment induces not only cis-trans isomerisation but also fission and fusion in the molecules. Furthermore, migration of the double bond in oleic and elaidic acids forming cis and trans isomers of the 18:1 acid was also observed. The heat-induced isomerisation in triolein follows a zeroth order reaction with an activation energy 41 ± 5 kcal/mol.

Full text of DOI:10.1016/j.chemphyslip.2008.12.002

Chemistry and Physics of Lipids 158 (2009) 22–31
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
Thermal degradation and isomerisation kinetics of triolein studied by infrared
spectrometry and GC–MS combined with chemometrics
∗
1
1
Alfred A. Christy , Zhanfeng Xu , Peter de B. Harrington
Department of Chemistry, Faculty of Engineering and Science, University of Agder, Serviceboks 422, N-4604 Kristiansand, Norway
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 July 2008
Received in revised form 21 October 2008
Accepted 10 December 2008
Available online 24 December 2008
Triolein, a triglyceride containing oleic acid as the only acid moiety in the glyceride molecules has been
isothermally treated at 280, 300, and 325 C in glass vials under nitrogen atmosphere. The products formed
during the thermal treatment at each temperature have been analysed both by infrared spectrometry and
GC–MS. The GC–MS analysis was performed after derivatisation of the fatty acids into their methyl esters
◦
(FAMEs).
Chemometric tools were used in determining the concentrations of the main products namely triolein
Keywords:
Triolein
Isomerisation
Infrared spectroscopy
Kinetics
and trieaidin in the thermally treated mixtures. The concentration profiles of the trielaidin formed during
thermal treatment at the above three temperatures were used in determining activation energy for the
cis–trans isomerisation of triolein.
The combined analysis reveals that the thermal treatment induces not only cis–trans isomerisation but
also fission and fusion in the molecules. Furthermore, migration of the double bond in oleic and elaidic
acids forming cis and trans isomers of the 18:1 acid was also observed. The heat-induced isomerisation
in triolein follows a zeroth order reaction with an activation energy 41 ± 5 kcal/mol.
Chemometrics
©
2008 Elsevier Ireland Ltd. All rights reserved.
1
. Introduction
published literature only deals with the chemical analysis of the
mixture formed during heating of pure fatty acids (Lithfield et al.,
1963; Grompone and Moyna, 1986; Grandgirard et al., 1984; Dutten,
1974), or methyl esters (Chipault and Hawkins, 1960). No kinetic
parameters presented.
During heating and frying of oils fatty acid moieties in glyceride
molecules can undergo several changes and lead to several differ-
ent products. The mono- and poly-unsaturated fatty acid molecules
can isomerise to trans fatty acids or undergo oxidation and degrade
into other products or undergo intracyclisation or polymerization
To study the isomerisation kinetics, the information from the lit-
erature as mentioned in the preceding paragraph had to be taken in
mind to plan the experimental and analytical methods for the study
of the process. Triolein is a glyceride containing only 18:1(9) cis fatty
acid units in the glyceride molecules. The heating process will lead
to the formation of elaidic acid units in the glyceride molecules.
Determination of kinetic parameters for the isomerisation requires
precise determination of the concentration of either reacted oleic
acids or formed elaidic acids in the reaction mixture at regular time
interval. There are two analytical methods that are used in this
paper for the evaluation of the concentration of trans isomer/s in
the mixtures. They are namely, infrared spectrometry and GC–MS.
The use of infrared spectrometry and gas chromatography to
determine the trans content in edible oils and fats have been exten-
sively investigated and they are standardized by The American
Oil Chemists Society (AOCS) (AOCS, 1989). Two of the methods
employ gas chromatography and the third one employs conven-
tional infrared spectrometry. The quantitative determination of
trans fatty acid concentration in fats and oils is based on the fact that
the trans isomers of monounsaturated fatty acids and trans isomers
of the polyunsaturated fatty acids containing isolated double bonds
(
Moreira et al., 1999; Rojo and Perkins, 1987; Fullana et al., 2004;
Umano and Shibamoto, 2001; Fujisaki et al., 2002). Some of the
products formed are toxic in higher concentrations. The processes
leading to these products are very complex and identifying the
precursors of different products can be difficult because of the num-
ber of fatty acids involved. Isomerisation of mono unsaturated and
poly-unsaturated 18 carbon fatty acids lead to all the different trans
isomers in the heated oil. Analysing such a complex mixture for its
total chemical composition is a daunting task.
Our intention in this work is to study the isomerisation kinet-
ics for the cis–trans isomerisation of triolein in inert atmosphere.
With several decades of research in the field of lipids, it is hard to
find any research work that is dealing with the determination of
kinetic parameters of lipids containing pure lone fatty acids. The
∗ Corresponding author. Tel.: +47 38141502; fax: +47 38141071.
E-mail address: alfred.christy@uia.no (A.A. Christy).
1
Department of Chemistry and Biochemistry, Clippinger Laboratories, OHIO Uni-
versity, Athens, OH 45701, USA.
−
1
give rise to one specific absorption at 969 cm . This absorption
0
009-3084/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2008.12.002

A.A. Christy et al. / Chemistry and Physics of Lipids 158 (2009) 22–31
23
arises due to the CH out of plane deformation vibration (Belton
et al., 1988; Dutten, 1974; Lancer and Emkem, 1988; Christy et al.,
end of each tube was then melted and sealed. It is important to keep
the glass tubes free from traces of air to avoid oxidation and poly-
merisation during the heating experiments. The sealed glass tubes
were then placed in a short 5 mL glass vial and placed in a chromato-
2
003).
However, the absorption band representing this absorption is
◦
broad with downward sloping baseline because of the overlap of
the broad absorption of triglycerides and of the C H out-of-plane
deformation bands arising from the conjugated linoleic acids in the
sample. The AOCS method involves the comparison of the peak
height of the trans band of the sample dissolved in carbon disul-
phide (CS2 2%, v/v) to the peak height of the trans band of elaidic
acid at the same concentration when trans content is higher than
graphic oven set at 280 C. The vial was placed deep inside the oven
on an inverted beaker. The same procedure was followed with sam-
◦
◦
ples heated at 300 C and 325 C. The glass tubes were removed at
regular time intervals and the cooled samples were used in infrared
and GC–MS analysis.
2.2. Infrared measurements and data treatment
15%. The comparison is made with the peak height of the trans band
of methyl elaidate when the trans content is less than 15%. This pro-
cedure requires sample preparation in a solvent that is considered
unpleasant and toxic. Furthermore, the oil or fat under analysis has
to go through saponification and methylation process to eliminate
the sloping glyceride band.
A PerkinElmer Spectrum One FT-IR spectrometer equipped with
a Harrick single reflectance attenuated total internal reflectance
(ATR) accessory and lead glycine sulphate detector was used in
measuring the infrared spectra. The accessory requires only a thin
layer of sample on the crystal to acquire the infrared spectrum.
Each glass ampoule was cut open and a small part of the sample
was spread on the ATR crystal using the blunt side of a capil-
lary glass tube. A background spectrum was scanned in the range
There has been progress made in the use of the trans band in the
quantification of trans fatty acids in edible oils and fats. Mossoba
et al. (1996) have summarised the developments in the sample
handling and computational techniques in quantifying the absorp-
−
1
of 4000–600 cm
before the application of a sample. A total of
−1
−1
tion around 669 cm . Developments in infrared instrumentation,
sampling techniques and multivariate data handling software were
responsible for the refinements in the determination of trans fatty
acids by infrared spectroscopy. Using the attenuated total internal
reflectance technique with the FTIR instrumental advantage, sam-
ples in neat form have been measured to determine the trans fatty
acids content (Dutten, 1974; Belton et al., 1988; Christy and Egeberg,
30 scans at a resolution of 4 cm were then made on each sample.
The ATR crystal was washed with dichloromethane and acetone
after each measurement.
The same procedure was repeated for samples heated at 300 and
◦
325 C. The sample tubes were sealed again by paraffin film.
The infrared spectra measured on the series were saved as
absorption spectra. Each infrared absorbance spectrum was trans-
formed to the second derivative and used further in the analysis
for the determination of the concentrations of cis and trans isomers
using multiple linear regression (MLR). The second derivatives of
spectra were calculated by using Savitzky–Golay method with a
smoothing window of 11 variables and a polynomial order of 3
(Savitzky and Golay, 1964). The use of second derivatives of spectra
aims to minimize the problem caused by baseline shift and overlap
among peaks (Windig and Stephenson, 1992).
2
006). It is clear from the enormous number of applications using
a combination of infrared spectroscopy and chemometrics, the
quantification of components formed during thermal treatment of
triolein using chemometrics will be superior to any method using
single peak-based quantification. Because, the infrared spectra of
pure triolein and trielaidin are available, we opted to use a method
based on the decomposition of sample spectral matrix into triolein
and trielaidin equivalents and rest. A description of this procedure
is given in Section 2.
Gas chromatography involves saponification and derivatisation
of the oil or fat involved and the separation of the methyl esters
using a high-resolution capillary column that is suitable for the sep-
aration of cis and trans isomers. In GC–MS (with electron impact
ionization), an additional dimension is available for confirmation
of the molecular structure of an analyte. However, the cis and trans
isomers do not show any differences between their mass spectra,
the mass spectra are useful for identifying the FAME congeners.
2.3. Derivatisation and GC/MS analysis
The triolein samples remaining in the tubes after infrared anal-
ysis were subjected to derivatisation. Each glass tube containing
the sample was cut just above the liquid mark and crushed inside a
15 mL test tube. The test tube was then added 2 mL of 0.5 M sodium
hydroxide in methanol. The test tubes containing the mixtures were
◦
then placed in a water bath at 60 C for 15 min. After cooling, each
test tube was added 2 mL of BF /methanol and placed in the water
3
bath again for 10 min. Each test tube was then added 2 mL of a sat-
urated solution of NaCl and 1 mL heptane. The tubes were shaken
to aid separation and dissolution of the FAMEs in the heptane layer.
The glass tubes were then allowed to stand for a few minutes and
the top heptane layer in each of the tubes was added anhydrous
magnesium sulphate. The heptane layers in the tubes were care-
fully extracted and placed in small brown vials and kept in dark
until GC/MS analysis.
2
. Experimental
2.1. Samples and heating experiments
Pure standards of triolein, methyl oleate and methyl elaidate
were purchased from Sigma–Aldrich. All the chemicals were GC
grade with over 99% purity. The samples were used without purifi-
cation. Methyl oleate and methyl elaidate were GC checked for their
purity.
The GC/MS analysis (electron impact MS) was carried out by
using a Hewlett-Packard 5890 gas chromatograph coupled to a
5971 quadrupole mass spectrometer. A 60 m capillary column
with 0.25 mm internal diameter coated with 0.25 ␮m thick (50%-
cyanopropyl)-methylpolysiloxane stationary phase was used in
the separation of cis and trans isomers of the 18:1 fatty acid
methyl esters. A temperature program with initial temperature
The heating experiments were carried out at 280, 300, and
◦
3
4
25 C in micro-glass ampoules. Several glass ampoules of length
cm were made from glass tubes with 1.5 mm internal diameter
and a wall thickness of 1 mm. Heating in micro-size glass ampoules
is an advantage experimentally. The glass tubes attain the tempera-
ture of reaction within a short time and cool quickly when the tubes
are removed from the oven. A propane–oxygen flame was used in
melting one of the edges of each glass tube. Fifteen glass tubes were
injected with 15 ␮l portions of triolein using a plastic syringe. Air in
the remaining part of each of the glass tubes was flushed by a weak
nitrogen flow and sealed by paraffin film. The paraffin film covered
◦
80 C with equilibration time of 1 min and then a temperature gradi-
◦
◦
ent 10 C/min with final temperature 220 C and final time of 10 min
was used. The GC–MS data was collected from 15 min run time. The
chromatographic peaks were integrated and the peak areas were
obtained as percentage.

24
A.A. Christy et al. / Chemistry and Physics of Lipids 158 (2009) 22–31
2
.4. Methodology—kinetic analysis
k
A
Ea
ln
= ln
−
[
A]0
[A]0
RT
When a reaction of the type A—>B + C is in progress, the rate of
the reaction can be given by the following general equation:
−1
should give a linear
A plot of ln (k/[A] ) with respect to T
0
relationship with slope of −Ea/R. Activation energy can be then
d[A]
dt
n
=
−k[A]
(1)
calculated from the slope.
If the reaction rates of the isomerisation are determined for at
where A is the starting material, k is the reaction rate, and n is the
order of the reaction.
Eq. (1) can be written as follows and integrated for the concen-
tration range and time interval as shown in the following equation:
least three different temperatures, the activation energy can be
−1
determined from the plot between ln k and T
.
Alternatively, if the concentration of the product B that is formed
during the reaction is monitored, similar expressions can be set up
to determine the rate and activation energy for the reaction.
The rate equation becomes:
d[A]
=
1−n)
−k dt
At
]Ao
n
[
A]
(2)
(
[
[A]
[B] = kt
(8)
t
0
t
=
−k[t]
(
1 − n)
This is for zeroth order reaction only. When the pure trielaidin
concentration (intensity of absorption) is known then the fraction
of trielaidin formed during the heating can be written as
where [A] , [A]t are concentrations of A at times 0 and t, respectively.
0
The integral is valid for n ≥ 2. When the order of the reaction is
zero, the result becomes,
ꢀ
ꢁ
[
B]t
k
=
t
[
B]trielaidin
[B]trielaidin
[
A] − [A] = kt
(3)
0
t
A plot between [B]t/[B]
and time t would give linear rela-
by dividing the equation by initial concentration [A] , the equation
becomes:
trielaidin
0
tionships for zeroth order reaction.
1
− ([A]
t
k
A]0
= [
t
(4)
2.5. Data handling and concentration estimation
[
A]
0
The spectral data collected from the samples during the heat-
ing experiments were imported into a data matrix using MATLAB
this becomes ([A] − [A]t)/[A] = (k/[A] )t.
0
0
0
When relative concentration is used [A] = 1 and [A]t is the frac-
0
2
007b with each row a spectrum and each column a spectral res-
tion that remains unreacted at time t. The left hand side of the
equation denotes the fraction of the starting material that reacted.
A plot of this quantity against time should give a straight line if
the reaction is of zeroth order. For a reaction of zeroth order, the
concentration change is independent of the concentration of the
reactant and it is only proportional to the time of the reaction.
For a first-order reaction, the result becomes,
olution element. The spectra absorbances were transformed to
their second derivatives to give a data matrix D. When the dou-
bly derivated spectra (S) of pure triolein and trielaidin are known,
then their concentrations (C) in the mixtures can be found by using
ordinary least squares (OLS):
D = CS + E
(10)
ˆ
ln[A]t
C = D/So
=
−kt
(5)
[A]0
The chemometric technique calculates the equivalent of
[B]t/[B]trielaidin which is used in the calculation of kinetic param-
eters.
For a reaction of first order, the reaction rate is proportional
to the concentration of the reactant in solution. The expression
ln ([A]t/[A] ) will be proportional to the time of the reaction. In
0
this work, the order of the reaction was determined by graphic
correlations.
3. Results and discussion
When the 18:1 fatty acids in the triglyceride isomerise to other
isomers, the relationship between concentration of the unreacted
fraction of the isomer and time can be investigated to determine
the rate and order of the reaction. The activation energy needed
to bring the molecules to an activated complex level during the
isomerisation tells us nothing about the product/products formed.
The product can be just one isomer or a mixture of several isomers
and other components. The end products formed and their amounts
depend on the mechanisms involved in the systems.
3.1. Infrared spectroscopy
Infrared spectra of the pure triolein, trielaidin, and a mixture
obtained after 5 days during the heating experiment at 280 C are
given in Fig. 1. The infrared band assignments for triolein and
trielaidin are given in Table 1. Partial second derivative profiles
◦
Table 1
Some of the infrared band assignments of triolein and trielaidin.
The activation energy for the reaction can be determined by car-
rying out the reaction at different temperatures. The reaction rate
k and temperature T of reaction are connected by the Arrhenius
equation:
Frequency (cm−1)
Functional group and mode of vibration
3025
3004
2953
CH trans stretching
CH cis stretching
CH (CH3) asymmetric stretch
CH ( CH2 ) asymmetric stretch
CH ( CH2 ) symmetric stretch
k = Ae−Ea/RT
2924
854
746
653
465
(6)
2
1
C
C
O ester Fermi resonance
C cis stretching
for which, A is Arrhenius constant, R is the gas constant and Ea is
activation energy.
1
1
CH ( CH2 , CH3) bending
For zeroth order reaction Eq. (6) can be written in the following
form:
1377
1238
CH (CH ) symmetric bending
3
C
C
C
O, CH2 stretching, bending
O, CH2 stretching, bending
O stretching
1
161
1118, 1097
67
ꢀ
ꢁ
e−Ea/RT
k
A
=
(7)
9
CH trans stretching
[
A]0
[A]0

A.A. Christy et al. / Chemistry and Physics of Lipids 158 (2009) 22–31
25
◦
Fig. 1. Infrared spectra of triolein, trielaidin and a sample of triolein after 5 days of heating at 280 C.
◦
−1
of the infrared spectra of the samples heated at 280 C are given
in Fig. 2. Another set of second derivative profiles from the sam-
for the wavenumbers at 3004, 1710, and 969 cm . The changes
in the absorptions are clearly visible in the second derivative pro-
◦
−1
ples treated at 300 C is presented in Fig. 3. The infrared spectra of
files given in Fig. 2. The absorption at 3004 cm is the stretching
the resolved components in the same sample mixture are given in
Fig. 4. The spectra shown in Fig. 1 have common features except
absorption of the C–H bond attached to the cis carbon atom in the
oleic acid moieties. This absorption is absent in the spectrum of
◦
−1
−1
Fig. 2. Second derivative profiles of the infrared spectra of the samples heated at 280 C in the regions (a) 1006–920 cm and (b) 3038–2979 cm .

2
6
A.A. Christy et al. / Chemistry and Physics of Lipids 158 (2009) 22–31
−
1
◦
Fig. 3. Second derivative profiles of the infrared spectra of heated triolein samples in the region 1790–1670 cm for samples (a) after 1 and 5 days of heating at 280 C and
◦
(
b) after 1 and 5 days of heating at 300 C.
trans isomer trielaidin. The absorption at 969 cm−1 is due to the
bending vibration of the C–H bond attached to the trans double
bonds in trielaidin. The cis isomer lacks this absorption. However,
the intensities of absorption between the two absorptions are com-
pletely different. This is because the extinction coefficient of the
The progress of isomerisation as determined by infrared spec-
troscopy is shown in Fig. 4 for the three temperatures. The trans
isomer concentration in the samples reached to a maximum of 35%
◦
◦
in 7 days at 280 C, 40% in 4 days at 300 C and 42% in 0.62 (15 h)
◦
days at 325 C.
−
1
CH stretching absorption (of the cis CH) at 3004 cm is ten times
smaller than the CH out of plane deformation vibration (of the
3.2. GC–MS analysis (electron impact MS)
−1
trans CH) at 969 cm (Fig. 1).
The carbonyl group in both isomers absorb at 1745 cm−1. The
The total ion chromatogram of the FAMEs obtained from three
◦
third spectrum in Fig. 1 shows absorption of carbonyl groups at
samples heated at 300 C (Fig. 5) show that there is baseline sepa-
−1
1
710 cm in the vicinity of the absorption of glyceride carbonyl
ration between the cis and trans isomers of the 18:1(9) fatty acids.
The FAMEs of the cis and trans isomers of the 18:1(9) fatty acids
were identified by pure standards of methyl oleate and methyl elai-
date. There is clear evidence that the concentration of trans isomer
increases with the heating time. Plots showing the progress in the
formation of trans isomers as determined by GC–MS for the three
group. A set of second derivative profiles in the carbonyl region for
◦
1
◦
samples heated at 280 C and 300 C are presented in Fig. 3. This
−
absorption at 1710 cm is very characteristic to aldehyde carbonyl
groups. Glycerides under thermal treatment at temperatures over
◦
1
80 C undergo oxidation and form aldehydes (Fullana et al., 2004),
◦
aldehyde acids, alcohols and hydrocarbons (Vann de Voort et al.,
994). According to Fullana et al. (2004) and Fujisaki et al. (2002),
temperatures are presented in Fig. 6. At 280 C, the concentration of
1
trans isomers reached a maximum concentration of 41% in 7 days of
◦
octanal, nonanal, decanal, 2-decenal, and 2-undecenal are aldehy-
des formed from oleic acid moieties in the glyceride molecules.
The chemical transformation of fatty acid moieties in the glyceride
molecules into aldehydes under thermal treatment is evident from
the Figs. 1 and 3.
heating. At 300 C, the concentration of trans isomers reached 64%
◦
in 5 days of heating and at 325 C, the concentration reached 56%
in 0.7 (17 h) days.
The concentrations of trans isomers determined by the two
methods differ significantly. This difference is caused by the con-

A.A. Christy et al. / Chemistry and Physics of Lipids 158 (2009) 22–31
27
Fig. 6. Plots showing the concentrations of methyl elaidate as determined by GC–MS.
concentrations between cis, trans isomers and other fatty acids
formed during the reaction. This result excludes the concentra-
tion of degradation products, aldehydes and other compounds
without an acidic group, because only the fatty acids in the
glyceride molecules and any other acid molecules formed dur-
ing degradation participate in the derivatisation process. In the
infrared spectroscopy/chemometric technique, the infrared spectra
of the heated samples were resolved into components repre-
senting the cis and trans isomers. Plots showing the correlation
◦
Fig. 4. Concentrations (as fractions) of trielaidin in the samples heated at (a) 280 C,
(
methodology described in the manuscript.
◦
◦
b) 300 C and (c) 325 C. These are concentrations determined by the chemometric
centration of trans isomers determined by infrared measures the
concentration of the reaction products which contains cis, trans
isomers of 18:1 fatty acids and degradation products. The con-
centrations determined by GC–MS of the FAMEs estimate relative
◦
Fig. 5. Total ion chromatograms of methyl ester derivatives of the samples heated at 300 C after (a) 1 day, (b) after 5 days and (c) after 9 days.

2
8
A.A. Christy et al. / Chemistry and Physics of Lipids 158 (2009) 22–31
FT-IR/chemometrics. The concentration of the degradation prod-
ucts reaches 8% in 8 days, 50% in 8 days, and 27% in 0.7 days at
◦
2
80, 300, and 325 C, respectively. It is very clear that the pro-
longed heating at higher temperatures leads to severe degradation
and oxidation of fatty acids in the glyceride molecules. The forma-
tion of several cis and trans 18:1 isomers start from the beginning
◦
but becomes prominent after 3 days at 300 C. The unsymmetrical
shape of the peaks representing the oleic and elaidic acid methyl
esters in the total ion chromatograms indicates the formation of
other positional isomers. As the double bond moves farther from
the 9 to 10 carbon–carbon double bond, the retention time of the
new isomers shift for longer. The GC–MS analysis of samples heated
◦
at 325 C gave peaks with unsymmetrical shape but the formation
of positional isomers was not prominent because of the short time
of the heating experiments.
The peaks in the total ion chromatograms shown in Fig. 5 indi-
cate the formation of several cis and trans isomers of the 18:1 fatty
acids. The appearance of peaks on both sides of the 18:1(9) trans and
cis fatty acids methyl esters show that the double bond migrate in
both directions during heating. The retention times of the positional
isomers follow the order of their double bond position (Wolf and
Precht, 1998).
The peak appearing at a retention time of 22.46 min in Fig. 5
is methyl ester of 18:0 fatty acid. The identity was confirmed by
GC/MS library search and by spiking the sample with 18:0 FAME.
The concentration seems to increase with time. Even though, the
accumulation is slow, the concentration becomes significant after
◦
9
days of heating at 300 C.
Fig. 7. Plots showing the correlations between concentrations (trielaidin) as deter-
3
.3. Reaction products and reaction mechanisms
mined by FT-IR/chemometrics and concentrations (methyl elaidate) as determined
◦
◦
◦
by GC–MS. (a) for 280 C, (b) for 300 C and (c) for 325 C. Here only the cis and trans
isomers are considered, i.e., the proportions of cis and trans isomers will sum to one.
There are several reactions that take place during thermal
treatment of triolein molecules. A sketch depicting the chemical
reactions and possible transformations into different products are
given in Figs. 8 and 9. The possibilities for the formation of reac-
tion products are: (1) the cis double bond can simply open up
and rotate freely along the 9, 10 C C bond and intraconvert to a
trans double bond, (2) with the opening of the double bond and
hydrogen transfer, the double bond can migrate in both directions
from the 9 to 10 position and form several cis and trans isomers
of the 18:1 fatty acids, or (3) the molecule can undergo oxidative
fission at different positions of the carbon chain back bone. The
first step in the fission reaction is the formation of a hydroperox-
ide at carbon number 8. The molecule is then cleaved by homolysis
into alkoxy and hydroxy radical (Fig. 9). The alkoxy radical then
undergoes ␤-scission of C C bond on either side of the oxygen
containing carbon atom. The scission leads to alkenes, aldehydes,
aldehydic acids, alcohols, etc. This process can continue with some
of the molecules formed due to the migration of the double bond
in the glyceride molecules or (4) exchange of hydrogen atoms can
lead to saturated and poly-unsaturated fatty acid moieties in the
system.
between the normalised trans isomer concentrations determined
by IR/chemometrics versus GC–MS method are presented in Fig. 7.
The concentrations presented in the plots are relative concen-
trations with only the cis and trans components taken into the
calculation. Very good linear correlations with slopes almost unity
◦ ◦ ◦
0.9947 for 280 C, 0.9807 for 300 C and 0.9923 for 325 C) shows
(
that the trans isomer concentrations in oils and fats can be precisely
determined by infrared spectroscopy/chemometrics if one knows
the fatty acid composition of the system well. The results clearly
illustrate that the infrared spectroscopy combined with chemomet-
rics gives relative concentrations (as fractions) of trans isomers in
treated oils and fats. The results also indicate that the concentra-
tions of trans isomers in thermally treated oils and fats determined
by GC–MS as FAMEs are overestimated. Without knowing the com-
position, the GC–MS determinations of trans isomers as methyl
esters can only lead to erroneous concentrations. Obviously, the
infrared method determines the real concentrations of the trans
isomers in the mixtures.
The concentrations (fractions) of other products formed during
the heating experiments were determined by subtracting the con-
centrations of cis and trans isomer concentrations determined by
However, apart from the isomerisation and hydrogen transfer
reactions, all the other reactions require the presence of oxygen
Fig. 8. Reaction pathways under heating of triolein.

A.A. Christy et al. / Chemistry and Physics of Lipids 158 (2009) 22–31
29
Fig. 9. Illustrations of cis–trans isomerisation and bond migration in the 18:1 fatty acid molecule.
in the reaction mixture. The heating experiments were carried out
in the presence of nitrogen. It is not very clear where these oxy-
gen atoms come from. One possibility is that they come from the
glyceride molecules.
formed can be used for this purpose. A diagram illustrating the
cis–trans isomerisation with an arbitrary energy scale is given in
Fig. 11. Plots showing the fraction equivalent of trielaidin formed
with respect to time are presented in Fig. 12. Only a few data
points representing the trans concentrations in the samples heated
◦
at 300 C are included in the plot. The reason is because the trans
3.4. Isomerisation kinetics and activation energy
isomers started decomposing and a decrease in the concentra-
tion of trans isomers observed (Fig. 4). Linear correlations indicate
that the thermally induced isomerisation reaction is of zeroth
order.
3
.4.1. Activation energy for the reaction
As discussed earlier, the activation energy for the heat-induced
reaction can be calculated by using the reaction rates k for the
reactions at three different temperatures. Triolein used in the heat-
ing experiments undergoes isomerisation and degradation (Fig. 10)
and therefore the change in the concentration of triolein cannot
be used in the determination of activation energy for the isomeri-
sation reaction. However, the concentration of the trans isomer
For a zeroth order reaction, the activation energy can be
determined by using the parameters from
a
plot between
−
1
ln (k//[B]trielaidin) and T
(Fig. 13). The activation energy Ea and
the frequency factor A calculated for the isomerisation reaction are
given in Table 2. The activation energy calculated, 41 ± 5 kcal/mol
for the cis–trans isomerisation lies within the values calculated
for C C double bonds in systems (41–63 kcal/mol) with dif-
ferent chemical environments (Rabinovitch and Michel, 1959;
Table 2
Kinetic parameters for the isomerisation.
◦
−1
)
Isomer
8:1(9) cis
Temp ( C)
k reaction rate (1/h)
Activation energy (kcal mol
1
280
300
325
0.510 ± 0.005
0.130 ± 0.013
0.799 ± 0.028
Fig. 10. Reaction mechanisms of oxidation and degradation of fatty acid molecules.
18:1(9) cis
41 ± 5
(The possible products presented in the figure follows a scheme presented in the
18:1(9) cis
web site http://www.cyberlipid.org/perox/oxid0009.htm).

3
0
A.A. Christy et al. / Chemistry and Physics of Lipids 158 (2009) 22–31
Fig. 11. Reaction progress on an arbitrary energy scale for the cis–trans isomerisation.
Fig. 13. An Arrhenius plot of ln (k/[B]trielaidin) with respect to T−1. k is the rate of
isomerisation at temperature T. The k/[B]trielaidin values are slopes from Fig. 12.
4. Conclusion
There are several interesting aspects of thermal degradation and
isomerisation reaction of triolein came to light after the analysis
of the mixtures using two different analytical techniques. First of
all, it is clear that thermal degradation takes place even within an
inert atmosphere. The source of oxygen in these reactions is not
known. Furthermore, the strain on the glyceride molecules under
prolonged heating is evident from the experiments conducted at
◦
3
00 C. Almost half of the original concentration had degraded into
other products. This result suggests that the frying oils heated at
elevated temperatures in the presence of oxygen in restaurants and
places that are open 24 h may actually contain a complex mixture of
dangerous chemicals in the frying oil bath. Low temperature frying
should be encouraged whenever possible.
The use of infrared spectroscopy in determining real concentra-
tion of trans isomers in fats and oils using chemometric multiple
linear regression methods has given us confidence in using the
technique in our future investigations involving fats and oils. Fur-
thermore, the technique may also be used in determining trans
concentrations in edible oils and fats, hydrogenated oils and other
situations where quantitative determination of trans fatty acid
content in a mixture is needed. The success of the estimation of
the concentration relies on the fact that there was no chemical
interactions between the molecules that may affect the extinction
coefficients of the components in the mixture.
Fig. 12. Correlation plots showing linear correlations between concentration of trans
isomers and time. (a) For 280 C, (b) for 300 C and (c) for 325 C.
◦
◦
◦
Rabinovitch and Hulatt, 1957; Butler and AcAlpine, 1963; Lin and
Laider, 1968). The activation energy determined for cis–trans iso-
merisation of triolein reflects that the environment where the
double bond is situated is more sterically hindered than in normal
alkenes.
The results clearly show that the GC–MS analysis of treated fats
and oils as FAMEs may not give correct picture of the products
formed in the systems during thermal treatment and may lead to
erroneous concentrations for trans isomers.

A.A. Christy et al. / Chemistry and Physics of Lipids 158 (2009) 22–31
31
The activation energy for the cis–trans isomerisation of triolein
has been determined for the first time and the value and order of
the reaction determined are now open for further investigation. We
believe that the activation energy determined for the isomerisation
is in the acceptable range for these types of systems.
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