RESEARCH ARTICLE | MAY 05 2023

Investigation the oxidative rancidity of soybean oil by NMR

spectroscopy
Sahri Yanti; Wei-Chih Chen; Wei-Jyun Chien 

AIP Conference Proceedings 2685, 020008 (2023)

https://doi.org/10.1063/5.0111708

CrossMark

 
View Export
Online Citation

Downloaded from http://pubs.aip.org/aip/acp/article-pdf/doi/10.1063/5.0111708/17354684/020008_1_5.0111708.pdf

Articles You May Be Interested In

Semiconductor Sensor Array Based Electronic Nose for Milk, Rancid Milk and Yoghurt Odors Identification
AIP Conference Proceedings (May 2009)

The effect of packaging type and storage temperature on the chemical and sensory properties of corn
getas
AIP Conference Proceedings (January 2023)

Effect of storage on fat hydrolysis system in pearl millet flour

AIP Conference Proceedings (May 2022)
 Investigation the Oxidative Rancidity of Soybean Oil by
NMR Spectroscopy
Sahri Yanti1, 2, Wei-Chih Chen1, Wei-Jyun Chien1, a)
1
Department of Applied Chemistry, Chaoyang University of Technology, Taichung, 413310, Taiwan
2
Faculty of Agricultural Technology, Sumbawa University of Technology, West Nusa Tenggara 84371, Indonesia

Downloaded from http://pubs.aip.org/aip/acp/article-pdf/doi/10.1063/5.0111708/17354684/020008_1_5.0111708.pdf

a)
Corresponding author: wjchien@gm.cyut.edu.tw

Abstract. Soybean oil (SBO) is common commercial vegetable oil. Reusing oil at high temperatures can cause rancidity,
and one of the indicators is the presence of free fatty acids. This study aims to investigate changes in the chemical
composition of SBO after heating using 1D NMR, 2D NMR, and TD NMR. Soybean oil was heated 8 h per day for 5
days at 190 oC. Total 6 samples were collated and consisted of SBO0 (unheated), SBO1 (heated on the first day) SBO2
(heated on the third day), SBO3 (heated on the third day), SBO4 (heated on the fourth day), and SBO5 (heated on the
fifth day). The sample was analyzed by using 1H NMR, PFG NMR, TD NMR. SBO0 and SBO4 by 13C NMR, DEPT 135,
and HSQC. 1H NMR spectra showed SBO after heating increase the amount of proton. However, linolenate was
decreased and SFA became higher than UFA and PUFA. 13C NMR spectra showed a reduced carbon chain. The
correlation between 13C NMR, DEPT 135 showed SBO contained secondary carbon was detected as a negative signal.
HSQC showed the carbon in the TAG group interacts strongly with the hydrogen in acyl and methyl groups. PFG NMR
and TD NMR results presented that the distribution coefficient decreased, and transverse relaxation times also decreased,
indicating a long period of heating increased the mass and viscosity of the oil. NMR is a quick method for analyzing the
physicochemical changes of SBO. The longer periods of heating make a different chemical composition and cause
oxidative rancidity.

Keywords: Soybean, Rancidity, HSQC, PFG NMR, TD NMR.

INTRODUCTION
33.5 % of vegetable oil consumption in the world comes from soybean oil with the demand in the US reaches
56.5 % [1]. Soybean oil can be found as a cooking oil product. Both oils and fats are fast heat conductors so that
food cooks faster [2,3]. Price, stability or shelf life, taste, and nutritional profile of cooking oil are the determinants
of selling in the market. SBO contains fatty acid methyl esters (FAME) which have benefits for health such as
palmitate (10í12 %), stearate (3í5 %), oleate (24 %), linoleate (54 %), and linolenate (8 %) [4,5]. Information of
trans FAs and SFAs compositions needs to be included in the packaging of cooking oil products.
Fresh vegetable oil is not rancidity and does not have FFAs [6,7]. Rancidity is the property of oil or fat that is
susceptible to oxidative reactions, causing unpleasant odors and discoloration. Other indicators of rancidity include
the presence of FFAs [8], aldehydes, and peroxides [9]. Heating oil at 150–190 o C can cause an oxidation reaction
and make the oil rancidity. The oil looks foamy, darkens with the viscosity and mass increased [10]. Heating oil also
causes hydrolysis, polymerization and produces hydroperoxide compounds, alcohols, aldehydes, and ketones
[10,11]. The accepted physical method for detecting oxidative rancidity is to detect conjugated dienes, as this
molecule is formed by the oxidation of PUFA [9]. The antioxidant content contributes to maintaining the stability of
the oil. Antioxidants can be added to delay the oxidation reaction [12,13].
Colored oil as a sample does not matter for NMR analysis [14]. 1HNMR has been reported to evaluate the
stability pattern of oil during storage [15] and the evaluation of FFAs in oil [7,14,16,17]. The correlation of 13C
NMR with DEPT-135 shows a clearer oil characterization owing to the higher ratio of signal than noise. The

The 4th International Annual Meeting on STEM Education (IAMSTEM 2021)

AIP Conf. Proc. 2685, 020008-1–020008-9; https://doi.org/10.1063/5.0111708
Published by AIP Publishing. 978-0-7354-4437-9/$30.00

020008-1
triacylglycerol fraction in olive oil is observed using DEPT 135 [18]. HSQC is a 2D NMR to investigate hydrogen
and carbon bonds in pyrolysis bio-oil products [19]. PFG NMR was used for lubricating oil analysis [20] and water-
in-oil separation [21]. PFG NMR has been applied to read DOSY. DOSY can be used to analyze the diffusion of
chemical compounds in a solution, showing the separation of intermolecular interactions and their size [22]. TD
NMR can detect adulteration of diesel oil based on molecular mobility [23,24]. The same method was applied for
the detection of adulteration in olive oil [25].
Soybean oil contains fatty acids that are beneficial for health. Reusing soybean oil repeatedly at high
temperatures has the potential to cause oxidative rancidity and change the chemical composition, formation of FFAs,
changes in viscosity, and mass. This study aims to investigate changes in the chemical composition of soybean oil
after heating using 1D NMR, 2D NMR, and TD NMR. The results of this investigation provide a way to prevent the
adverse effects of waste cooking oil so that consumers are more careful and stay healthy.

METHODS AND MATERIALS

Chemicals and NMR Specification

Downloaded from http://pubs.aip.org/aip/acp/article-pdf/doi/10.1063/5.0111708/17354684/020008_1_5.0111708.pdf

Soybean oil from Taiwan sugar Co. Ltd, dichloroform (Sigma Aldrich) and tetramethylsilane (Sigma Aldrich).
Sealed oil heater (diameter 15 cm, wide 15 cm), NMR Bruker Avance 500 MHz model, Bruker SpectroSpin 11.7
Tesla, standard diameter 54 mm, UltraShield shielded superconducting magnet, constant temperature system B-VT
3000, Probe 5.0 mm, operating system Topspin 2.1. TD NMR model Bruker mq20 BioSpin minispec NF4733,
Probe H20-10-3 3 RH2GX, constant temperature system BL-3001, operating system minispec NF.

Sample Preparation and NMR Analysis Procedure

1 L soybean oil heated 8 hours per day for 5 days at 190oC. A total of 6 collated samples were stored in the
refrigerator consisting of SBO0 (unheated), SBO1 (heated on the first day) SBO2 (heated on the third day), SBO3
(heated on the third day), SBO4 (heated on the fourth day) and SBO5 (heated on the fifth day). Each 200 μl of
dissolved into 500 μl of dichloroform is added a little to tetramethylsilane. The mixture was shaken for 5 minutes
and then analyzed using 1H NMR, PFG NMR, TD NMR. SBO0 and SBO4 were analyzed by 13C NMR, DEPT 135,
and HSQC.

RESULT AND DISCUSSION

The internship project is to foster students from an intern to becoming an experienced aircraft mechanic – a
highly-skillful profession. In addition to providing basic salary for internship, EGAT also provides lunch and work
clothes. The interns will get to facilitate their hands-on skills by working on the country’s largest, state of art fleet.
Nurtured by experienced task force in industry, they’ll learn and harvest what would be the first step on their
aviation maintenance engineering career. Students have a semester off-campus internship at industry. Through their
comprehensive assistance to aircraft maintenance. Students are trained to be able to provide collaboration with
company the high-quality internship. The job descriptions are as following:

Analysis of Fatty Acid by 1H NMR, 13C NMR, DEPT 135, and HSQC
1
HNMR showed that SBO contained a mixture of SFA and UFA. UFA had a proton signal at į 0.98 ppm (B), į
2.04 ppm (E), į 2.73 -2.86 ppm (G) and į 5.36 ppm (J) (Figure 1 and Table 1). As a UFA, PUFAs have more proton
signals at J and G. The total integral of proton in SBO increases with increasing temperature. It can be assumed that
the double bonds of UFA become a single bond due to hydrolysis and oxidation. As the hydrolysis depends on the
type of vegetable oil and the temperature, it can be accelerated by catalyst [26]. Hydrogenation effort to maintain
stability or shelf life and control the taste of cooking oil [27].

020008-2
 Downloaded from http://pubs.aip.org/aip/acp/article-pdf/doi/10.1063/5.0111708/17354684/020008_1_5.0111708.pdf
FIGURE 1. 1H NMR, 13C NMR and DEPT 135 of SBO0 at 298 K, 1H operation conditions p1=15s (90° pulse width), pL1= -
3.70 dB (power level pulse intensity), d1=1.2 s (delay relaxation time), ns=128 (number of scans scan time). 13C operation
conditions p1=10.3 s, pL1= -1.10 dB, d1=1.2 s, ns=1024.

TABLE 1. Identification of functional groups

1
H NMR 13
C NMR Multiplicity and Integral of SBO
Peak Functional group
į (ppm) į (ppm) 0 1 2 3 4 5
A 0.83-0.92 14.05 – 14.10 íCH2íCH2íCH2íCH3 m,4 m,4 m,4 m,5 m,5 m,5
B 0.93-1.03 14.09 – 14.14 íCH=CHíCH2íCH3 t,1 t,1 t,1 d, 1 d,1 s,1
C 1.25-1.35 29.0 – 29.80 í(CH2)n m,21 m,20 m,20 m,23 m,23 m,23
m,2 m,3
D 1.57-1.67 22.5 -22.71 ROOCíCH2íCH2í m,3 m,3 m,4 m,4
m,1 br.s,1
br.s,1
E 2.00-2.09 31.5 -31.9 íCH2íCH=CHí m,4 m,4 m,4 m,5 m,4
m,4
F 2.30-2.40 33.9 -34.21 ROOCíCH2- dt,3 m,3 m,2 td,3 td,4 td,4
m,1 m,1
G 2.73-2.86 24.8 – 27.22 íCH=CHíCH2íCH=CH2í t,2 m,1 s,1 s,1
br.s,1 br.s,1
H 4.12-4.22 62.0-62.13 íCH2íOíCOíR dd,1 dt,1 m,1 dd,1 d,1 d,1
I 4.27-4.35 76.8-77.4 íCHíOíCOíR dd,1 m,1 dd,1 dd,1 d,1 d,1
J 5.31-5.41 127.7-130.2 íCH=CHí m,4 m,4 m,4 m,4 m,3 m,3
1 172.5-173.2 íCOO
s (singlet), br.s (broadened singlet), d (doublet), dd (double doublet), td (triple doublet), t (triplet), dt (double triplet,) m (multiplet)

020008-3
 The reduction of double bonds decreases the amount of UFAs. Biodiesel degradation was observed through
changes in the triglyceride backbone proton signal at į 4.12–4.36 ppm and correlated with the proton signal at į
5.24–5.6 ppm [28]. Based on the integral in SBO after heating, the proportion of proton in triglyceride structure was
stable, but the proton environment was changed. The original splinting caused multiplet changes to a doublet (Table
1). The coupling constants at į 5.25 and 5.30 ppm (C=C) also indicated the structure of the isomer [29]. The cis
isomer of fatty acid has a coupling constant of less than 10 Hz. All samples had a coupling constant of less than 10.0
Hz, so the structure was still in the cis isomeric form. Interaction of hydrogen in į 5.79 ppm with carbon į 123.74
showed the molecule had cis isomers [30], which was found in SBO0 and SBO4 (J/J) (Figure 2). The increase in
iodine value was directly proportional to the percentage of protons in the olefin functional group (C=C), while iodin
values were at unsaturated degrees [31]. Based on the integral of the proton, the percentage of olefin functional
groups in SBO were 9.09 % (SBO0), 9.30 % (SBO1), 9.52 % (SBO2), 8.33 % (SBO3), 6.38 % (SBO4), and 6.25 %
(SBO5), respectively. Iodine values increased on the second day and then decreased until the fifth day. This shows
that the occurring process is the evaporation of water and then oxidation. The hydrolysis during heating of the oil is
a reversible reaction, initially slow and then quickly. Evaporation of water and increasing temperature
simultaneously cause triacylglycerol to be decomposed into diacylglycerol and monoacylglycerol [10].

Downloaded from http://pubs.aip.org/aip/acp/article-pdf/doi/10.1063/5.0111708/17354684/020008_1_5.0111708.pdf

Soybean oil has been reported to contain linolenate [4,32]. Heating at 240 o C resulted in the formation of trans
linolenate and trans linoleate [32]. Linoleate and linolenate are PUFAs. The only linolenate has a proton signal at į
0.98 ppm (B) due to the proton in the methylene functional group directly adjacent to the double bond. Based on the
comparison of integrals methyl of B with total methyl (A dan B), each SBO with linolenate were respectively 20 %
(SBO0), 20 % (SBO1), 20 % (SBO2), 16 % (SBO3), 16 % (SBO4), and 16% (SBO5). The percentage of linolenate
decreased with an increased heating period. Soybean oil and canola oil have moderate levels of linolenate and are
susceptible to oxidative reactions [33].
Chemical shift at 2.38 ppm with multiplet (m) belongs to the ROOíCH2 functional group. When soybean oil has
a triplet and is mixed with oleate, the peak becomes a quartet [7,17]. FFA testing generally uses oleate as the
standard, and improper storage of the oil for a long period will increase the FFA [34]. The oleate content in soybean
oil was 3.4–10.6 %. The percentage depends on genetic factors [35]. The amount of oleate in soybean oil has the
same range as rapeseed oil, sunflower oil, and corn oil, which is 0.03–0.08 g/100 g [36]. It is close to peanuts [37].
Oils rich in oleate are more heat resistant and produce less toxic aldehydes [38]. 1HNMR analysis result shows an
increase in proton signal intensity at į 2.29–2.33 ppm (F) during heating. SBO0 to SBO5 has multiplicity double
triplet, multiplet, multiplet, triple doublet, and triple doublet, respectively. The multiplicity of peak F is thought to
initially overlap as triglycerides and FFAs and then slowly separated. According to Di Pietro [17], the dissolving
effect of protons in the carboxylic group is stronger than ester, so the proton of the carboxylic acid group is seen in
the downfield area. FFA products were formed in SBO3, SBO4, and SBO5. The qualitative test using pH paper
showed that SBO3, SBO4, and SBO5 had a pH above the minimum standard. Compared with the temperature effect,
the long period of heating has more effect on acid number value [39].
1
H NMR, 13C NMR, and DEPT 135 were correlated in evaluating the TAG. The isomers cis and trans structures
can also be identified by 13C NMR (Garcia et al. 2019). SBO has primary carbon (–CH3) at į 14.05í14.10 ppm (A
and B) and two kinds of secondary carbon, first at į 22.5–34.21 ppm (C, D, E, F, G) and the second (–CH2) appear
at į 62.0–62.1 ppm (H) (Table 1). Tertiary carbon (–CH) appears in į 68.9–68.94 ppm (I). both peak H and I were
TAG functional groups. Primary, secondary, and tertiary carbons had peaks that overlap at į 24í34 ppm, besides
that the signals also overlapped glycerol carbon signals at į 10í35 ppm [18]. COOCH2 functional group in
linolenate appears at į 51-52 ppm [40]. Primary, secondary, and tertiary carbon signals can be distinguished by
DEPT 135. The secondary carbon has a negative signal, while the primary and tertiary carbons have a positive
signal in different chemical shifts (Figure 1). DEPT-135 provided information on primary, secondary, and tertiary
[18], [19]. Based on the DEPT 135 spectra, the secondary carbon with a negative signal is the acyl group (C, D, E, F,
and G). The chemical shift at 127.7–130.2 ppm belongs to carbon signal J (all UFAs). Carbon signals at į 14.1, 22.7,
29.7, and 31.9 ppm belong to SFA while 128í132 ppm belong to UFA [41]. C16 and C2 of linoleate appear at į
31.5 and 34.0 ppm [42]. Based on that, peak E belongs to C16, and C2 belongs to F in SBO. The chemical shift (į)
is based on the interaction of carbon in different environments, so that nucleus of carbon in C=O and CíO is
unprotected even the signal is not visible in DEPT135. The chemical shift depends on the basicity of the lewis
functional group, the strength of the interaction decreases with amines, alcohols, ketones, aldehydes, ethers, esters,
nitriles [30].

020008-4
 Downloaded from http://pubs.aip.org/aip/acp/article-pdf/doi/10.1063/5.0111708/17354684/020008_1_5.0111708.pdf
FIGURE 2. HSQC of SBO0 and SBO4 operation conditions p1=15.0 s (1H 90° pulse width pulse width), pL1= -3.70 dB
(1H power level pulse power), p3=10.3 s (13C 90° Pulse width), pL2= -1.10 dB (power level pulse intensity 13C), d1=1.2
s (relaxation delay time), ns=128 (number of scan times).

Hydrogenation transforms the FA profile from the liquid into solid and producing glycerol, while triglycerides
become hydrogen donors [43]. The triglyceride ester functional group was found at į 172.5 – 173.2 ppm in 13C
NMR and the signal was detected in the downfield. The carbon signal in the carbonyl group is located at į 200 ppm
[44]. Carbonyl is seen at į 172 – 174 ppm [18]. Ketones, aldehydes, esters, and acids are seen at į 165 - 215 ppm
and esters of oil pyrolysis have a chemical shift at 165-180 ppm [19]. Proton signals belonging to aldehydes were
found at į 9.74 -9.77 ppm, signals seen in SBO3, SBO4, and SBO5. Aldehyde is the second product after
hydroperoxide formation in oil [45] appeared in į 8.3 -10 ppm [19]. The lipid peroxides were aldehydes (hexanal,
propanal) and malondialdehyde [1, 9,14,46,47]. The aldehyde or hydroxyl group compounds found in oil were non-
volatile groups. The oil which has been stored for a long time has decreased proton signal intensity of phenolic
compounds, and a low-intensity proton signal owns by saturated aldehyde [15]. Before heating, the total carbon in
SBO was reduced from 61 to 34 after heating, indicating that the long-chain carbon transformed into a sort chain.
Signal carbon of FFA can be detected at carbon signal į 68.91 ppm and proton signal at į 4.0 – 4.5 ppm [48].
SBO had a carbon signal į 62–77 ppm that belongs to H and I, while proton signals at į 4.0 and 4.5 belong to H.
HSQC is used to investigate the carbon and hydrogen bonds [19]. The signal of FFA appeared in SBO4 by carbon
peak H and proton peak H (H/H). Decreased signal of I/I in HSQC showed FFA by TAG functional group
interaction. Overall, the signal intensity was reduced in SBO4, especially in the TAG functional group, indicating
that TAG has been converted to diacylglycerides and monoacylglycerides. Fatty free radicals and peroxide (E/G,
G/D) are formed on the hydrogen closest to the double bond [13] (Figure 2). SBO4 had C=O and CíO, both of
functional group belongs to ester TAG. The functional group interacts with hydrogen belonging to A, B, C, D, E, F,
G, I, and J (Figure 2). Strong interactions occur at hydrogen A and C belonging to the methyl (FA terminal) and acyl
functional groups (all FA). The intensity of the internal interaction of carbon with hydrogen in the acyl FA
functional group decreased (C/C) in SBO4, followed by a decrease in the intensity of the interaction between carbon
and hydrogen in the UFA functional group (G/D, E/G, F/F, I/I, and J/J) (Figure 2). It is assumed that during heating,
UFA transformed into SFA, opened double bond in carbon chains, and also the carbon of the TAG functional group
released hydrogen. The longer periods of heat, the proportion of SFA increased. From peak C, split into two parts
and decrease of the peak at the downfield area were observed. Oxidative rancidity is characterized by the appearance
of conjugated dienes resulting from the breakdown of PUFA [9]. The double bonds in oleate, linoleate, and
linolenate are chemically active sites and make the oil susceptible to oxidation [13].

020008-5
 Analysis of Transverse Relaxation Times (T2) and Distributions Coefficients (D) by PEG
NMR and TD NMR

D = 4.5 x 10-10 D = 2.05 x 10-10

-10.5 -10.5

/s) (ppm)
/s)(ppm)
-10.0 -10.0

Shift
Shift
2

2
log D (m

log D (m
F1 Chemical
F1 Chemical
-9.5 -9.5

-9.0 TMS -9.0

Downloaded from http://pubs.aip.org/aip/acp/article-pdf/doi/10.1063/5.0111708/17354684/020008_1_5.0111708.pdf

TMS

5 4 3 2 1 0 5 4 3 2 1 0
1
1
F2 Chemical Shift (ppm)
H NMR F2 Chemical Shift (ppm)
H NMR
SBO0 SBO4

FIGURE 3. PFG NMR of SBO0 and SBO4 operation conditions the maximum gradient BBFO probe 50.0 G/cm, the
magnetic field recovery time gradient (D16) 100.0 sec, D21 5 msec, GPZ7 -17.13, GPZ8 -13.17, P19 600.0 sec, linear
magnetic field gradient from 2% to 95% in 16 steps, adjusting pulse gradient time (p30) and diffusion time (d20).

In addition to GC-MS, the estimated weight of pyrolysis biomass has been studied using DOSY by plotting 1H
NMR spectra [49]. DOSY also has been used to measure viscosity through D values. The DOSY integration plot
shows the volume (mass) of oil droplets down to the micron scale [22,50]. The DOSY visualization illustrates how
fast the distribution of glycerol in water. The D standard of glycerol in water at 25 oC was 1.025×10-5 cm2 s-1 [50].
The D is measured in units of m2 s-1. The results showed that the D values of SBO0 to SBO5 were 4.5×10-10,
4.35×10-10, 6.25×10-10, 3.51×10-10, 2.05×10-10, 2.70×10-10 m2s-1. The pattern of D values of SBO0 to SBO5
decreased due to the reduction in the number of carbons. The reduction of antioxidants in oil is also triggered by
heating [13]. Soybean oil has been reported to contain antioxidants [13]. The moisture of soybeans has been reported
as 8í16 % [51]. Using mechanical screw processing can produce the maximum moisture of 5.81 % from seeds [52].
SBO2 had the highest distribution coefficient as the molecule opened the double bond in the chain, and the addition
of oxygen or the formation of peroxide radicals (ROO*) then became hydrogen peroxide (ROOH). According to
Buck [13], a series of products such as fatty free radicals (R*), then peroxide free radicals (ROO*), hydrogen
peroxide (ROOH) form when oil is heated. The last step of hydrolysis breaks down hydrogen peroxide into small
molecules.
The combination of PEG NMR and DOSY can identify molecules that have overlapping chemical shifts but have
different distribution coefficients [20]. The small size of the molecule has a small signal and is distributed rapidly.
Large molecules have a wide and slowly distributed signal. The PFG NMR image shows that SBO0 and SBO4 have
9 types of protons (Figure 3). The proton signals at SBO4 are weaker, smaller, and wider, while the proton signals at
SBO0 appear to be more concentrated. This indicates that SBO0 has a more structured proton with a small
molecular mass, faster distribution, more dilute viscosity than other samples. On the other hand, SBO4 has a heavy
molecular mass, slow distribution, and higher viscosity. TD NMR shows how long the transverse travel time (T2)
changes. SBO0 has a T2 of 167 ms, SBO1 has that of 150 ms, SBO2 has 149 ms, SBO3 has 106 ms, SBO4 has 85
ms, and SBO5 has 68 ms. The long period of heating decreases T2. As molecules in biodiesel have a lower T2 value
than before, molecule migration becomes slow and the viscosity increases as it is adulterated with kerosene [23].

CONCLUSION
Soybean oil characterization has been carried out using 1D NMR and 2D NMR analyses. The results showed that
4 times heating oil for 8 h at 190oC caused oxidative rancidity profiles. The peak had aldehyde in the 1HNMR

020008-6
spectra. The double bond chain of UFA increased the proton proportions, the percentage of linolenate reduced, and
SFA was higher than UFA. The change in multiplicity at 2.29–2.33 indicates that the FFA is starting to form. 13C
NMR shows a reduced carbon chain, and HSQC shows the TAG group interacts strongly with the protons of the
acyl and methyl groups. PFG NMR result showed that the distribution coefficient decreased, while TD NMR result
showed that T2 of the oil decreased. This indicates that a long period of heating increases the mass and viscosity of
the oil. We suggest reusing the soybean oil for cooking a maximum of three times and not mixed with waste oil.

ABBREVIATION
NMR (Nuclear Magnetic Resonance), 1H NMR (proton NMR), PFG NMR (Pulse Field Gradient NMR), TD
NMR (time-domain NMR), 13C NMR (carbon NMR), DEPT 135 (Distortionless enhancement by polarization
transfer), HSQC (Heteronuclear Single-Quantum Correlation spectroscopy), DOSY (Diffusion-ordered
spectroscopy), SFA (saturated fatty acid), UFA (unsaturated fatty acid), PUFA (polyunsaturated fatty acid), FFA
(free fatty acid), TAG (triacylglycerol), FAME (fatty acid methyl esters).

Downloaded from http://pubs.aip.org/aip/acp/article-pdf/doi/10.1063/5.0111708/17354684/020008_1_5.0111708.pdf

ACKNOWLEDGMENT
The authors would like to thank the support of the Department of Applied Chemistry, Chaoyang University of
Technology, and the partial funding by the Ministry of Education, Taiwan.

REFERENCES
1. M. Messina, G. Shearer, and K. Petersen, “Soybean oil lowers circulating cholesterol levels and coronary heart
disease risk, and has no effect on markers of inflammation and oxidation,” Nutrition, May 2021, pp. 111343,
2. Z. Liefeng et al., “Improving the stability of insulin in solutions containing intestinal proteases in vitro,” Int. J.
Mol. Sci., vol. 9, no. 12, 2008, pp. 2376–2387.
3. A. Alvis, C. Vélez, M. Rada-Mendoza, M. Villamiel, and H. S. Villada, “Heat transfer coefficient during deep-
fat frying,” Food Control, vol. 20, no. 4, April. 2009. pp. 321–325.
4. N. Bellaloui, K. N. Reddy, and A. Mengistu, “Drought and Heat Stress Effects on Soybean Fatty Acid
Composition and Oil Stability,” Process. Impact Act. Components Food, January. 2015, pp. 377–384.
5. H. K. Woodfield and J. L. Harwood, “Oilseed Crops: Linseed, Rapeseed, Soybean, and Sunflower,” in
Encyclopedia of Applied Plant Sciences, 2016, pp. 34–38.
6. H. Ako, N. Kong, and A. Brown, “Fatty acid profiles of kukui nut oils over time and from different sources,”
Ind. Crops Prod., vol. 22, no. 2, 2005, pp. 169–174.
7. J. K. Satyarthi, D. Srinivas, and P. Ratnasamy, “Estimation of free fatty acid content in oils, fats, and biodiesel
by 1H NMR spectroscopy,” Energy and Fuels, vol. 23, no. 4, 2009, pp. 2273–2277.
8. S. Naz, M. A. Shabbir, M. R. Khan, and M. Shahid, “Comparison of flaxseed oil characteristics of three
Pakistani varieties obtained by supercritical CO2 and two conventional extraction methods,” Int. Food Res. J.,
vol. 26, no. 5, 2019, pp. 1599–1607.
9. M. Pignitter and V. Somoza, “Critical Evaluation of Methods for the Measurement of Oxidative Rancidity in
Vegetable Oils,” vol. 20, no. 4, 2012, pp. 772–777.
10. Q. Zhang, A. S. M. M. Saleh, J. Chen, and Q. Shen, “Chemical alterations taken place during deep-fat frying
based on certain reaction products: A review,” Chem. Phys. Lipids, vol. 165, no. 662, 2012, pp. 681.
11. A. Le Gresley, G. Broadberry, C. Robertson, J. M. R. Peron, J. Robinson, and S. O’Leary, “Application of pure
shift and diffusion NMR for the characterization of crude and processed pyrolysis oil,” J. Anal. Appl. Pyrolysis,
vol. 140, June 2019, pp. 281–289.
12. A. J. Ali Alsaad, A. B. Altemimi, S. N. Aziz, and N. Lakhssassi, “Extraction and Identification of Cactus
Opuntia dillenii Seed Oil and its Added Value for Human Health Benefits,” Pharmacogn. J., vol. 11, no. 3,
2019, pp. 579–587.
13. D. F. Buck, “Antioxidants in soya oil,” J. Am. Oil Chem. Soc., vol. 58, no. 3,1981, pp. 275–278.
14. C. Skiera, P. Steliopoulos, T. Kuballa, U. Holzgrabe, and B. Diehl, “Determination of free fatty acids in edible
oils by 1H NMR spectroscopy,” Lipid Technol., vol. 24, no. 12, 2012, pp. 279–281
15. R. M. Alonso-Salces et al., “1H–NMR fingerprinting and supervised pattern recognition to evaluate the
stability of virgin olive oil during storage,” Food Control, vol. 123, no. 107831, May 2021, pp. 1–13.

020008-7
16. A. L. Mihai, M. Negoi‫܊‬ă, A. C. Adascălului, V. Ionescu, and N. Belc, “Evaluation of Fatty Acids Composition
of Some Food Samples by Using GC-MS and NMR Techniques,” “Agriculture Life, Life Agric. Conf. Proc.,
vol. 1, no. 1, 2018, pp. 548–554.
17. M. E. Di Pietro, A. Mannu, and A. Mele, “NMR determination of free fatty acids in vegetable oils,” Processes,
vol. 8, no. 4, 2020, pp. 410.
18. R. Garcia, A. Pires, N. Martins, T. Carvalho, A. J. Burke, and M. J. Cabrita, “Assessment of the triacylglycerol
fraction of olive oil by 1D-NMR spectroscopy: exploring the usefulness of DEPT tool on the peak assignments
of 13C NMR spectra,” Eur. Food Res. Technol., vol. 245, no. 11, 2019, pp. 2479–2488.
19. N. Hao, H. Ben, C. G. Yoo, S. Adhikari, and A. J. Ragauskas, “Review of NMR Characterization of Pyrolysis
Oils,” Energy and Fuels, vol. 30, no. 9, 2016, pp. 6863–6880.
20. G. S. Kapur and S. Berger, “Pulsed-field gradient (PFG) NMR spectroscopy: An effective tool for the analysis
of mixtures of lubricating oil components,” Tribo Test, vol. 6, no. 4, 2000, pp. 323–336.
21. D. S. Marques, G. Sørland, S. Less, and R. Vilagines, “The application of pulse field gradient (PFG) NMR
methods to characterize the efficiency of separation of water-in-crude oil emulsions,” J. Colloid Interface Sci.,
vol. 512, February 2018., pp. 361–368,

Downloaded from http://pubs.aip.org/aip/acp/article-pdf/doi/10.1063/5.0111708/17354684/020008_1_5.0111708.pdf

22. G. Pagès, V. Gilard, R. Martino, and M. Malet-Martino, “Pulsed-field gradient nuclear magnetic resonance
measurements (PFG NMR) for diffusion ordered spectroscopy (DOSY) mapping,” Analyst, vol. 142, no. 20,
2017, pp. 3771–3796.
23. D. A. Cunha, L. F. Montes, E. V. R. Castro, and L. L. Barbosa, “NMR in the time domain: A new
methodology to detect adulteration of diesel oil with kerosene,” Fuel, vol. 166, February 2016, pp. 79–85,
24. D. A. Cunha, Á. C. Neto, L. A. Colnago, E. V. R. Castro, and L. L. Barbosa, “Application of time-domain
NMR as a methodology to quantify adulteration of diesel fuel with soybean oil and frying oil,” Fuel, vol. 252,
2019, pp. 567–573.
25. P. M. Santos, F. V. C. Kock, M. S. Santos, C. M. S. Lobo, A. S. Carvalho, and L. A. Colnago, “Non-invasive
detection of adulterated olive oil in full bottles using time-domain NMR relaxometry,” J. Braz. Chem. Soc., vol.
28, no. 2, 2017, pp. 385–390.
26. J. Salimon, B. M. Abdullah, and N. Salih, “Hydrolysis optimization and characterization study of preparing
fatty acids from Jatropha curcas seed oil,” Chem. Cent. J., vol. 5, no. 1, 2011, pp. 67.
27. S. O. Serna-Saldivar and C. Chuck-Hernandez, Food uses of lime-cooked corn with an emphasis in tortillas
and snacks, 3rd ed., no. 2018, Elsevier Inc., pp 469-500
28. A. Biswas, A. Adhvaryu, D. G. Stevenson, B. K. Sharma, J. L. Willet, and S. Z. Erhan, “Microwave irradiation
effects on the structure, viscosity, thermal properties and lubricity of soybean oil,” Ind. Crops Prod., vol. 25,
no. 1, January 2007, pp. 1–7,
29. P. Li et al., “Identification of polyunsaturated triacylglycerols and C=C location isomers in sacha inchi oil by
photochemical reaction mass spectrometry combined with nuclear magnetic resonance spectroscopy,” Food
Chem., vol. 307, March 2020, pp. 125568,
30. E. Alexandri, R. Ahmed, H. Siddiqui, M. I. Choudhary, C. G. Tsiafoulis, and I. P. Gerothanassis, “High-
resolution NMR spectroscopy as a structural and analytical tool for unsaturated lipids in solution,” Molecules,
vol. 22, no. 10, 2017, pp. 1–71.
31. M. D. Guillén and A. Ruiz, “Rapid simultaneous determination by proton NMR of unsaturation and
composition of acyl groups in vegetable oils,” Eur. J. Lipid Sci. Technol., vol. 105, no. 11, 2003, pp. 688–696.
32. A. Li, Y. Ha, F. Wang, W. Li, and Q. Li, “Determination of Thermally Induced trans-Fatty Acids in Soybean
Oil by Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy and Gas Chromatography
Analysis,” Agric. Food Chem., vol. 60, no. October 2012, pp. 10709í10713, 2012.
33. F. Hashempour-Baltork, M. Torbati, S. Azadmard-Damirchi, and G. P. Savage, “Vegetable oil blending: A
review of physicochemical, nutritional and health effects,” Trends in Food Science and Technology, vol. 57.
Elsevier Ltd, November 2016, pp. 52–58, 01-.
34. R. Dand, “Cocoa bean processing and the manufacture of chocolate,” in The International Cocoa Trade,
Elsevier, 2011, pp. 268–289.
35. M. Kanai, T. Yamada, M. Hayashi, S. Mano, and M. Nishimura, “Soybean (Glycine max L.) triacylglycerol
lipase GmSDP1 regulates the quality and quantity of seed oil,” Sci. Rep., vol. 9, no. 1, 2019, pp. 1–10
36. Y. Chen et al., “The analysis of trans fatty acid profiles in deep frying palm oil and chicken fillets with an
improved gas chromatography method,” Food Control, vol. 44, October 2014, pp. 191–197,
37. S. Yanti, D. S. Saputri, H.-Y. Lin, Y.-C. Chou, D. C. Agrawal, and W.-J. Chien, “Fatty Acid Evaluation of
Seeds and Nuts by Spectroscopy and Chromatography,” Food Sci. Technol., vol. 9, no. 3, 2021.

020008-8
38. M. D. Gullén and A. Ruiz, “Monitoring of heat-induced degradation of edible oils by proton NMR,” Eur. J.
Lipid Sci. Technol., vol. 110, no. 1. 2008, pp. 52–60.
39. K. Suri, B. Singh, A. Kaur, M. P. Yadav, and N. Singh, “Impact of infrared and dry air roasting on the
oxidative stability, fatty acid composition, Maillard reaction products and other chemical properties of black
cumin (Nigella sativa L.) seed oil,” Food Chem., vol. 295, April 2019, pp. 537–547.
40. N. A. Chira, A. Nicolescu, R. Stan, and S. Rosca, “Fatty acid composition of vegetable oils determined from
13
C-NMR spectra,” Rev. Chim., vol. 67, no. 7, 2016. pp. 1257–1263.
41. D. Palu, A. Bighelli, J. Casanova, and M. Paoli, “Identification and Quantitation of Ursolic and Oleanolic
Acids in Ilex aquifolium L. Leaf Extracts Using 13 C and 1 H-NMR Spectroscopy,” molecules, vol. 24, no.
December 2019, pp. 4413.
42. M. F. Díaz and J. A. Gavín, “Characterization by NMR of Ozonized Methyl Linoleate,” J. Braz. Chem. Soc,
vol.18, no. 3 2007. pp 513-518
43. G. C. Díaz, R. S. Perez, N. de la C. O. Tapanes, D. A. G. Aranda, and A. A. Arceo, “Hydrolysis -
Hydrogenation of soybean oil and tallow,” Nat. Sci., vol. 03, no. 07, 2011, pp. 530–534.
44. U. A. Jenie, L. B. S. Kardono, M. Hanafi, R. J. Rumampuk, and A. Darmawan, Teknik Modern Spektroskopi

Downloaded from http://pubs.aip.org/aip/acp/article-pdf/doi/10.1063/5.0111708/17354684/020008_1_5.0111708.pdf

NMR: Teori dan Aplikasi dalam Elusidasi Struktur Molekul Organik. Jakarta, Indonesia: Lembaga Ilmu
Pengetahuan Indonesia (LIPI), Pusat Penelitian Kimia, 2014.
45. P. P. Lankhorst and A. N. Chang, “The application of NMR in compositional and quantitative analysis of oils
and lipids,” Mod. Magn. Reson., 2018, pp. 1743–1764.
46. D. Yang et al., “Comparative analysis of the effects of novel electric field frying and conventional frying on
the quality of frying oil and oil absorption of fried shrimps,” Food Control, vol. 128, October 2021.
47. G. Wu et al., “Effect of the phenolic extract of Camellia oleifera seed cake on the oxidation process of soybean
oil by 1H nuclear magnetic resonance during frying,” Lwt, vol. 150, no. June, October. 2021, pp. 111900,
48. N. A. Gomez, R. Abonia, H. Cadavid, and I. H. Vargas, “Chemical and Spectroscopic Characterization of a
Vegetable Oil used as Dielectric Coolant in Distribution Transformers Neffer,” J. Braz. Chem. Soc., vol. 22, no.
12, 2011, pp. 2292–2303.
49. C. A. Mullen, G. D. Strahan, and A. A. Boateng, “Characterization of Biomass Pyrolysis Oils by Diffusion
Ordered NMR Spectroscopy,” ACS Sustain. Chem. Eng., vol. 7, no. 24, 2019, pp. 19951–19960.
50. S. M. Patil et al., “A Simple and Noninvasive DOSY NMR Method for Droplet Size Measurement of Intact
Oil-In-Water Emulsion Drug Products,” J. Pharm. Sci., vol. 108, no. 2, 2019, pp. 815–820.
51. S. Seth, Y. C. Agrawal, P. K. Ghosh, and D. S. Jayas, “Effect of moisture content on the quality of soybean oil
and meal extracted by isopropyl alcohol and hexane,” Food Bioprocess Technol., vol. 3, no. 1, 2010, pp. 121–
127.
52. G. Singh, A. K. Singh, and P. Singh, “Effect of Moisture Content on the Mechanical Oil Extraction of Canola
Seeds,” Int. J. Curr. Microbiol. Appl. Sci., vol. 8, no. 03, 2019, pp. 965–977.

020008-9