Scheme 1 LSO omega-3 PUFA-rich thermal aging process
Numerous chemical and physical analytical methods have been developed to
asses lipid oxidation such as conjugated diene value, peroxide value
(PV), alcohols, epoxides, p -anisidine assay, HBR titration,
iodometric titration, xynol orange, total polar components (TPC), high
performance liquid chromatography (HPLC), fatty acid composition
determined by gas chromatography-mass spectrometry (GC-MS), Fourier
transformation infrared spectroscopy (FTIR), volatile product
determination by gas chromatography, dimer/polymers by size exclusion
chromatography (SEC), and electron spin resonance (ESR) (Jacobsen, 2015;
Hwang et al., 2017, Velasco et al., 2005). There is however a lack of
consistency in many of the results, because most of these analytical
methods are designed to detect one type of oxidation product while lipid
oxidation is a very complicated process producing numerous products at
different times of oxidation. Hence, as suggested in Hwang et al. (2017)
comprehensive review, the development of methods that combine the
concomitant detection of different types of oxidation products is
necessary for the consistent assessment of lipid oxidation. Two
important questions were raised by this researcher: which oxidation
product best represents a given stage of the lipid’s oxidation? And
which analytical method should be used? In this respect, as described
below LF-1H-NMR spectroscopy technology has
significant potential, as shown in this paper, in elucidating molecular
structures of oxidation products from lipids and in revealing the
mechanisms of lipid oxidation.
The field of 1H LF-NMR energy time relaxometry is a
powerful tool for identifying molecular species and to study their
dynamics even in complex materials (Berman et al., 2013a; 2015; Wiesman
et al., 2018; Resende et al., 2019ab; Rudszuck et al., 2019). This
relates to the measurement of energy time relaxation values as a
consequence of interactions among nuclear spins and between spins and
their surroundings (matrix). Longitudinal magnetization returns to
equilibrium following application of a radio frequency pulse because of
energy transferred to the lattice (spin-matrix interactions), and
transverse relaxation arises from spin-spin interactions following a 90°
pulse. The time constants for longitudinal and transverse energy
relaxations are T 1 and T 2 respectively
(Berman et al., 2013b). Relaxation time distribution experiments range
from simple and rapid one dimensional (1D) tests (T1 or T2) to more
complicated multidimensional ones (e.g., T1 vs.
T2). 1D tests use constant intervals between pulses,
allowing for either longitudinal or transverse relaxation to be
evaluated, whereas in multidimensional experiments, the signal is
measured as a function of two or more independent variables, allowing
the spin system to evolve under different relaxation mechanisms (Song et
al., 2002, Berman 2013b). By assuming a continuous distribution of
exponentials, a relaxation time distribution of exponential coefficients
is achieved with components appearing as peaks. This is an ill-posed
Inverse Laplace Transform (ILT) problem. The common mathematical
solution implemented today, for both 1D and 2D data, is based
on L2 -norm regularization
(Song
et al., 2002; Graham, et al., 1996; Berman et al., 2013b, Campisi-Pinto
et al., 2018).
Current technologies are not effective in characterizing the
morphological and chemical structural domains of saturated,
monounsaturated fatty acids (MUFA) and PUFA materials, or how the
morphological structures of fatty acids, at the meso, nano, and
molecular levels, affect their oxidation mechanisms.1H LF-NMR energy relaxation time technology consisting
of L1/L2 norm regularization (Campisi et al. 2018, 2019; Resende
et al., 2019a,b), is proposed as a tool to analyze PUFA oils undergoing
thermal oxidation. This technology can generate two‐dimensional (2D)
chemical and morphological spectra using a recently modified and
developed primal‐dual interior method for the convex objectives (PDCO)
optimization solver for computational processing of the energy
relaxation time signals T1 (spin–lattice)
and T2 (spin–spin): With carefully chosen
reconstruction parameters, the data signals can be reconstructed into 2D
graphics of the different energy relaxation times assigned to the
mobility of different chemical structures, and their adjacent
environments (Wiesman et
al., 2018).
This reconstruction of LF‐NMR signals into two and three dimensional (2D
and 3D) T1 vs. T2 graphs is able to
effectively characterize the chemical and morphological domains of
complex materials (Wiesman et
al., 2018).
The 2D graphical maps
of T1 vs. T2 generated for butter,
rapeseed oil, soybean oil, and linseed oil show that the different
degrees of unsaturation of fatty‐acid oils affect their chemical and
morphological domains, which influences their oxidative susceptibility
(Resende et al., 2019a). The technology of the 1H
LF‐NMR energy relaxation time proved to be an effective tool to
characterize and monitor PUFA oxidation (Resende et al., 2019a). The use
of 2D graphic reconstruction
of T1 vs. T2 as compared to only one
dimensional (1D) has the ability to increase peak separation on the
diagonal (T1 = T2) and when new
polymerized oxidation products new peaks appear below the diagonal (same
constant T1 but decreased T2) during
later stages of the oxidation process (Resende et al., 2019a,b).
Methods using high field 1H NMR relaxation were found
by Sun and Moreira (1996), Hein et al (1998), Sun et al. (2011) and
Bakota et al (2012) to correlate well with various parameters associated
with lipid oxidation (e.g., free fatty acid; polar materials in heated
oils; solid fat content (SFC approved as AOCS Cd 16b-93)). It was
proposed by Hwang et al. (2017) that ”there are molecular structure and
composition changes in oil during oil oxidation and degradation process
affecting the chemical environment surrounding the protons. Thus the
proton mobility affecting the NMR energy relaxation time values changes
as oil degrades”.
High field 1H NMR was also used to analyze aldehydes
produced in various heated oils (Guillen and Uriarte, 2009) .
These researchers reported on the ability to analyze a list of aldehyde
products in linseed oil heated at 190oC for 20 h, and
also determined acyl groups’ iodine value and polar compounds. Merkx et
al. 2018 reported a broad band selective 1H NMR method
for determination of both hydroperoxides and aldehydes in oxidized oils.
Furthermore, based on electron spin resonance (ESR) system, combined
with free radical standard and trapping agents (TEMPO and PBN) was
released for determination of peroxides in the early fast initiation
phase of oil oxidation (Velasco et al., 2005). Blumich (2016) developed
compact 1H LF -NMR systems and Guilleux et al. (2016)
developed an automate LF-NMR system. However, one of the remaining
problems of 1H LF -NMR and especially 2D
T1-T2 systems is the relatively long
experimental and data processing time required to finalize the results.
Therefore these systems are not yet suitable for high throughput
applications such as real-time reaction monitoring or rapid screening of
oil oxidation (Hwang et al., 2017).
In the present study the objective is to develop a non-sample modifying1H LF-NMR energy time relaxation sensitive application
supported with other techniques (e.g., HF NMR, GC-MS and viscosity) to
evaluate LSO oxidative aging processes, based on monitoring the main
chemical and structural changes occurring during thermal oxidative
reactions. As for example by following the alkyl tails
T2 values it is possible to correlate the degree of
olefin functionality and the ratio of the olefin components with the
different degrees of functionality that control the onset of
crosslinking polymerization and gel structure formation. We demonstrate
below the capability of using the rapid 1H
T2 energy relaxation time technology to monitor LSO
molecular segments mobility. In particular the monitoring of the LSO
aliphatic tail’s relaxation was used to follow the chemical and
structural changes in all the autoxidation aging phase; starting from
the initiation phase (abstraction of hydrogen, fatty acid chain
rearrangement and oxygen uptake yielding of hydroperoxides production),
the propagation phase (chain reactions resulting in tail cleavage to
form alkoxy radicals, and alpha, beta-unsaturated aldehydes formation),
and the termination phase (cross linking formation of polymerized end
products).