3.2. NMR
Peak assignments of the triglyceride structure of soybean oil in the1H-NMR spectra are shown in Fig. 3a (each proton in a different chemical environment has been numbered from 1-10 in blue), corresponding to: 1) Methyl terminal group, 𝛿 (ppm) 0.8-0.9;2) Methyl terminal group for linolenic acid, 𝛿 (ppm) 0.9-1.0;3) Methylene protons -CH2-CH2 -CH2-, 𝛿 (ppm) 1.2-1.4; 4) Methylene protons –C(=O)-CH2-CH2 -CH2- 𝛿 1.5-1.7; 5) Methylene protons -CH2-CH2 -CH=CH-, 𝛿 1.9-2.1;6) Methylene protons –C(=O)-CH2 -CH2-CH2-𝛿 2.2-2.3; 7) Allylic group= CH-CH2 -CH=, 𝛿 (ppm) 2.7-2.9;8) Glycerol protons n-1 and n-3, doublet: 𝛿 (ppm) 4.0-4.3;9) Glycerol protons n-2, 𝛿 (ppm) 5.2-5.3; 10) Olefinic protons CH2-CH=CH- CH2, 𝛿 (ppm) 5.3-5.4. As can be seen in Fig. 3a, untreated, and treated samples have the same number of peaks with different intensities as well as the partially hydrogenated sample of soybean oil (PHO). This result supports the proposed mechanism shown in Fig. 3 where all the allylic protons with a chemical shift at 2.7-2.9 ppm, have a decreasing tendency concerning treatment time. Soybean oil-treated for 6h showed a reduction of double bonds (peak 10) from 7.4% to 4.7% and an increasing percentage of single bonds (peak 3) from 53.5% to 59.5%. This reduction can be attributed not only to hydrogenation but also to dimerization by the Diels Alder reaction (Koehler, Anseth, & Bowman, 2013; Smith, 2017) between the conjugated double bonds formed after the HVACP treatment with the double bond of another triglyceride’s fatty acid unit. So, each Diels Alder reaction (Smith, 2017) decrease two double bonds. The proposed mechanism for Diels Alder reaction is presented in Fig. 4. Ionescu and Petrovic patented the cationic polymerization of soybean oil (Ionescu & Petrovic, 2009). They have reported that the polymerization initiates with the formation of conjugated double bonds on the hydrocarbon chain of linoleic and linolenic acid units (Ionescu & Petrovic, 2009). Once the conjugated double bonds are formed, the polymerization goes through a pericyclic reaction like Diels-Alder (Aragonès et al., 2016; Ionescu & Petrovic, 2009; Smith, 2017). The results that we have obtained are consistent with the results published by the authors aforementioned. We have proposed a similar polymerization/dimerization reaction with one difference, radical aided conjugated double bond formation, and following Diels-Alder reactions (Fig. 2 and 4) (Smith, 2017).
Moreover, peaks 5 and 7 of the 1H-NMR spectra related to single bonds close to double bonds will reduce if double bonds are modified, as it occurs with treated samples. The reduction of peak integration area for 5 and 7 may correlate with the increased amount of peak 3, because of possible hydrogenated double bonds or Diels-Alder reaction (Smith, 2017) proposed (Fig. 4). The glycerol moiety did not show significant modifications, peaks 8 and 9. Suggesting that treated samples maintain a triglyceride structure.
Figure 4 – about here
Oxidation products include hydroperoxides (chemical shift 𝛿 (ppm) 5.7, 8.3-8.9), aldehydes (chemical shift 𝛿 (ppm) 9.5-9.8), alcohols (chemical shift 𝛿 (ppm) 3.43-3.62), epoxides (chemical shift 𝛿 (ppm) 2.63, 𝛿 (ppm) 2.88-2.90, 𝛿 3.1), ketones (chemical shift 𝛿 (ppm) 6.08, 𝛿 6.82), or conjugated double bonds (chemical shift 𝛿 5.7-6.4) (Gunstone et al., 2007). Treated samples showed an increased peak only for conjugated double bonds, as can be seen in the expanded spectra in figure 3a, corresponding to 𝛿 (ppm) 5.7-6.3. Moreover, spectra from 2D-NMR (HSQC) of the treated sample was used to identify a cross-peaks between J-coupled protons in the spin of the conjugated double bonds and each carbon involved in this network. From this analysis, two points were identified on treated samples: (𝛿 (ppm) 6.2;126.3) and (𝛿 (ppm) 5.9;128.7), as can be seen in figure 3c. These peaks are related to the ’double bonds’ region of the hydrocarbon chain in the13C-NMR spectra. These results suggest that double bonds can migrate to a conjugated arrangement during HVACP treatment (Fig. 2), forming a ring structure that can result in the dimerization of triglycerides (Fig. 4) (Biswas et al., 2007).
The assignments of carbon resonances of the triglyceride molecules of soybean oil are characterized by four main signals: carbonyl carbons (𝛿 (ppm) 170-174), olefinic carbons (𝛿 (ppm) 120-140), glyceride backbone carbons (𝛿(ppm) 60-80), aliphatic and methyl carbons (𝛿 (ppm) 10-40), as shown in Fig. 3b. The carbon spectra for untreated and treated samples (6h, gel fraction) showed the same number of peaks, and it indicates that the primary structure of soybean oil treated with HVACP is maintained as a triglyceride. A shift to the left was observed in the spectra (Fig. 3b), mostly in the olefinic carbons. A shift may indicate changes in the electron density near the carbon that affect the whole molecule, with a higher shielding of the external magnetic field. These changes may be related to the formation of the modified double bond of the dimer.