# _Sponias mombin_ Seed Oil Compounds Identification by Raman Spectroscopy and NMR
## 3 Results and Discussion
In each batch of fruit seed oil, the frequencies associated with carotenoids were observed (Lutein, \(\alpha\)-carotene and \(\beta\)-carotene) at 1006, 1158 and 1523 cm\({}^{-1}\)[30, 40, 41]. NMR is a powerful and widely analytical method used in structure elucidation protocols. NMR spectroscopy constitutes an accurate, sensitive method to supplement chemical information for structural identification. Here, we obtained the 500 MHz \({}^{1}\)H-NMR spectra of the crude oil, showing a characteristic signal pattern found in other seed oils [42] (Table 2). The olefinic protons for all unsaturated fatty acids appeared at $ 5.30-5.41 ppm. In the low-field region of the spectra, a multiplet at $ 5.25-5.29 ppm was assigned to the proton attached to C-2 of the glycerol. The chemical shifts and multiplicities of coupling constants for the signals at $ 4.14-4.19 ppm (\(J_{1}\) = 9 Hz, \(J_{2}\) = 6 Hz, dd) and $ 4.29-4.33 ppm (\(J_{1}\) = 9 Hz, J\({}_{2}\) = 3 Hz, dd) are characteristic for H-1 and H-3 protons of glycerol system. Bis-allylic and allylic proton signals appeared as a triplet at $ 2.77-2.80 ppm (\(J\) = 3 Hz) and a complex multiplet at $ 2.00-2.09 ppm. A triplet at $ 2.31-2.37 ppm (\(J\) = 6.0 Hz) was designated to methylene H-2 protons adjacent to the acyl group, a multiplet at $ 1.59-1.66 ppm was assigned to H-3, while the other methylene protons appeared between $ 1.25 and 1.41 ppm. The terminal methyl protons appeared at $ 0.87-0.92 ppm. The downfield-shifted proton signal at $ 6.25 indicated the presence of linoleic acid [43]. The relative % of _SFA__PUFA_ present in the sample was calculated following the method described by [PERSON] et al. (Figure 3) [25]. The chemical shift differences between some fatty acids depend on substitution on glycerol backbone. The \({}^{13}\)C NMR spectra (Table 3) showed signals at \(\delta\) 173.25 and 173.21 ppm assigned to carbonyl carbons attached to 1,3-glycerol positions, and a signal at \(\delta\) 172.79 ppm corresponding to a fatty acid esterified at position two of glycerol, while no signal was observed for free fatty acids (\(\delta\) 177.0-182.0 ppm). The C-1 and C-3 terminal carbons for the triglyceride moiety were found at the same chemical shift, 62.09 ppm, whereas signal for C-2 was present at 68.91 ppm. The olefinic carbons appeared in the range from \(\delta\)127.88 to 130.20 ppm. For most components, such as oleic acid, \(\alpha\) and \(\beta\) carbon signals could be distinguished, such as C-10 at \(\delta\)\(\alpha\) = 129.98 and \(\delta\)\(\beta\) = 129.99 ppm. As expected, most of the signals were observed at the low field region (\(\delta\) 14.02-34.17 ppm), corresponding to methyl, methylene and allylic carbons. All of the above NMR signals were in agreement with the structure of a triacylglycerol bearing unsaturated fatty acid chains. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Chemical Shift (ppm)** & **Proton** & **Functional Group** \\ \hline
0.87–0.92 & –CH\({}_{3}\) & Terminal methyl protons of saturated and unsaturated chains \\
1.25–1.41 & CH\({}_{2}\) & Protons of methylene envelop \\
1.59–1.66 & CH\({}_{2}\)–CH\({}_{2}\)–COO & H-3 protons of acyl moieties in \\
2.00–2.09 & CH\({}_{2}\)–CH = CH & Alyllic methylenes \\
2.31–2.37 & CH\({}_{2}\)–COO & H-2 protons of acyl moieties in \\
2.77–2.80 & C = C–CH\({}_{2}\)–C = C & Protons attached to bis allylic carbon \\
4.14–4.19 & CH\({}_{2}\)O & H-1 and H-3 protons of glycerol \\
4.29–4.33 & CH\({}_{2}\)O & H-2 of the glycerol backbone \\
5.25–5.29 & CHO (\(\beta\)) & H-2 of the glycerol backbone \\
5.30–5.41 & CH\({}_{2}\)–CH & Olefinic protons of unsaturated Fatty acids \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Chemical shifts and assignments of the characteristic resonances in the \({}^{1}\)H-NMR spectrum of crude seed oil. Figure 3: Relative % of saturated (SFA), monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA) as determined using \({}^{1}\)H-NMR.