# Raman Spectroscopy Analysis of Free Fatty Acid in Olive Oil
## 3 Results and Discussion
In the Raman study, the analysis of relative intensity ratios would be easier and more accurate than absolute intensity, since absolute Raman intensity depends on various factors such as alignment of the Raman system, laser power stability, focus of the laser, absorption coefficient of the sample, and so forth. We show below that the study of relative intensity ratio of vibrational modes at 1525 cm\({}^{-1}\) and 1655 cm\({}^{-1}\) presents a good and rapid analysis of FFA content in extra virgin olive oil, which would be helpful for quick quality evaluation of extra virgin olive oil. Figure 3 presents the relative intensity ratios of I\({}_{1525}\)/I\({}_{1655}\) and I\({}_{1155}\)/I\({}_{1655}\) as a function of FFA content in extra virgin olive oil samples; for comparison, the relative intensity ratio of I\({}_{1440}\)/I\({}_{1655}\) is also presented in the figure. Seven different FFA contents from 0.1% to 0.8% are included in Figure 3. Samples A, B, C, D, and E had FFA content of 0.1%, 0.15%, 0.4%, 0.6%, and 0.8%, respectively. Mixtures of C and D, and D and E with volume ratio 1:1 were prepared to obtain FFA content of 0.5% and 0.7%, respectively. Also, a mixture of C and E with volume ratio 1:1 was prepared to obtain FFA content of 0.6%, for comparison with sample D. As can be seen in Figure 3, the relative intensity ratios of I\({}_{1525}\)/I\({}_{1655}\) and I\({}_{1155}\)/I\({}_{1655}\) decrease linearly with increasing FFA contentin extra virgin olive oils. The intensity ratio of I\({}_{1525}\)/I\({}_{1655}\) shows a better linear correlation with FFA content in extra virgin olive oil than that of I\({}_{1155}\)/I\({}_{1655}\). This would be due to the vibrational mode at 1525 cm\({}^{-1}\) being less affected by its surrounding peaks than that of 1155 cm\({}^{-1}\), consistent with the result in Table 1. Therefore, the relative intensity ratio of I\({}_{1525}\)/I\({}_{1655}\) would be a good parameter for rapid analyses of FFA content in extra virgin olive oil. In the above analysis, the mode at 1655 cm\({}^{-1}\) was chosen as a reference for calculation of relative intensity ratios. This would be valid for the Raman study of same type of vegetable oils. For comparison of different types of vegetable oils, the mode at 1440 cm\({}^{-1}\) could be chosen as a reference [30]. In our recent study, the relative intensity ratio of 1655 cm\({}^{-1}\) to 1440 cm\({}^{-1}\) has been systematically investigated in various types of vegetable oils and three different types of pure unsaturated fatty acids (oleic acid, linoleic acid, and \(\alpha\)-linolenic acid) [31]. This study showed that the mode at 1440 cm\({}^{-1}\) can be chosen as a reference for first order differentiation of different types of vegetable oils; and for more accurate analysis of same type of vegetable oils, the mode at 1655 cm\({}^{-1}\) can be chosen as a reference. In addition, a systematic density functional theory (DFT) calculation of the Raman spectra of unsaturated fatty \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline
**Sample** & **C** & **C:F (2:1)** & **C:F (3:2)** & **C:F (1:1)** & **C:F (2:3)** & **C:F (1:2)** & **F** \\ \hline I\({}_{1440}\)/I\({}_{1525}\) & 4.58 & 8.30 & 9.35 & 10.97 & 15.83 & 24.91 & - \\ I\({}_{1440}\)/I\({}_{1655}\) & 1.47 & 1.22 & 1.22 & 1.12 & 1.11 & 1.08 & 0.92 \\ I\({}_{1655}\)/I\({}_{1525}\) & 3.11 & 6.78 & 7.64 & 9.33 & 14.29 & 23.08 & - \\ \(x_{1525}\) & - & 0.84 & 0.75 & 0.73 & 0.65 & 0.48 & - \\ \(x_{1155}\) & - & 0.89 & 0.74 & 0.70 & 0.72 & 0.44 & - \\ \hline \hline \end{tabular}
\end{table} 
Table 1: The experimental relative intensity ratios: I\({}_{1440}\)/I\({}_{1525}\), I\({}_{1440}\)/I\({}_{1655}\), I\({}_{1655}\)/I\({}_{1525}\); and the calculated intensity decrease factors: \(x_{1525}\), \(x_{1155}\). acids up to six carbon-carbon double bonds, is currently underway, which also supports the above method for choosing the reference mode. The DFT result will be presented elsewhere.