# Raman Spectroscopy Analysis of Free Fatty Acid in Olive Oil
## 3 Results and Discussion
These observations indicate that when FFA content increases in the mixture, FFA would have more significant degradation of carotenoid, whose characteristic vibrational modes are 1155 cm\({}^{-1}\) and 1525 cm\({}^{-1}\). For a clear representation of the intensity decrease of the two vibrational modes at 1155 cm\({}^{-1}\) and 1525 cm\({}^{-1}\) in the mixture, an intensity decrease factor \(x\) is introduced as follows. When samples C and F are mixed, for example with a volume ratio of 1:1, we assume that the intensities of 1155 cm\({}^{-1}\) and 1525 cm\({}^{-1}\) modes in the mixture would be \(x\)/2 (\(x\) should be smaller than 1) of their intensities in sample C, and the intensities of all the other modes in samples C and F would be half of their intensities. Then in the mixture (M), the relative intensity ratio of I\({}_{\text{M},1440}\)/I\({}_{\text{M},1525}\) and I\({}_{\text{M},1655}\)/I\({}_{\text{M},1525}\) can be estimated by (I\({}_{\text{C},1440}\)/_x\({}_{\text{C},1525}\)_ + I\({}_{\text{F},1440}\)/_x\({}_{\text{F},1525}\)) and (I\({}_{\text{C},1655}\)/_x\({}_{\text{C},1525}\)_ + I\({}_{\text{F},1655}\)/_x\({}_{\text{F},1525}\)), respectively. Therefore, through the analysis of relative intensity ratios, the intensity decrease factor \(x\) of 1525 cm\({}^{-1}\) mode can be calculated with the ratio of \(\{(\)I\({}_{\text{C},1440}\)/I\({}_{\text{C},1525}\)\()\) - (I\({}_{\text{C},1655}\)/I\({}_{\text{C},1525}\))-(I\({}_{\text{F},1440}\)/I\({}_{\text{F},1655}\)) to \(\{(\)I\({}_{\text{M},1440}\)/I\({}_{\text{M},1525}\)) - (I\({}_{\text{M},1655}\)/I\({}_{\text{M},1525}\))-(I\({}_{\text{F},1440}\)/I\({}_{\text{F},1655}\))]. The calculated values for intensity decrease factor \(x\) of 1525 cm\({}^{-1}\) mode are presented in Table 1. Similarly, the intensity decrease factor \(x\) of 1155 cm\({}^{-1}\) mode can be calculated; the values are also included in Table 1. The intensity decrease factor for 1155 cm\({}^{-1}\) and 1525 cm\({}^{-1}\) modes should be same, the small difference between the two \(x\) values would be mainly due to analysis error from measuring the intensities of the vibrational modes. The mode at 1525 cm\({}^{-1}\) is less affected by its surrounding peaks than that of 1155 cm\({}^{-1}\), thus the intensity decrease factor \(x\) of 1525 cm\({}^{-1}\) mode would be more accurate than that of 1155 cm\({}^{-1}\). As can be seen in Table 1, the intensity decrease factor  
Figure 2: Raman spectra of the mixtures of sample C and F with volume ratios of 2:1, 3:2, 1:1, 2:3, and 1:2. For comparison, the spectra of sample C and F are included in the figure. The intensities of all the spectra are normalized using the intensity of the vibrational mode at 1655 cm\({}^{-1}\) as a reference. Each spectrum is y-shifted for clarity. \(x\) is indeed smaller than 1 and it decreases systematically with increasing FFA content in the mixture. This further confirms the FFA degradation of carotenoid in olive oil. The above results show that relative Raman intensity analysis is very helpful for investigating FFA content in extra virgin olive oil. The analysis confirmed that the two vibrational modes at 1155 cm\({}^{-1}\) and 1525 cm\({}^{-1}\) are characteristic Raman modes for analyzing FFA content in extra virgin olive oil, and their intensities decrease systematically with increasing FFA content in extra virgin olive oil. This is consistent with the reported result that the spectral region of 945-1600 cm\({}^{-1}\), which includes the carotenoid Raman modes, is important for predicting the FFA content in olive oil [6]. In the study, absolute Raman intensities of various FFA content extra virgin olive oil samples were analyzed, and it was found that the absolute Raman intensity of the spectral window 945-1600 cm\({}^{-1}\) increases systematically with decreasing FFA content in extra virgin olive oils.