## 3 Results and Discussion
### Spectroscopy Analysis
#### 3.4.3 FT-Raman  
While Raman spectroscopy often complements FTIR by providing more detailed vibrational data, the samples in this study gave weaker Raman signals compared to FTIR-ATR due to their dark colour. The FT-Raman spectra obtained from the algae analysed are presented in Figure 8.  
Figure 7: Principal component analysis to discriminate between different samples analysed using FTIR-ATR spectral information (**left** side) and the corresponding loads of spectral information (**right** side) using derivative Savitzky–Golay transformation with 15 smoothing points. GMB—_Gracilaria multipartite_, UB—_Ulva_ sp, SMB—_Sargassum muticum_ from Buarcos, SMSM—_S. muticum_ from San Marcos. The sharp and well-defined band at 1529 cm\({}^{-1}\) is attributed to C=C in the plane band attributed to carotenoids [92; 93] and is particularly abundant in _S. muticum_ from S. Marcos beach, closely followed by _S. muticum_ from Buarcos. This is not surprising given the high content of fucoxanthin in this taxon. The large band at 1156 cm\({}^{-1}\) in Raman is typically associated with C-O-C stretching vibrations of glycosidic bonds in polysaccharides, such as cellulose, pectins, agar, alginate, fucoidan, ulvan, or other phycocolloids. This is corroborated by the small band at 1088 cm\({}^{-1}\), which is a specific polysaccharide signature and, in _G. multipartita_, indicates the presence of agar. Furthermore, the 966 cm\({}^{-1}\) band can also be linked to S=O stretching in sulphated polysaccharides [93; 94]. In FT-RAMAN, the wavenumber of 1608 cm\({}^{-1}\) is again characteristic of C=C stretching vibrations in aromatic rings, which in algae is often associated with phenolic compounds, flavonoids, and aromatic amino acids present in proteins [94]. The wavenumber at 1277 cm\({}^{-1}\) also indicates the C-H bending or C-C stretching of phenolic compounds. The small band at 1009 cm\({}^{-1}\) is often associated with amide linkages and C-N spatial stretching in proteins and peptides. The band at 2924 cm\({}^{-1}\) is commonly associated with the C-H stretching vibrations in aliphatic hydrocarbons, which indicates the presence of methylene (CH\({}_{2}\)) and methyl (CH\({}_{3}\)) groups. The Raman bands at 1340 and 1346 cm\({}^{-1}\) have been assigned to CCH, COH, and OCH deformations in the plane of pyramid rings with CH\({}_{2}\) and CH\({}_{3}\), a contribution of galactose and fucose, respectively [94]. The small bands at 1445 cm\({}^{-1}\), 1388 cm\({}^{-1}\), and 1277 cm\({}^{-1}\) are commonly attributed to the presence of lipids and fatty acids [30]. Figure 9 shows the PCA plot, where the samples of different species and locations are distributed according to two principal components that together explain 99% (97% for PC1 and 2% of PC2) of the total variation. Although PC2 makes a much smaller contribution, _Ulva_ sp., separated by both PC1 and PC2, indicates distinct characteristics compared to the other species. The two _S muticum_ samples show only a slight separation between locations along the PC1, suggesting some degree of chemical similarity between them. Figure 8: FT-Raman spectra of the species of macroalgae samples selected: _Gracilaria multipartita_, _Ulva_ sp., and _Sargassum muticum_ (from Buarcos and San Marcos). Despite the higher values observed for the variation explained by PC1+PC2, the peaks that contribute most to discrimination in the PCA are located at 1529 cm\({}^{-1}\) and 1156 cm\({}^{-1}\) for PC1 and at 1156 cm\({}^{-1}\) and 2924 cm\({}^{-1}\) for PC2 (representing only 2 per cent of the total variance) (Figure 9, right side). This weak contribution from the spectral regions, combined with the low signal strength, indicate that for the same material, FT-Raman spectroscopy appears to be the least effective technique for distinguishing between the algae species studied. In this case, additional pre-treatment may be necessary as long as it does not interfere with the analysis of this matrix or its chemical composition. This will be the aim of future research in this area.