*3.9. FTIR-ATR Spectroscopy of Pollen*

The FTIR-ATR spectra of 24 pollen samples, recorded in absorbance mode in the mid-infrared region, is presented in Figure 1. The broad band around 3290 cm–1 that was observed in all pollen samples corresponded to O–H stretching vibration due to the presence of water [52,53]. As it was previously reported that the moisture content of fresh pollen varies between 21 and 30% [9], the presence of a broad band in this spectral region was expected. Between 3000 and 2850 cm–1, two peaks were identified in all the samples analyzed: a peak around 2920 cm–1 and one around 2850 cm–1, both assigned to C–H stretching, and mainly CH2 and CH3 vibrations of lipids, proteins and carbohydrates [52]. For one pollen sample (sample 4) a small peak was found at 2360 cm–1 and was also attributed to C–H stretching vibrations of lipids [54]. Furthermore, another signal corresponding to lipids, and namely the peak at 1740 cm–1, which is characteristic of stretching vibrations of C=O groups, was prominent in some pollen samples; previous research found that this signal shows large variation within pollen samples of related plant species [55]. In the spectra of all pollen samples, peaks were observed at 1650 cm–1 and 1540 cm–1 that were attributed to stretching vibrations of amide I and II [56]. Characteristic of all samples was also the peak at 1414 cm–1 that was assigned to asymmetric in-plane bending of the –CH3 group [53]. The peak in the region between 1350 and 1200 cm–1 was assigned to amide III, and more precisely an in-phase combination of N–H deformation vibrations and C–N stretching vibrations [56]. All samples had high absorption peaks around 1030 cm–1 that corresponded to stretching vibrations of saccharides and proteins, and were also reported for crude pollen and defatted pollen samples in our previous study on the extraction of polyphenols from crude pollen [17]. In the spectral range between 1200 and 500 cm–1, which is considered the fingerprint region of pollen, the peaks observed for the analyzed samples were due to C–O and C–C stretching vibrations, and their variation among pollen samples indicated differences in the saccharide, protein and lipid composition. This overlapped the region of 1500–800 cm–1, where characteristic signals were attributed to C–O and C–C stretching vibrations of flavonoids and phenolic compounds [57].

**Figure 1.** Bee pollen FT-IR spectra in the region 4000–650 cm<sup>−</sup>1.
