## 3 Results
### FTIR-ATR Spectral Profiling

Table 5: _Cont._The spectral region between 1800 and 700 cm\({}^{-1}\) (fingerprint region) is populated by a series of absorption bands that reflect unique spectral pattern of bee pollen. A weak absorption band peaking at 1740 cm\({}^{-1}\) occurs primarily due to the carbonyl group C=O stretching vibrations of the of the ester bond [39] in lipid-based bee pollen constituents. A medium intense absorption band observed at 1645 cm\({}^{-1}\) is characterized by overlapping effects of several vibrations related to lipids, proteins and water. The lipids are represented by absorption due to COO- and C=C stretching given that COO- asymmetric stretching (RCOO-group) typically absorb between 1610 and1550 cm\({}^{-1}\), and C=C stretching between 1680 and1600 cm\({}^{-1}\)[39]. This band position can also be assigned to amide I, an intense absorption band in proteins (\(\beta\)-sheet) primarily governed by the stretching vibrations of the C=O (70-85%) and C-N groups (10-20%) that can be attributed to the protein fraction of bee pollen. Absorption at 1645 cm\({}^{-1}\) also belongs to well-known molecular vibrations of water, H-O-H deformation vibration, namely. The band at 1645 cm\({}^{-1}\) is followed by the lower intensity analyte signals occurring at 1545 and 1515 cm\({}^{-1}\) that are attributed to C=C stretchings of aromatic structures, primarily lipid-based. It can be assumed that protein-related amide II band that comprises N-H bending and C-N stretching vibrations and typically absorb between 1510 and 1580 cm\({}^{-1}\), is overlapped by the former C=C stretchings. The spectral region between 1490 and 1190 cm\({}^{-1}\) is populated by a series of low intensity analyte signals primarily attributed to proteins and sugars. The absorption bands peaking at 1413, 1370 and 1340 cm\({}^{-1}\) can be linked to the protein side chain COO- stretching vibrations, while the absorption at 1240 cm\({}^{-1}\) occurs due to the amide III band, which comprises 30% of C-N stretching, 30% of N-H bending, 10% of C-O stretching, and 10% of O=C-N bending vibrations (the rest belongs to other vibrations) [39]. In this spectral region, mentioned protein-related absorptions overlap less intensive sugar vibrations: weak bands peaking at 1413 and 1340 cm\({}^{-1}\) that are assigned to C-O-H deformation of  
Figure 1: Characteristic FTIR-ATR spectrum of bee pollen with the assignation of major underlying molecular vibrations. glucose and fructose, respectively, while the band at 1240 cm\({}^{-1}\) occurs due to CH\({}_{2}\) bending of glucose [41; 42; 43]. The most prominent absorption envelope observed in the FTIR-ATR spectrum of bee pollen occurs between 1170 and 950 cm\({}^{-1}\), and it is followed by less intensive vibrations occurring between 950 and 750 cm\({}^{-1}\). These spectral regions are also characterized by the overlapping spectral effects covering both sugar and protein related functional group vibrational modes. A weak absorption band observed at 1100 cm\({}^{-1}\) (along with shoulder at 1077 cm\({}^{-1}\)) can be assigned to the stretching vibration of endo C-O bonds of the most predominant sugars in bee pollen, glucose and fructose [41; 43], as well as C-H and N-H in-plane deformation vibrations of aromatic structures related to proteins. The most prominent absorption bands observed in the bee pollen spectrum arising at 1052 and 1032 cm\({}^{-1}\) are attributed to numerous overlapping signals of sugars and proteins, stretching C-O vibrations, peaking at 1052 cm\({}^{-1}\) for fructose, and at 1032 cm\({}^{-1}\) for glucose [43], as well as N-H and C-H deformation vibrations in relation to proteins. Less intensive absorption occurring at 993 cm\({}^{-1}\) is known as the characteristic sucrose bend related to glycosidic linkage [39]. This was additionally confirmed by a comparative analysis of unifloral bee pollen containing high vs. low amount of sucrose presented in the latter Figure 2c. There are five weak absorptions occurring in the spectral range from 950 to 750 cm\({}^{-1}\).