## 3 Results
### Fluorescence Properties of Minor Compounds Present in Honey  
The emission/excitation properties of several fluorescent minor compounds typical of honey are here reported. As observed in Section 3.1, honey samples obtained from sulla and sunflower plants, contain pigments, such as \(\beta\)-carotene, in large amounts. Carotenoids are known to have small fluorescence yield [52] and, due to their large molar extinction coefficient, they are usually responsible for self-absorption and quenching phenomena. For instance, \(\beta\)-carotene has a typical near UV-Vis absorption band centered from 380 to 480 nm (Figure 4a), while a large emission band with two relative maxima at 520 nm and 570 nm is obtained by exciting at wavelengths in the near UV-Vis region (350-420 nm) (Figure 4b). Among other pigments, chlorophylls and their derivatives have very strong fluorescence emission, with a relative sharp peak in the interval 670-690 nm, and despite their concentrations are usually small; almost all honey samples present sharp emission peaks in this region [53]. Among antioxidant phenolic compounds, ellagic acid, which was suggested as a fluorescent marker of Belgian polyfloral honey [54] and also found in very small amounts in some Tuscan honey samples [47], has a strong and characteristic emission band with a maximum at 420 nm (by exciting with \(\lambda_{\text{ex}}\) = 255 or 360 nm; see Figure 5A). As reported in several papers about FFF investigations of honey [23; 24; 35; 36], the typical fluorescence emission of honey samples centered around 340-380 nm (with excitation at \(\lambda_{\text{ex}}\) = 270-290 nm) is partially due to amino acids, such as tryptophan, tyrosine, and phenylalanine. In particular, the emission spectrum of tryptophan, which are the amino acids with higher fluorescence yield, is reported in Figure 5B. Figure 4: (**a**) Absorption spectrum of a solution of \(\beta\)-carotene in n-hexane (C = 0.2 mg/mL). (**b**) A selection of FFF emission and excitation spectra of the same solution of \(\beta\)-carotene. Intensity of emission and excitation spectra are scaled and reported in arbitrary units. Other minor compounds responsible for a fluorescence emission band with a maximum between 320 and 380 nm (with excitation at \(\lambda_{\text{ex}}\) = 270 nm) are the hydroxybenzoic bicida, such as those indicated in Figure 6A. The 4-hydroxybenzoic acid has indeed a maximum at 320 nm, while the vanillic and gallic acids have a maximum at \(\sim\)350 nm, and the syringe acid has a maximum at 360 nm. As discussed in Section 4, these hydroxybenzoic bicida were found in some of the honey samples analyzed in this work [17], and they were suggested as possible markers of some botanic origins. Another class of fluorescent minor compounds present in honey is that of hydrocinnamic phenolic acids. When excited at wavelengths around 270-290 nm, a strong emission band centered between 410 and 440 nm is observed (see Figure 6B). These compounds were found in most of the samples investigated here [17], and their concentrations, in particular those of ferulic, p-coumaric, and caffe acids, change depending on the botanic origin; this was also reported in several papers focusing on characteristic phenolic profiles [14; 16; 17; 19; 55]. Figure 5: (a) FFF emission spectrum (\(\lambda_{\text{ex}}\) = 360 nm) of a solution of ellagic acid in water/methanol (90% v/v) (C = 0.02 mg/mL). (b) FFF emission spectrum (\(\lambda_{\text{ex}}\) = 280 nm) of a solution of tryptophan in water (C = 0.02 mg/mL). Intensity of emission spectra is scaled and reported in arbitrary units. Figure 6: FFF emission spectra of solutions of hydroxybenzoic (a) and hydroxycinnamic (b) phenolic acids in water/methanol (90% v/v) (C = 0.02 mg/mL). (a) FFF emission spectra (\(\lambda_{\text{ex}}\) = 270 nm) of gallic acid (gallic a. ), syringe acid (syringic a. ), vanillic acid (vanillic a. ), and 4-hydroxybenzoic acid (4-hb a.). (b) FFF emission spectra (\(\lambda_{\text{ex}}\) = 290 nm) of ferulic acid (Fer), caffe acid (Caf), and p-coumaric acid (p-cum).