## 3 Results
As can be seen in Figure 11, the data are fully explained by Equations (3) and (4). In particular, at pH values lower than 3, \(Z_{\mathrm{H}}\) tends to be \(-1\) for all the investigated systems, confirming that a protonation occurs, according to Equations (3) and (4). Furthermore, as expected, the proteolysis equilibria of quercetin and luteolin occur in the alkaline range (\(-\log\)\([\mathrm{H}+]\) > 8) at the expenses of the hydroxyl groups (see Figure 11a,b). In contrast, for L-ascorbic acid, the dissociation starts at a pH higher than 3 (Figure 11c) with the proteolysis of -OH in position 3 (see Figure 1c). Accurate values for the constants of proteolysis equilibria (Equation (4)) were determined for quercetin and luteolin only. By performing a back titration, whose points are depicted in Figure 11c as orange crosses, we have verified that L-ascorbic acid undergoes a degradation in an alkaline medium (i.e., pH higher than 7.5), which occurs faster than the potentiometric measurement time. Therefore, we have performed UV-vis spectrophotometric titrations at different \(C_{\mathrm{L}}\) values (i.e., between 0.01 and 0.04 mM). UV-vis absorption spectrophotometry is a simple and accurate method for determining acidic constants, whose determination is based on a change in the shape and intensity of the absorption spectra with a change in the pH of the test solutions. The region of pH close to the \(\mathrm{p}K_{\mathrm{a}}\) values of corresponding acids was supposed to be optimal to obtain accurate values, in the choice of the pH range for all the compounds. Additionally, the spectral range for all compounds was designated according to the variations in the spectra at both acidic and basic solutions. Consequently, the hydrogen ion concentration was varied by adding small and exactly known volumes of a standard NaOH solution (\(C_{\mathrm{B}}\)), from 1.0 \(\times\) 10\({}^{-5}\) M to 1.0 \(\times\) 10\({}^{-12}\) M for quercetin and luteolin and from 1.0 \(\times\) 10\({}^{-2}\) M to 1.0 \(\times\) 10\({}^{-11}\) M for L-ascorbic acid, and the absorbance values were recorded at 93 wavelengths between 230 and 457 nm for quercetin and luteolin and at 50 wavelengths between 211 and 330 nm for L-ascorbic acid. The typical recorded spectra are depicted in Figure 11a-c, for quercetin, luteolin and L-ascorbic acid, respectively. The UV-vis spectra of quercetin and luteolin (Figure 11a,b, respectively) show two main absorption bands, which are comparable to signals observed for most flavonoids [12; 14]: band I in the 350-400 nm range, as a result of the conjugation between the B and C ring (cinnamoyl system), and band II in the 240-270 nm range, deriving from the A-C ring (benzoyl system). Moreover, two other less-intense absorption bands at 303 and 280 nm for quercetin and at 295 and at 267 nm for luteolin were observed. The UV-vis spectrum of L-ascorbic acid exhibits a single band centered at 260 nm, in accordance with the literature [11; 15]. Figure 11a shows that the maximum of band I was blue shifted (i.e., from 367 to 320 nm) by moving from pH 5.5 to pH 6.5. The absorption profile changed negligibly upon raising the pH from 6.5 to 12.0, indicating, according to [35], that the acid-base sites of quercetin were not appreciably rearranged during the dissociation in this pH range. In contrast, for luteolin and L-ascorbic acid (Figure 11b,c) the maxima of the principal absorption bands were red shifted (i.e., from 352 to 402 nm and from 243 to 267 nm, for luteolin and L-ascorbic acid, respectively) by moving from acidic to basic pH, attesting to the formation of a new spectrally active species in the solution. The numerical elaboration of spectrophotometric data was carried out by simulating each titration from the knowledge of the total concentrations, \(C_{\mathrm{B}}\) and \(C_{\mathrm{L}}\).