]>

(a)

(b)

(c)

(d)

(e)

(f)

Figure 2: The nonlinear regression analysis of the protonation equilibria model and factor analysis of silybin: (a) Absorption spectral dependence on pH at ${25}^{°}$C, (b) Cattel’s scree plot of the Wernimont-Kankare procedure for the determination of the number of light-absorbing species in the mixture $k^{*}=5$ leads to the actual instrumental error of the spectrophotometer used $s_{\text{inst}}(A)=0.30$ mAU (INDICES in S-Plus), (c) The absorbance versus pH curves for 243 nm, 250 nm, 288, and 327 nm in dependence on pH at ${25}^{°}$C, (d) Detecting influential outlying spectra with the use of the goodness-of-fit test and the plot of the residual standard deviation $s(e)$ versus pH for 20 spectra in dependence on pH at ${25}^{°}$C and $I=0.03$, (e) Pure spectra profiles of molar absorptivities versus wavelengths for variously protonated species $\text{L}\text{,}$$\text{LH}\text{,}$$\text{L}{\text{H}}_2\text{,}$$\text{L}{\text{H}}_3$, and $\text{L}{\text{H}}_4$, (f) Distribution diagram of the relative concentrations of both variously protonated species $\text{L}\text{,}$$\text{LH}\text{,}$$\text{L}{\text{H}}_2\text{,}$$\text{L}{\text{H}}_3$, and $\text{L}{\text{H}}_4$, of silybin as a function of pH at ${25}^{°}$C is calculated from estimates by SQUAD and SPECFIT: pKa 1 = 6.898(0.022) and 6.897(0.002); pKa 2 = 8.666(0.021) and 8.667(0.012); pKa 3 = 9.611(0.010) and 9.611(0.004); pKa 4 = 11.501(0.008) and 11.501(0.007), (SQUAD and SPECFIT, ORIGIN).