To interpret these spectroscopic data, we focus on the change in protonation states of 8AQ upon change in pH. The examined molecule can exist in three forms: neutral (8AQ, 1), as a monocation in which the aromatic nitrogen or amine nitrogen atoms are protonated (2a or 2b, respectively; see Figure ) and also as a di-cation in which both nitrogen atoms are protonated (8AQH2 2+ ). The equilibrium of these three forms depends strongly on the pH of the medium. 8AQ has two pKa values: pKa1 = -0.52 and pKa2 = 3.99 On the basis of recorded spectra, we calculated the upper pKa value and obtained pKa2 = 4.00. On the basis of both pKa values reported in the literature, we plotted the species distribution diagram (Figure ), which confirmed the monoprotonated form's dominance at pH=3 (~ 90%). Our calculations show that the energy difference between the 2a and 2b forms of monoprotonated species is 0.7 kcal/mol, with the aromatic nitrogen protonation more favoured. Based on these results, we can expect both forms to be present in the solution -assuming Boltzmann distribution, this would yield ca. 77% of 2a and 23% of 2b. Indeed, the UV-Vis spectrum at low pH contains an intense band at 260 nm (2a) and a less intense band at 220 nm, characteristic of 2a. The relative intensity of these bands reflects the dominance of the 2a form. Moreover, the lack of significant absorption >300 nm for 2b confirms that experimentally observed changes in 331 nm and 383 nm bands are associated with deprotonation of the 2a form to 1. It is worth noting that the agreement between experimentally obtained and calculated spectra is excellent, especially taking into account the positions of isosbestic points (exp. 370 and 254 nm, calc. 375 and 250 nm). Note that each spectrum was normalized so that the maximum for each spectrum has an intensity of one. According to calculations, 2a and 2b exist in 77%:23% equilibrium; therefore, the experimental absorbance of 2b will be at least two times smaller than 2a.