To observe the tautomer transition, we used f reshly purchased FAM prepared f rom a completely dehydroxylated state (FAM1, shown in red in f ig. 3) without stabilization of the buff er (0.5μM solution in KCl 100mM). Figure shows the time dependent curves of the voltage (black), current (red) and the f luorescence (green) f rom the cell containing the WE. The interpretation of the cyclic voltammograms does not change, as there is no inf luence of the state of compounds on the electrolyte. All the peaks were f itted to extract Q(t) f rom the Faradic currents. However, for this preparation of the f luorophore in the electrolyte, we observed a diff erent behavior. Figure (b) zooms into the f luorescence of cycle 2 (extended f or a time slightly before and af ter), which is representative of the behavior observed. The most notorious f eature is the relative minimum of the f luorescence that can be observed around 150 s occurring at the middle of the oxidation cycle. As the voltammogram continued to complete the oxidation, the descending f luorescence with the oxidation cycle was retrieved to achieve the global minimum at the end of the oxidation cycle and then the maximum of the f luorescence was recovered af ter the reduction (in this cycle af ter 210 s). We interpreted the intermediate minimum with the f ormation of a medium tautomer FAM2, less f luorescent than the species generated at this instant in the solution with a limited lif etime (f ew microseconds). This sudden drop in the f luorescence can be explained based on the absorption spectra (f ig. 3(c)) of the diff erent FAM species. The starting species FAM1 (shown in red) transition f rom one acid/base pair to another with a similar in absorbance at our excitation wavelength (470nm). However, the lower absorbance of the neutral lactone N (FAM2 show in green in f ig. 3(c)) at the excitation wavelengths translates into lower emission fluorescence. At this specif ic moment we recorded the tautomerization of the keto/spiro form (Lactone FAMH 3 ) into the zwitterionic equivalent (one of them being less f luorescent than the other). The colored band at the bottom of the f luorescence spectra represents the pH dependence of the diff erent FAM acid/base pairs present in the solution and its evolution with time. Later the transition to cationic FAM3 recovers the normal behavior. Figure shows the calculated charge exchanged as f unction of time (Q(t)) extracted f rom the Faradaic currents during the oxidation as detailed bef ore. It shows that the pH was varying in the solution via electrochemical generated acid f rom 7 to 2 passing theref ore through all the pK a values of the FAM. Thus, as the generation of protons during the oxidation protonates f irst the triaonic FAM 3-it passes by the neutral (zwitterionic) FAMH 3 leading at the end to the cationic nonf luorescent FAMH 4 + . During the reduction of the 4-A TP, the protons are re-adsorbed leading to the reverse exchange phenomenon (f ig 3(a)), but the tautomerization is no longer observed, showing the instability of the neutral Tautomer N.