In the case of (b), i.e., calibration in the absence of vesicles, we have noticed that depending on the salt (NaX) employed, the ionic strength of the solution, and the HPTS concentration, the pKa of the fluorophore changes, meaning that the calibration equation is modified. Temperature is also a crucial variable. All these factors must be controlled to avoid inconsistencies between raw data provided by the spectrofluorometer, and pH values, resulting from applying the calibration equation.