Determination of the Acidity Constants of Serotonin in the Ground and Excited States Using Spectroscopic Methods

Serotonin (5-hydroxytryptamine, 5-HT) is a biologically important neurotransmitter whose photophysical behavior depends on its protonation state. This study investigates the acid-base properties of serotonin in aqueous solution using UV-visible absorption and fluorescence spectroscopies. The ground-state acidity constant (pK<sub>a</sub>(S<sub>0</sub>)) was determined spectrophotometrically, while the excited-state acidity constant (pK<sub>a</sub>*(S<sub>1</sub>)) was estimated using the Förster thermodynamic cycle. Absorption spectra revealed an isosbestic point, indicating a simple two-state equilibrium between the protonated (R-OH) and deprotonated (R-O⁻) forms. The results show that serotonin behaves as a weak acid in the ground state (pK<sub>a</sub>(S<sub>0</sub>) ≈ 10.5–10.6), whereas its acidity increases significantly in the excited state (pK<sub>a</sub>*(S<sub>1</sub>) ≈ 4.7) due to electronic redistribution within the indole chromophore. Excited-State Proton Transfer (ESPT) occurs efficiently, influencing both fluorescence intensity and emission wavelength. These findings provide a comprehensive understanding of serotonin’s photophysical behavior and support its use as an intrinsic fluorescent probe for monitoring local pH variations in aqueous or cellular environments. The combination of UV-Vis and fluorescence measurements, with triplicate statistical validation, ensures reproducibility and accuracy of the determined acidity constants. This work contributes to a better understanding of neurotransmitter acid-base behavior under physiologically relevant conditions and demonstrates the potential application of serotonin in fluorescence-based pH sensing and molecular studies.