In Situ Light‐driven pH Modulation for NMR Studies

Abstract Proton exchange is a fundamental chemical event, and NMR provides the most direct readout of protonation events with site‐specific resolution. Conventional approaches require manual titration of sample pH to collect a series of NMR spectra at different pH values. This requires extensive sample handling and often results in significant sample loss, leading to reduced signal or the need to prepare additional samples. Here, we introduce a novel approach to control pH in NMR samples using water soluble photoacids, which alters the pH of the solution from near neutral to acidic pH upon in situ photo illumination. We show that the solution pH can be precisely controlled by choice of illumination wavelength and intensity, and sufficient protons are released from the photoacid to achieve meaningful pH change in samples where the molecule of interest has significant buffering capacity, such as a >100 µM protein sample. The pH is monitored in situ using internal standards with pH‐sensitive chemical shifts. This method enables precise, calibrated, and noninvasive change of sample pH within an NMR magnet, dramatically reducing the necessary sample handling. These findings highlight the potential of light‐induced pH control in NMR experiments and increase the robustness and reliability of pH‐dependent studies.