On the Chemistry of Aqueous Ammonium Acetate Droplets during Native Electrospray Ionization Mass Spectrometry

Ammonium acetate (NH4Ac) is a widely used solvent additive in native electrospray ionization (ESI) mass spectrometry. NH4Ac can undergo proton transfer to form ammonia and acetic acid (NH4+ + Ac- → NH3 + HAc). The volatility of these products ensures that electrosprayed ions are free of undesired adducts. NH4Ac dissolution in water yields pH 7, providing "physiological" conditions. However, NH4Ac is not a buffer at pH 7 because NH4+ and Ac- are not a conjugate acid/base pair (Konermann, L. J. Am. Soc. Mass Spectrom.2017, 28, 1827-1835.). In native ESI, it is desirable that analytes experience physiological conditions not only in bulk solution but also while they reside in ESI droplets. Little is known about the internal milieu of NH4Ac-containing ESI droplets. The current work explored the acid/base chemistry of such droplets, starting from a pH 7 analyte solution. We used a two-pronged approach involving evaporation experiments on bulk solutions under ESI-mimicking conditions, as well as molecular dynamics simulations using a newly developed algorithm that allows for proton transfer. Our results reveal that during droplet formation at the tip of the Taylor cone, electrolytically generated protons get neutralized by Ac-, making NH4+ the net charge carriers in the weakly acidic nascent droplets. During the subsequent evaporation, the droplets lose water as well as NH3 and HAc that were generated by proton transfer. NH3 departs more quickly because of its greater volatility, causing the accumulation of HAc. Together with residual Ac-, these HAc molecules form an acetate buffer that stabilizes the average droplet pH at 5.4 ± 0.1, as governed by the Henderson-Hasselbalch equation. The remarkable success of native ESI investigations in the literature implies that this pH drop by ∼1.6 units relative to the initially neutral analyte solution can be tolerated by most biomolecular analytes on the short time scale of the ESI process.