On the Origin of Protons in Electrospray Ionization

In electrospray ionization (ESI), analyte molecules are protonated in the positive ion mode to form molecular ions. However, little is known about the origin of these protons. To investigate, an enclosed ESI ion source was used to control the type of gas introduced into the source. First, when only inert gas such as N2 or CO2 was used, protonated molecular ions disappeared. Second, when gas phase D2O was used, [M+D]+ became the predominant peak in the mass spectrum. However, when gas phase H2O was used, [M+H]+ became dominant even though caffeine was dissolved in pure D2O. Furthermore, when mixtures of D2O/H2O with different ratios were used in the gas phase, the percentages of [M+D]+ peak calculated from the mass spectra strictly follow the percentages of D2O in the gas phase mixture. Based on these observations, we conclude that the source of protonation in ESI could originate entirely from the gas phase H2O molecules, independent of the solvent the analyte molecules are dissolved in. When pure H2O was sprayed out, protonated small water clusters (PSWC) were formed, which comprise of a single proton surrounded by 2 to 5 water molecules. These PSWC may have a wide range of biological functions.