What exactly is causing analyte protonation in electrospray ionization MS?
[ This is a short answer, as a complete answer would require going through multiple references and books on the topic. ]
First of all, the ESI process does not require the pH to be acidic : positive ESI is routinely done in ammonium acetate or ammonium bicarbonate media, which have a pH of 7.4 or 8.1. But indeed, to produce protonated analytes it indeed requires a source of protons. So what is the source of protons?
One should go back to the initial stage of ESI, which is the production of positively (or negatively) charged droplets under the influence of a strong electric field. Let's assume that we focus on positive ESI and that the droplets carry an overall positive charge. These charges are often simply represented as + signs, but their molecular nature is important. In the case were an electrolyte is present in high concentration and in its ionic form, the major charge carrier will be the positive charge of this electrolyte. So when considering an organic acid at low pH, the positive charges will be mostly solvated protons. If the electrolyte was ammonium acetate at neutral pH, it would be ammonium ions. Even at higher pH, if the electrolyte is ammonium hydroxide, the droplets would still bear a net positive charge borne by an excess of ammonium ions.
Then come the evaporation / Coulombic fission stages whereby smaller droplets are formed from the larger one, with an overall concentration step. The concentration effect of this stage will affect the overall pH of the droplet if initially acidic, but it is not required to lead to protonated species. The important point being that within the droplets, there is still an excess of positive charges, mostly in the form of solvated proton. At some stage, as concentrations of both the analyte and the solvated proton increase, the proton will be placed in close proximity with the analyte (both not being volatile do not evaporate). At this stage, the system is no more in the bulk solution thermodynamic conditions, but not really either in the gas phase thermodynamic conditions.
But we can still at this stage approximate the situation as &quot;in the middle&quot; by considering the thermodynamic parameters for both ends: on the bulk side, we have pKa of the analyte, with decreasing pH, and on the gas phase side, we have the proton affinity (PA) of the analyte that should be compared to the PA of the other species competing for the proton. The two scales are related but not strictly: for instance water has a quite low proton affinity (692 kJ/mole) compared to most polar molecules (ketones are in the 800 kJ/mole range). Thus is the proton located on the analyte is more stable than on molecules from the mobile phase, it will end up protonating the molecule, without considderation of the pKa.