The Effects of Analyte Concentration on the Protonation Sites of 4-Aminobenzoic Acid Upon Atmospheric Pressure Chemical Ionization as Revealed by Gas-phase Ion-molecule Reactions.

The most basic site of 4-aminobenzoic acid in aqueous solution is the amino nitrogen, while the carbonyl oxygen is calculated to be the most basic site in the gas phase. However, the preferred protonation site of 4-aminobenzoic acid upon electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) depends upon the ionization solvent and ion source parameters. The influence of the concentration of the analyte on the manifested protonation sites upon APCI has not been investigated and is reported here. Gas-phase ion-molecule reactions of trimethoxymethylsilane were used to identify the protonation sites of 4-aminobenzoic acid ionized using APCI with methanol or acetonitrile-water as the solvent. The nitrogen-protomer was found to be about twice as abundant as the oxygen-protomer at low analyte concentrations (10(-9)-10(-6) M) in methanol solvent. This finding was rationalized on the basis of a previous finding that when the O-protomer is surrounded by more than eight methanol molecules in the gas phase it starts behaving as if it were in an aqueous solution and converts to the N-protomer. At greater analyte concentrations (>= 10(-4) M), the amino group was predominantly protonated, which was rationalized based on the formation of a particularly stable proton-bound dimer of 4aminobenzoic acid that preferentially dissociates to form the N-protomer. The above findings suggest that solution processes are much more important in APCI than commonly assumed, in agreement with recent literature. Indeed, when 1:1 (v/v) acetonitrilewater was used as the solvent system for 4-aminobenzoic acid, the N-protomer was predominantly generated at all analyte concentrations.