Proton-bound Dimers Research Articles

Investigation of the Proton-Bound Dimer of Dihydrogen Phosphate and Formate Using Infrared Spectroscopy in Helium Droplets.

Understanding the structural and dynamic properties of proton-bound complexes is crucial for elucidating fundamental aspects of chemical reactivity and molecular interactions. In this work, the proton-bound complex between dihydrogen phosphate and formate, and its deuterated counterparts, is investigated using IR action spectroscopy in helium droplets. Contrary to the initial expectation that the stronger phosphoric acid would donate a proton to formate, both experiment and theory show that all exchangeable protons are located in the phosphate moiety. The experimental spectra show good agreement with both scaled harmonic and VPT2 anharmonic calculations, indicating that anharmonic effects are small. Some H-bending modes of the nondeuterated complex are found to be sensitive to the helium environment. In the case of the partially deuterated complexes, the experiments indicate that internal dynamics leads to isomeric interconversion upon IR excitation.

Location of the Shared Proton in Proton-Bound Dimer Compound of Hydrogen Sulfate and Formate: Path Integral Molecular Dynamics Study.

The structure of the proton-bound dimer compound of hydrogen sulfate and formate has been studied by considering nuclear quantum effects (NQEs) using the path integral molecular dynamics method. This study unveiled the location of the shared proton and answered the following question: "Is the shared proton localized on either an anion or located around the center of two anions?" We have elucidated that the shared proton is distributed in the region beyond the transition state due to the NQEs, even though the shared proton did not completely overcome the transition state for the proton shuttle.

The impact of side-chain fluorination on proton-bound phenylalanine dimers: a cryogenic infrared spectroscopic study.

The incorporation of fluorine into amino acids is an important strategy to produce tailored building blocks with unique properties for peptide-based materials. Phenylalanine is frequently modified due to its role in cation-π interactions and the formation of amyloid fibres. Previous studies have utilized gas-phase vibrational spectroscopy to study interactions between canonical amino acids. In this study, we employ a combination of cryogenic gas-phase infrared spectroscopy and density functional theory to study the interactions in proton-bound dimers of side-chain fluorinated phenylalanines. Our findings reveal how the position and number of fluorine atoms affect the interactions and structures of the dimers. Monofluorinated phenylalanines adopt charge-solvated structures in which the two amino acids interact via their ammonium and amine functions (NH3+⋯NH2). The dimer with the perfluorinated side chain forms multiple charge-solvated and salt-bridged structures with varying interaction types. These structural changes are attributed to the significant reduction of electron density in the aromatic systems.

Protonation of gabapentin: Ion mobility spectrometry and computational study.

Protonation of gabapentin was studied using corona discharge ion mobility spectrometry with two types of reactant ions at temperature of 200°C. It was found that at elevated temperatures, ionization of gabapentin proceeds via two channels, including the protonation and reactant ion attachment, in the presence of both hydronium and ammonium reactant ions. The effect of the sample concentration on the relative intensity of product ion peaks was also studied. It turned out that in high concentrations, in addition to the protonation of gabapentin, binding of the reactant ion and the formation of proton-bound dimer also occurred, while in low concentrations, the only product of the ionization process is protonated gabapentin. Density functional theory (DFT) with B3LYP and M062X functionals employing the same basis set 6-311++G(d,p) was used to extend the experimental findings. The structures of the several conformers of neutral and protonated gabapentin were obtained. Based on them, topical proton affinity and topical gas-phase basicity of gabapentin were calculated for selected conformers. The attachment of reactant ions to neutral gabapentin and formation of proton-bound dimer were thermodynamically studied.

Gas-Phase External Solvation of Protonated Benzonitrile by Eight Methanol Molecules.

The gas-phase sequential association of methanol onto protonated benzonitrile (C6H5CNH+) and the proton-bound dimer (C6H5CN)2H+ have been examined experimentally by equilibrium thermochemical measurements and computationally by density functional theory (DFT). The bonding enthalpy (ΔH°) for the association of methanol with protonated benzonitrile (25.2 kcal mol-1) reflects the strong electrostatic interaction provided by the formation of an ionic hydrogen bond in the C6H5CNH+OHCH3 cluster in excellent agreement with a DFT-calculated binding energy of 24.9 kcal mol-1. The sequential bonding enthalpy within the (C6H5CN)H+(OHCH3)n clusters decreases from 25.2 to 10.6 kcal mol-1 for the eighth solvation step (n = 8), which remains more than 25% above the enthalpy of vaporization of liquid methanol (8.4 kcal mol-1). The nonbulk convergence of ΔH°n-1,n with eight solvent molecules is attributed to the external solvation of a benzonitrile molecule by an extended hydrogen bonding network of protonated methanol clusters H+(CH3OH)n. In the external solvation of protonated benzonitrile by methanol, the proton resides on the methanol subcluster and the neutral benzonitrile molecule remains outside and bonded to the surface of the protonated methanol cluster. The bonding enthalpy of methanol to the proton-bound benzonitrile dimer (C6H5CN)H+(NCC6H5) is measured to be 18.0 kcal mol-1, in good agreement with a DFT-calculated value of 17.1 kcal mol-1, which reflects the association of the proton with the lower proton affinity methanol molecule, thus forming a highly stable structure of protonated methanol terminated by two ionic hydrogen bonds to the two benzonitrile molecules. The external solvation of benzonitrile by methanol ices in space allows benzonitrile to remain on the ice grain surface rather than being isolated inside the ice. This could provide accessibility for reactions with incoming ions and molecules or for photochemical processes by UV irradiation, leading to the formation of complex organics on the surface of ice grains.

Ultrasensitive Ion Source for Drift Tube Ion Mobility Spectrometers Combining Optimized Sample Gas Flow with Both Chemical Ionization and Direct Ionization.

Efficient ionization of analyte molecules is a crucial step for the outstanding sensitivity of ion mobility spectrometers (IMS) used for trace gas detection. Here, we present a new ion source that combines the previously published extended field switching ion shutter with two switchable ionization sources and an optimized sample gas flow that leads to a focused laminar stream through the reaction region of the ion source. The X-ray ionization source allows for chemical gas phase ionization of analyte molecules, while the UV ionization source allows for direct ionization of analyte molecules. The optimized sample gas flow not only allows for quickly washing out analyte molecules from the reaction region but also has improved sensitivity by a factor of about 5 for protonated monomers, 20 for proton-bound dimers, and over 100 for the proton-bound trimer of 1-octanol. The resulting limits of detection using chemical X-ray ionization are in the subpptv-range for protonated monomers and in the low pptv-range for proton-bound dimers, while the limits of detection using direct UV ionization are in the subppbv-range. Especially, a direct comparison between chemical and direct ionization of ketones using this ultrasensitive ion source reveals a stepwise conversion from directly ionized monomers to proton-bound dimers via protonated monomers during direct UV ionization.

Helium Nanodroplet Infrared Action Spectroscopy of the Proton-Bound Dimer of Hydrogen Sulfate and Formate: Examining Nuclear Quantum Effects.

The proton-bound dimer of hydrogen sulfate and formate is an archetypal structure for ionic hydrogen-bonding complexes that contribute to biogenic aerosol nucleation. Of central importance for the structure and properties of this complex is the location of the bridging proton connecting the two conjugate base moieties. The potential energy surface for bridging proton translocation features two local minima, with the proton localized at either the formate or hydrogen sulfate moiety. However, electronic structure methods reveal a shallow potential energy surface governing proton translocation, with a barrier on the order of the zero-point energy. This shallow potential complicates structural assignment and necessitates a consideration of nuclear quantum effects. In this work, we probe the structure of this complex and its isotopologues, utilizing infrared (IR) action spectroscopy of ions captured in helium nanodroplets. The IR spectra indicate a structure in which a proton is shared between the hydrogen sulfate and formate moieties, HSO4–···H+···–OOCH. However, because of the nuclear quantum effects and vibrational anharmonicities associated with the shallow potential for proton translocation, the extent of proton displacement from the formate moiety remains unclear, requiring further experiments or more advanced theoretical treatments for additional insight.

Sensitivity of secondary electrospray ionization mass spectrometry to a range of volatile organic compounds: Ligand switching ion chemistry and the influence of Zspray™ guiding electric fields.

Secondary electrospray ionization (SESI) is currently only semi-quantitative. In the Zspray™ arrangement of SESI-MS, the transfer of ions from near atmospheric pressure to a triple quadrupole is achieved by guiding electric fields that partially desolvate both reagent and analyte ions which must be understood. Also, to make SESI-MS more quantitative, the mechanisms and the kinetics of the reaction processes, especially ligand switching reactions of hydrated hydronium reagent ions, H3 O+ (H2 O)n , with volatile organic compound (VOC) molecules, need to be understood. A modified Zspray™ ESI ion source operating at sub-atmospheric pressure with analyte sample gas introduced via an inlet coaxial with the spray was used. Variation of the ion-guiding electric fields was used to reveal the degree of desolvation of both reagent and analyte ions. The instrument sensitivity was determined for several classes of VOCs by introducing bag samples of suitably varying concentrations as quantified on-line using selected ion flow tube MS. Electric field desolvation resulted in largely protonated VOCs, MH+ , and their monohydrates, MH+ H2 O, and for some VOCs proton-bound dimer ions, MH+ M, were formed. There was a highly linear response of the ion signal to the measured VOC sample concentration, which provided the instrument sensitivities, S, for 25 VOCs. The startling results show very wide variations in S from near 0 to 1 for hydrocarbons, and up to 100, on a relative scale, for polar compounds such as monoketones and unsaturated aldehydes. The complex ion chemistry occurring in the SESI ion source, largely involving gas-phase ligand switching, results in widely variable sensitivities for different classes of VOCs. The sensitivity is observed to depend on the dipole moment and proton affinity of the analyte VOC molecule, M, and to decrease with the observed fraction of MH+ H2 O, but other yet unrecognized factors must play a significant role.

IRMPD Spectroscopy of Homo- and Heterochiral Asparagine Proton-Bound Dimers in the Gas Phase.

We investigate gas-phase structures of homo- and heterochiral asparagine proton-bound dimers with infrared multiphoton dissociation (IRMPD) spectroscopy and quantum-chemical calculations. Their IRMPD spectra are recorded at room temperature in the range of 500–1875 and 3000–3600 cm–1. Both varieties of asparagine dimers are found to be charge-solvated based on their IRMPD spectra. The location of the principal intramolecular H-bond is discussed in light of harmonic frequency analyses using the B3LYP functional with GD3BJ empirical dispersion. Contrary to theoretical analyses, the two spectra are very similar.

Ligand Switching Ion Chemistry: An SIFDT Case Study of the Primary and Secondary Reactions of Protonated Acetic Acid Hydrates with Acetone.

A study was performed of the reactions of protonated acetic acid hydrates, CH3COOHH+(H2O)n, with acetone molecules, CH3COCH3, using a selected ion flow-drift tube (SIFDT). The rationale for this study is that hydrated protonated organic molecules are major product ions in secondary electrospray ionization mass spectrometry (SESI-MS) and ion mobility spectrometry (IMS). Yet the formation and reactivity of these hydrates are only poorly understood, and kinetics data are only sparse. The existing SIFDT instrument in our laboratory was upgraded to include an octupole ion guide and a separate drift tube by which hydrated protonated ions can be selectively injected into the drift tube reactor and their reactions with molecules studied under controlled conditions. This case study shows that, in these hydrated ion reactions with acetone molecules, the dominant reaction process is ligand switching producing mostly proton-bound dimer ions (CH3COCH3)H+(CH3COOH), with minor branching into (CH3COCH3)H+(H2O). This switching reaction was observed to proceed at the collisional rate, while other studied hydrated ions reacted more slowly. An attempt is made to understand the reaction mechanisms and the structures of the reaction intermediate ions at the molecular level. Secondary switching reactions of the asymmetric proton-bound dimer ions lead to a formation of strongly bound symmetrical dimers (CH3COCH3)2H+, the terminating ion in this ion chemistry. These results strongly suggest that, in SESI-MS and IMS, the presence of a polar compound, like acetone in exhaled breath, can suppress the analyte ions of low concentration compounds like acetic acid thus compromising their quantification.

Evolution of the linker in microhydrated hydrogen dinitrate anions: From H+ to H5 O2.

Hydrogen dinitrate anion, HNO3 (NO3- ), is a proton-bound dimer with a very strong hydrogen bond. By employing ab initio molecular dynamics (AIMD) method, we studied the effects of the proton transfer and the rotation of the nitrates on the vibrational profiles of HNO3 (NO3- )(H2 O)n (n =0-2). The AIMD results indicate that the structure of the n =0 cluster is very flexible, even though its hydrogen bond is quite strong. Significant rotations around the hydrogen bond and frequent transfers of proton from HNO3 to NO3- are observed in AIMD simulations. Dynamic changes are therefore an important factor in understanding the broadening of vibrational features. For n =1, the extent of structural fluctuation increases further, as H2 O could move around the anion while the HNO3 (NO3- ) core also goes through structural changes. Its vibrational spectrum can be understood as a mixture of many isomers visited during AIMD simulations. By n =2, the structure is stabilized around one isomer, with the linker between the two nitrates being H5 O2+ , rather than H+ . Due to strong hydrogen bonds between nitrates and water molecules, this H5 O2+ takes the extraordinary structure with the H+ localized on one H2 O, rather than being shared. While this novel structure is stable during AIMD simulations, the dynamic fluctuations in hydrogen bond distances still produce significant broadening in its vibrational profile.

Toward Compact High-Performance Ion Mobility Spectrometers: Ion Gating in Ion Mobility Spectrometry.

Printed circuit board (PCB) based drift tube ion mobility spectrometers enable the use of state-of-the-art production techniques to manufacture compact devices with excellent performance at minimum cost. The new PCB ion mobility spectrometer (PCB-IMS) presented here is equipped with either a 140 MBq tritium or a 95 MBq nickel-63 ionization source and consists of a combination of horizontally arranged 6-layer PCBs for the drift and reaction regions and vertically arranged PCBs for interfacing the ionization source, ion shutter, and detector. The design allows the reproducible manufacturing and thus comparison of different IMS topologies. Here, we investigate different ion shutters, field-switching, Bradbury-Nielsen, and tristate and their effects on resolving power and limits of detection considering two different ionization region geometries and ionization sources, tritium and nickel-63. It is shown that the high resolving power of RP > 80 at low drift voltage of 3 kV and short drift length of 50 mm can be achieved independent of the used ion shutter mechanism and reaction region geometry. While the resolving power of all ion shutters is excellent, the Bradbury-Nielsen shutter shows a pronounced discrimination of slow ion species when using short shutter opening times for small initial ion cloud widths, as required for high resolving power. Thus, the intensity of the proton-bound dimer of 2-pentanone is reduced by 30% compared to the signal intensity obtained with both the field-switching and tristate shutter. The detection limits employing the Bradbury-Nielsen shutter and a 50 mm reaction region as required for nickel-63 are 58 pptv for the protonated monomer and 3.4 ppbv for the proton-bound dimer of 2-pentanone. The detection limits achieved with the tristate shutter utilizing the same reaction region are slightly higher for the protonated monomer at 68 pptv, but lower for the proton-bound dimer at 2 ppbv due to the advanced ion shutter principle not discriminating slow ions. However, the lowest detection limits of 13 pptv and 301 pptv can be achieved with the field-switching shutter and a 2 mm reaction region, sufficient for a tritium ionization source.

Fermi resonance switching in KrH+Rg and XeH+Rg (Rg = Ne, Ar, Kr, and Xe).

Matrix isolation experiments have been successfully employed to extensively study the infrared spectrum of several proton-bound rare gas complexes. Most of these studies have focused on the spectral signature for the H+ stretch (ν3) and its combination bands with the intermolecular stretch coordinate (ν1). However, little attention has been paid to the Fermi resonance interaction between the H+ stretch (ν3) and H+ bend overtone (2ν2) in the asymmetric proton-bound rare gas dimers, RgH+Rg'. In this work, we have investigated this interaction on KrH+Rg and XeH+Rg with Rg = (Ne, Ar, Kr, and Xe). A multilevel potential energy surface (PES) was used to simulate the vibrational structure of these complexes. This PES is a dual-level comprising of second-order Møller-Plesset perturbation theory and coupled-cluster singles doubles with perturbative triples [CCSD(T)] levels of ab initio theories. We found that when both the combination bands (nν1 + ν3) and bend overtone 2ν2 compete to borrow intensity from the ν3 band, the latter wins over the former, which then results in the suppression of the nν1 + ν3 bands. The current simulations offer new assignments for the ArH+Xe and KrH+Xe spectra. Complete basis set (CBS) binding energies for these complexes were also calculated at the CCSD(T)/CBS level.

On the Proton-Bound Noble Gas Dimers (Ng-H-Ng)+ and (Ng-H-Ng')+ (Ng, Ng'= He-Xe): Relationships betweenStructure, Stability, and Bonding Character.

The structure, stability, and bonding character of fifteen (Ng-H-Ng)+ and (Ng-H-Ng’)+ (Ng, Ng’ = He-Xe) compounds were explored by theoretical calculations performed at the coupled cluster level of theory. The nature of the stabilizing interactions was, in particular, assayed using a method recently proposed by the authors to classify the chemical bonds involving the noble-gas atoms. The bond distances and dissociation energies of the investigated ions fall in rather large intervals, and follow regular periodic trends, clearly referable to the difference between the proton affinity (PA) of the various Ng and Ng’. These variations are nicely correlated with the bonding situation of the (Ng-H-Ng)+ and (Ng-H-Ng’)+. The Ng-H and Ng’-H contacts range, in fact, between strong covalent bonds to weak, non-covalent interactions, and their regular variability clearly illustrates the peculiar capability of the noble gases to undergo interactions covering the entire spectrum of the chemical bond.

Ligand effects in gold-carbonyl complexes: Evaluation of the bond dissociation energies using blackbody infrared radiative dissociation

In a previous work, the ability of gold-carbonyl bond dissociation energies (BDEs) to describe ligand effects in gold(I) complexes was probed using collision-induced dissociation measurements. Despite the large experimental error of the technique, results agreed with theoretical description of these effects. We propose here another experimental approach to evaluate the BDEs of [LAu-CO]+ complexes, by using Blackbody Infrared Radiative Dissociation (BIRD) method which is able to provide absolute energetic measurements. To this aim, the dissociation of 8 gold-carbonyl complexes was studied with this approach to obtain their Arrhenius activation energies. Because these complexes do not reach the rapid energy exchange (REX) limit conditions, critical energies of these dissociations were then determined using the kinetic data and an Equilibrium Truncated Thermal (ETT) internal energy distribution model implemented in the RRKM-QET MassKinetics software. To validate our approach, the known critical energies of proton-bound amino acid dimers were first reinvestigated using this model. The results are in excellent agreement with those reported in the literature. This indicates that the assumptions of this simple model are reasonable and allow the determination of accurate dissociation energies. For [LAu-CO]+ complexes, even though a shift (from 0.18 to 0.26 eV) is observed between binding energies obtained and calculated values, a linear tendency between these two sets of data is still obtained. These results validate the use of BIRD in combination with equilibrium truncated thermal internal energy distributions model to obtain dissociation energies for relatively small ions. It is also shown herein that the measure of the gold-carbonyl bond dissociation energies is a good descriptor of ligand effects in gold(I) complexes that may be reliably evaluated using the BIRD technique.

Mass spectral and theoretical investigations of the transient proton-bound dimers on the cleavage processes of the peptide GHK and its analogues.

Fragmentation mechanisms of the singly protonated peptides GHK, GHKH and HGHK have been investigated by mass spectrometry and theoretical calculations. Fragmentation behavior of the protonated H–K amide bond in GHK was changed completely when a histidinyl residue was introduced into the C-terminus of GHK. The H–K amide bond breaking was a predominant pathway in the case of GHK and GHKH. For HGHK, the histidinyl residue at the N-terminus hampered significantly breaking of the H–K amide bond resulting in a high potential energy barrier; calculations indicated that this histidinyl effect played a vital role for the H–K amide bond fragmentation. Subsequent analysis of the fragmentation mechanism revealed that recombination processes of the hydrogen bonding for the intermediate products were all exergonic. Formation of a proton-bound dimer (PBD) lowering the energy barriers from a thermodynamic perspective for all the designed fragmentation pathways was demonstrated to be feasible by our systematic calculations. Moreover, the involvement of different PBDs was further confirmed by analyses of the reduced density gradient (RDG) isosurfaces and scatter maps. A dynamically favored pathway was likely via six-membered ring or nine-membered ring structures generated by the diketopiperazine as revealed by atom-in-molecules (AIM) analyses, since the steric interaction energies in the newly formed ring were estimated to be relatively small when compared to the products generated from a lactam and/or an oxazolone pathway. This is the first feasibility investigation from a dynamic viewpoint for formation of different rings involved in the lactam, oxazolone or diketopiperazine pathways in the fragmentation mechanisms proposed.

Infrared multiple photon dissociation action spectroscopy of protonated unsymmetrical dimethylhydrazine and proton-bound dimers of hydrazine and unsymmetrical dimethylhydrazine.

The gas-phase structures of protonated unsymmetrical 1,1-dimethylhydrazine (UDMH) and the proton-bound dimers of UDMH and hydrazine are examined by infrared multiple photon dissociation (IRMPD) action spectroscopy utilizing light generated by a free electron laser and an optical parametric oscillator laser system. To identify the structures present in the experimental studies, the measured IRMPD spectra are compared to spectra calculated at the B3LYP-GD3BJ/6-311+G(d,p) level of theory. These comparisons show that protonated UDMH binds the proton at the methylated nitrogen atom (α) with two low-lying α conformers probably being populated. For (UDMH)2H+, the proton is shared between the methylated nitrogen atoms with several low-lying α conformers likely to be populated. Higher-lying conformers of (UDMH)2H+ in which the proton is shared between α and β (unmethylated) nitrogen atoms cannot be ruled out on the basis of the IRPMD spectrum. For (N2H4)2H+, there are four low-lying conformers that all reproduce the IRMPD spectrum reasonably well. As hydrazine and UDMH see usage as fuels for rocket engines, such spectra are potentially useful as a means of remotely monitoring rocket launches, especially in cases of unsuccessful launches where environmental hazards need to be assessed.

Pyridine and Methylpyridines: Calculations of the Structure, Proton Affinity, Gas-Phase Basicity, and Mobility of Protonated Molecules and Proton-Bound Dimers

Accurate calculations of the structure and properties of analytes and their ions are of great interest to the theory and practice of mass spectrometry, ion mobility spectrometry, and related methods. In this work, using accurate quantum chemical methods, we computed the structure of neutral and protonated pyridine, 2-methyl-, 4-methyl-, 2,4-dimethyl-, 2,6-dimethyl-, and 2,4,6-trimethylpyridine molecules and also of proton-bound dimers of pyridine and 2,4-dimethylpyridine. Two stable conformers of the 2,4-dimethylpyridine proton-bound dimer are found. An accurate and economical method is proposed for calculating proton affinity and gas-phase basicity with the calculation error close to the experimental one. The values of proton affinity and gas-phase basicity for 2,4,6-trimethylpyridine are computed. Reduced mobilities of protonated molecules and proton-bound dimers are calculated by the trajectory method in different versions. A calculation version is proposed that allows mobility computation with the error close to the experimental one and adequately reproduces small differences in the mobility of isomers. The versatility of the chosen B3PW91−D3BJ/def2−TZVP method of the density functional theory with the inclusion of dispersion correction is shown. The method (in combination with B2GP−PLYP−D3BJ/def2−TZVPPD calculations) ensures accurate calculations of the molecular structure, proton affinity, and gas-phase basicity, and also charges on atoms for computing ion mobility.

Strong Fermi Resonance Associated with Proton Motions Revealed by Vibrational Spectra of Asymmetric Proton-Bound Dimers.

Infrared spectra for a series of asymmetric proton-bound dimers with protonated trimethylamine (TMA-H+ ) as the proton donor were recorded and analyzed. The frequency of the N-H+ stretching mode is expected to red shift as the proton affinity of proton acceptors increases. The observed band, however, shows a peculiar splitting of approximately 300 cm-1 with the intensity shifting pattern resembling a two-level system. Theoretical investigation reveals that the observed band splitting and its extraordinarily large gap of around 300 cm-1 is a result of strong coupling between the fundamental of the proton stretching mode and overtone states of the two proton bending modes, that is commonly known as Fermi resonance (FR). We also provide a general theoretical model to link the strong FR coupling to the quasi-two-level system. Since the model does not depend on the molecular specification of TMA-H+ , the strong coupling we observed is an intrinsic property associated with proton motions.

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 4-aminobenzoic 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) acetonitrile-water was used as the solvent system for 4-aminobenzoic acid, the N-protomer was predominantly generated at all analyte concentrations.