Effect of Electrospray Ionization Source Conditions on the Tautomer

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Effect of Electrospray Ionization Source Conditions on Tautomer Distribution of Deprotonated p-Hydroxybenzoic Acid in Gas Phase Hanxue Xia, and Athula B. Attygalle Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01230 • Publication Date (Web): 10 May 2016 Downloaded from http://pubs.acs.org on May 10, 2016

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Analytical Chemistry

Effect of Electrospray Ionization Source Conditions on Tautomer Distribution of Deprotonated p-Hydroxybenzoic Acid in Gas Phase

Hanxue Xia, and Athula B. Attygalle* Center for Mass Spectrometry, Department of Biomedical Engineering, Chemistry, and Biological Sciences, Stevens Institute of Technology, Hoboken, NJ, 07030, USA ABSTRACT: The deprotonation site of p-hydroxybenzoic acid upon electrospray ionization has been a subject of fervent debate in several articles in the Journal of the American Chemical Society and elsewhere. General consensus is that electrospray-ionization mass spectrometry (ESI-MS) experimental results reflect the situation in solution to a considerable extent. Our research, using ion-mobility mass spectrometry challenges the notion that ESI-MS results directly reflects solution-phase structures and demonstrates that the relative populations of the thermodynamically less favored gaseous carboxylate tautomer or the thermodynamically more favored gaseous phenoxide tautomer, generated from the same aqueous solution of phydroxybenzoic acid by ESI, can be varied back and forth by changing the probe position, capillary voltage, desolvation-gas temperature, sample infusion flow rate, and cone voltage. In other words, solvent effects are not the primary criteria that determines the relative population distributions of tautomeric carboxylate (C-) and phenoxide (P-) ions (m/z 137) generated by electrospray ionization of p-hydroxybenzoic acid. In addition, we propose that the observed ratio of the P- and C- forms indirectly reflects the relative contribution of the charge-residue or ion-evaporation process that occur during the electrospray ion generation process.

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1.

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INTRODUCTION

The deprotonation site of p-hydroxybenzoic acid upon electrospray ionization has been a subject of fervent debate in the Journal of the American Chemical Society and elsewhere. 1-5 A single deprotonation of p-hydroxybenzoic acid can generate either the carboxylate ion (C-) or the phenoxide ion (P-) (Scheme 1).

Scheme 1. Deprotonation of p-hydroxybenzoic acid.

Unlike in the aqueous phase, where solvation effects stabilizes the more polarized carboxylate tautomer, the phenoxide structure, with higher charge distribution owing to delocalization, is the more stable form in the gas phase. Schröder et al.5 have carried out detailed experimentation to confirm that the carboxylate tautomer6 is the preferred form in solution 2 ACS Paragon Plus Environment

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irrespective of the nature of the solvent. Moreover, their study concluded that the tautomer distribution in the gas phase depends on the nature of the actual solvent used for electrospray, and the total concentration and pH value of the solution. An analogous solvent dependence on the two populations of N- and O-protonated forms (protomers6) generated by electrospray ionization of p-aminobenzoic acid7 and benzocaine (the ethyl ester of p-aminobenzoic acid) have been reported.8-10 A significant degree of research effort is focused on the localization of protonation or deprotonation sites of multifunctional molecules: such knowledge is vital to the understanding of gas-phase ion fragmentation processes and to the interpretation of mass spectra from unidentified compounds. Particularly under positive-ion generating conditions, protonation processes that take place under ESI and APCI conditions have been thoroughly investigated. Although the preferred protonation site of a polyfunctional molecule is the group with the highest proton affinity, because a thermodynamically favored product is formed, sometimes kinetically controlled protonation takes place at a less preferred site due to steric, temperature or other factors.11,12 On occasion, intramolecular proton transfers can produce thermodynamically less favored species upon collisional activation. For example, three decades ago Wood et al.13 described that the product-ion spectra recorded from the mass–isolated molecular ion of 2pentanone depend on the ion source temperature. The changes were attributed to the increase of the enol form abundance at higher temperatures. Recently, Pan et al. reported that APCI source conditions such as vaporizer and drying-gas temperatures exert a significant influence on the preferential formation of the thermodynamically favored protonated product from p(dimethylamino)chalcone.11 Nevertheless, source thermal effects in mass spectrometry are considered to be trivial. Many publications even overlook to report parameters such as source and desolvation-gas temperatures. However, in a detailed study on spatial effects on electrospray ionization, Janusson et al. recently reported that source parameters have powerful effects on peak intensities of ions generated from an equimolar mixture of two different ions.14 In our explorations on atmospheric-pressure ionization mechanisms, we found that the ESI ion-source conditions exert a dramatic effect on the abundance ratio of the gaseous anions generated from p-hydroxybenzoic acid. Herein, we report that the relative abundance of the two populations depends not only on the solvent composition, but also on the ESI probe spatial



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orientation, the capillary voltage, the source temperature, the solvent flow rate, and the cone voltage.

2.

METHODS

Chemicals. Acetonitrile was purchased from PHARMCO-AAPER (Brookfield, CT, USA). All chemicals, including p-hydroxybenzoic acid, were purchased from Sigma-Aldrich Chemical Co. (St Louis, MO) and used without further purification. Ultrapure water was obtained from a MilliQ purification system (Millipore Corporation, USA). A 100-ppm solution (7.2 x 10-5 M) of phydroxybenzoic acid was prepared in 0.1% aqueous ammonium hydroxide.

Mass Spectrometry. All experiments were conducted on a Synapt G2 HDMS (Waters, MA, USA) mass spectrometer equipped with an ESI, ESCi, and ASAP source. Nitrogen was used as the nebulizer, desolvation, and cone gas. Mass calibration (m/z 20 to 1500) was performed using a solution of sodium formate (100 ppm). Mass spectra were acquired in the negative-ion mode over a range of m/z 10–1200. For ion-mobility experiments, the ions of interest were mass selected by the quadrupole analyzer (Q) and transferred to the Tri-wave cell. A mass-selected ion is briefly accumulated in the Trap region of the Tri-wave cell and released to the travellingwave-ion-mobility cell via a short chamber filled with helium.15 After the ion-mobility separation, the ions pass the Transfer cell and undergo acceleration by the pusher before they are mass separated by the time-of-flight analyzer. Ion-mobility separation experiments were carried out on the m/z 137 ion generated by electrospraying a 100-ppm solution of p-hydroxybenzoic acid in 0.1% aqueous NH4OH, or acetonitrile containing 0.1% Et3N, under following instrumental conditions: the trap collision energy 4 eV, transfer collision energy 2 eV, IMS wave velocity 1500 m/s, IMS wave height 40.0 V, scroll pump pressure 4.34 mbar, source pressure 3.43 × 10-3 mbar, helium-cell pressure 1.40 × 103 mbar, IMS cell pressure 3.98 mbar (N2), TOF analyzer pressure 8.36 × 10-7 mbar, trap pressure 3.10 × 10-2 mbar (Ar), and transfer pressure 3.38 × 10-2 mbar (Ar). Unless otherwise stated, the general ESI source conditions for most experiments were: capillary voltage 2.22 kV, sample cone 11.0 V, extraction cone 1.5 V, desolvation-gas flow rate 370 L/Hr, sample infusion flow rate 10 µL/min, and Vernier-probe-adjuster position 5.92 mm. 4 ACS Paragon Plus Environment

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The source and desolvation-gas temperatures were held at 80 and 100 °C, respectively. To investigate individual effects, the magnitude of a specific source parameter was varied while other parameters were kept constant. The following source parameter were varied discreetly: 1) the Vernier-probe-adjuster position between 4.92-9.92 mm, 2) the capillary voltage between 2-3.75 kV, 3) the desolvation-gas temperature between 100-500 °C, 4) the desolvation-gas flow rate between 200-400 L/Hr, 5) the sample infusion rate was between 3-50 µL/min, and 6) the cone voltage between 5-45 V. To study the effect of volatile solvents on the arrival-time profiles of the m/z 137 ion, pure water or methanol was sprayed into the ESI source via the lockspray capillary at a flow rate of 30 µL/min with the mechanical barrier baffle was set at “sample” position to avoid the direct mixing of the additional solvent droplets with those of the spray-analyte solution. The other parameters were as follows: The ESI probe capillary voltage 2.22 kV, lockspray voltage 0.0 V, sampling cone 11.0 V, extraction cone 1.5 V, desolvation-gas flow rate 370 L/Hr, sample infusion flow rate 10 µL/min, and Vernier-probe-adjuster position 5.92 mm. The source and desolvation-gas temperatures were held at 80 and 100 °C, respectively. The instrument parameters for the ESCi experiment were as follows: the corona voltage was set at 3.5 or 5.0 kV, ESI capillary voltage 0 kV, sampling cone 11 V, extraction cone 1.5 V, desolvation-gas flow rate 370 L/Hr, sample infusion flow rate 10 µL/min, and Vernier-probeadjuster position 5.92 mm. The source and desolvation-gas temperatures were held at 80 and 100 °C, respectively. The instrument parameters for the HePI experiment were as follows: the capillary voltage was set at 2.0 kV, sampling cone 15 V, extraction cone 1.3 V, desolvation-gas flow rate 370 L/Hr, HePI-gas flow rate 30 mL/min, and Vernier-probe-adjuster position 5.92 mm. The source and desolvation-gas temperatures were held at 80 and 100 °C, respectively. A solid sample of phydroxybenzoic acid was placed in a 2 mL vial and the vial was attached close to the cone and capillary tip within the ion-source chamber. For the ASAP experiment, the open end of a melting point tube was inserted and removed from the 100-ppm p-hydroxybenzoic acid solution with 0.1% aqueous NH4OH, after which the melting point tube was secured into the ASAP probe. The corona voltage was set at 5.0 kV, sampling cone 11 V, extraction cone 1.5 V, desolvation-gas flow rate 380 L/Hr and



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Vernier-probe-adjuster position of 5.92 mm. The probe, source and desolvation-gas temperatures were held at 100, 80 and 100 °C, respectively. For the sample flow-rate experiments, the ESI source conditions were: capillary voltage 2.76 kV, sampling cone 7 V, extraction cone 1.3 V, desolvation-gas flow rate 370 L/Hr of N2 and Vernier-probe-adjuster position of 5.92 mm.. The source and desolvation-gas temperatures were held at 80 and 200 °C, respectively. To investigation the effect of adding oxygen to the ion source chamber, O2 gas (Praxair, Inc.) was introduced into the ion source though the nesting hole for the PEEK union of the lockspray capillary via a plastic tube (I.D.~1.5 mm). Other conditions were: capillary voltage 3.0 kV, sampling cone 25 V, extraction cone 2.5 V, desolvation-gas flow rate 200 L/Hr of N2, O2 flow rate 400 L/Hr, sample infusion flow rate 10 µL/min, and Vernier-probe-adjuster position of 5.92 mm.. The source and desolvation-gas temperatures were held at 80 and 100 °C, respectively.

3. RESULTS AND DISCUSION Even though the solvent composition has been considered to be the most important parameter that determines the relative ratio of the carboxylate and phenoxide ions (C-/P-), our investigations show that in fact several instrumental parameters exert dramatic effects on the gas-phase abundance of the two species.

Effect of Probe Position To start with, gaseous negative ions were generated from p-hydroxybenzoic acid by electrospraying a 100-ppm aqueous solution containing 0.1% NH4OH. The negative-ion mass spectrum recorded from this solution showed an intense signal at m/z 137 for deprotonated phydroxybenzoic acid. The m/z 137 ion generated in this way was then mass selected by the quadrupole analyzer (Q) and transferred to the travelling-wave ion-mobility (TWIM) cell of a Synapt G2 HDMS instrument. The power of ion-mobility mass spectrometry to separate structurally closely related ions is well documented.15-17 In addition to size and shape, the charge distribution of an ion also plays an important role on its relative mobility thorough a gasfilled mobility cell.5 As recognized by Bowers et al., it is the strength of interaction of the charged species with the neutral mobility gas that determines the travel time of an ion in a mobility cell.18 6 ACS Paragon Plus Environment

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Similar to results reported by Schröder et al.,5 we observed two peaks with different arrival times for the carboxylate and phenoxide species upon ion mobility separation of the massselected the m/z 137 ion (Figure 1). The peak at 5.20 ms represented the phenoxide, which is known to be more mobile than the carboxylate. The computations carried out by Schröder et al. show that the charge of the carboxylate tautomer is primarily localized on the carboxyl group. Consequently, the carboxylate tautomer manifests a longer travel time because it undergoes more induced-dipole interactions with the mobility gas. We observed that the mere spatial position of the ESI capillary relative to the entrancecone orifice of the mass analyzer exerts a dramatic effect on the P-/C- abundance ratio. The ESI probe of the Synapt G2 HDMS instrument has a built-in Vernier probe adjuster that enables pivoting the spray tip so that the physical distance between it and the entrance orifice can be controlled (Figure S1). When the Vernier adjuster is set at a high value (e.g. 10 mm), the probe is nearly vertical, and the probe tip is at its farthermost position from the mass spectrometer cone orifice (Figure S1). Figure 1 depicts the observed effect of the probe position on the P-/Crelative abundance ratio. When the probe is tilted closer to the entrance orifice, the carboxylate becomes the dominant species. However, when the spray head is moved away from the entrance orifice, then the phenoxide becomes the predominant species (Figure 1, Inset B). In other words, the population ratio can be changed drastically and manipulated in a reproducible manner, by a simple “turn of a screw,” without changing the spray solvent composition. Even though relative peak intensity differences of specific ion clusters, 14,19 and signal intensity enhancements20 due to changes of the distance between the spray capillary and the entrance orifice under pneumatically assisted ESI conditions have been noted, variations in the population densities of tautomeric forms due to electric field effects have not been recognized previously. Hiraoka et al. was one the first groups to report on the discriminatory ion-evaporation from charged droplets due to the capillary position.19 Janusson et al.14 reported that the intensities of signals recorded from an equimolar mixture of tetramethylammoniun (TMA) and bis(triphenylphosphine)iminium (PPN) ions were dependent on the probe position. These researchers rationalized that TMA ions, being small and spherical, are more readily extracted from droplets than PPN ions with larger collisional cross section. On the other hand, when the



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probe tip was closer to the cone aperture, i.e. under high-field conditions, they observed that the PPN peak dominated the spectrum.

Figure 1. A plot of the natural logarithm of average P−/C− peak intensity ratios against different Vernier-probe adjuster settings (N = 3). The m/z 137 ion generated by ESI at capillary voltage of 2.22 kV, from a sample of p-hydroxybenzoic acid in water containing 0.1% NH4OH was subjected to ion-mobility separation. The sampling cone was set to 11 V, extraction cone 1.5 V, sample infusion flow rate 10 µL/min and desolvation-gas flow rate 370 L/Hr. The source and desolvation-gas temperatures were held at 80 and 100 °C, respectively. The arrival times of the m/z 137 anions were recorded at different probeposition settings. Insets A and B show arrival-time profiles recorded at Vernier-probeadjuster position of 4.92 or 9.92 mm, respectively (the small peak at 4.2 ms represents the phenoxide ion, a product of decarboxylation of m/z 137).

To rationalize our experimental observations, we must first consider the mechanism of ion generation by electrospray ionization, which is still a highly debated question. 20-22 Ion evaporation (IEM) from the charged droplets is one of the models that has been proposed. 23-25 After charged droplets are ejected from the Taylor cone, the droplets undergo shrinkage and


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Coulombic explosions. Assuming that the IEM theory holds, solvated ions leave the droplets and become gaseous ions even before the Rayleigh limit is reached. The applied electric field is believed to be sufficient to distort the potential energy distribution at the liquid surface to enable the direct emission of solvated ions present at the surface to the gas phase.26 Presumably, under high-field conditions ions are dragged from levitated droplets by electrostatic forces to the mass analyzer without any significant changes to their chemical structure. In practice, the ESI head position is usually adjusted to obtain the best spray stability and signal intensity. As the probe tip is moved away from the mass spectrometer entrance orifice, the electric field at the tip of the capillary is lowered. When the distance between the spray capillary and its counter-electrode is short, then the electric field at the surface of a droplet becomes sufficiently high to extract ions from charged droplets by the IEM mechanism. According to Touboul et al., the distance between the spray capillary and its counterelectrode (d) has no effect on the survival yield of gaseous ions.20 However, the Coulombic repulsion between charged droplets and gaseous ions ejected under high-field conditions induces an increase in the kinetic energy of ions.27 Kinetically activated ions undergo energetic collisions with residual molecules and are subjected to vibrational excitation. As emphasized by Campbell et al.,28 high translational and vibrational energies of ions reduce the likelihood of forming associative bimolecular complexes. Thus, ions extracted under high-field conditions are dragged into the mass analyzer without significant changes to their chemical structure even though such structures are thermodynamically disfavored. In other words, populations of ions dragged out by the field effect mostly preserve their solution-based ionic structures. Using this model, we can rationalize why the carboxylate structure is predominantly found among the gasphase ions extracted under high-field conditions, even though it is not the preferred structure in the gas phase. In contrast, when d is increased, the electric field becomes weaker. Moreover, a longer distance between the spray needle and the entrance orifice affords more efficient solvent evaporation rate.29 In other words, the ion evaporation mechanism appears to play a lesser role under low-field conditions, and the charge-residue mechanism (CRM) may become the prominent ion-generation method as the spray capillary is moved away from the spectrometer orifice.20 Thus, under low-field conditions, the solvent continues to evaporate and the charge9 ACS Paragon Plus Environment


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residue mechanism contributes more to the gaseous-ion generation process. The ions generated by charge-residue mechanism are kinetically less activated. Consequently, such ions are able to interact with residual water within the atmospheric-pressure outer source in an associative manner. According to the conclusions made by Tian et al. the gaseous carboxylate ions are able to transform to the more stable phenoxide form by interacting with solvent molecules by a relay mechanism.1,2 We propose that kinetically less activated carboxylate ions generated under lowfield conditions interact with residual water molecules in the outer ion source, and transform into the thermodynamically more stable phenoxide by a relay mechanism similar to that described by Tian et al.1,2 A simplified illustration to depict this mechanism is given in Scheme 2. The presence of water molecule dimers to pentamers at atmospheric pressure has been demonstrated.30 Presumably, the relay mechanisms proceeds via an oligomer rather a single water molecule.

Scheme 2. Proposed relay mechanism for the formation of phenoxide from carboxylate of deprotonated p-hydroxybenzoic acid.31 To confirm that the C- → P- transformation is mediated by a bimolecular relay mechanism, we introduced an additional nebulized spray of water or methanol (30 µL/min) via the lock-spray-device needle of the instrument. The mechanical baffle of the lock-spray device was used to prevent direct mixing of additional solvent droplets with those from the analyte solution spray. The purpose of the additional flow was to increase the vapor pressure of the added solvents in the source enclosure. Arrival-time profiles recorded for the m/z 137 confirmed that the population of phenoxide increases when the vapor pressure of water or methanol is increased in the source (Figure S2). Our results are similar to observations made by Schröder et al.5 However, under our experimental conditions, we were able to render the phenoxide deprotomer to be the dominant species that reached the detector. Moreover, water was more efficient than methanol as the relay mediator for the transformation (Figure S2B). 10 ACS Paragon Plus Environment

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Effect of Capillary Voltage In 1993, Hiraoka et al. noted that ions are extracted discriminately when ESI capillary voltage is changed.19 Herein, we report that in addition to the position of the spray capillary, the voltage applied to it also plays a significant role on the relative ratio of the deprotomer populations of the m/z 137 ion. As the capillary voltage was increased from 2.0 kV to 3.75 kV, the intensity of the peak for the more mobile deprotomer also increased, and beyond a voltage of 2.75 kV that peak became dominant (Figure 2). Evidently, at low capillary voltages (ca. 2.0 kV), the carboxylate deprotomer is the predominant species, whereas at high capillary voltages (ca. 3.8 kV) the more abundant form is the phenoxide (Figure 2). At first look, this trend appeared to contradict the results from the probe-position experiments because there we concluded that ions transferred under high-field conditions are predominantly the carboxylate. Since at high capillary voltages the electric field is high, why should the phenoxide predominate?

Figure 2. A plot of the natural logarithm of average P−/C− peak intensity ratios against different capillary voltage settings (N = 3). The m/z 137 ion generated by ESI at Vernierprobe-adjuster setting of 5.92 mm, from a sample of p-hydroxybenzoic acid in water containing 0.1% NH4OH was subjected to ion-mobility separation. The sampling cone was set to 11 V, extraction cone 1.5 V, sample infusion flow rate 10 µL/min and desolvation-gas flow rate 370 L/Hr. The source and desolvation-gas temperatures were 11 ACS Paragon Plus Environment


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held at 80 and 100 °C, respectively. The arrival times of the m/z 137 anion were recorded at different capillary voltage settings. Insets A and B show arrival-time profiles recorded at capillary voltage settings of 2 and 3.75 kV, respectively. It is well known that electrical discharges occur at the ESI needle tip when the capillary voltage is high.32 It is also known that discharges occur at much lower capillary voltages when the system is operated at negative polarities.33 In fact, at higher capillary voltages, an electrospray ion source behaves more like an Atmospheric-Pressure Chemical Ionization (APCI) source. Ikonomou et al. reported at a very early stage that simultaneous corona discharges occur at high capillary voltages when water is used as the solvent.34 We recorded ion-mobility profiles from the m/z 137 ion, generated from a sample of p-hydroxybenzoic acid in water containing 0.1% NH4OH, using an ESCi35 (a combined ESI and APCI) source. When the APCI corona voltages were set between 3.5-5.0 kV, we noted not just that the phenoxide form is the predominant deprotomer but also that the population distribution profiles were not drastically affected by the corona-voltage value (Figure S3). Analogously, the m/z 137 ion, generated by warming a solid sample of p-hydroxybenzoic acid under Helium-Plasma Ionization (HePI) conditions36,37 (Supplementary Figure S4), or by Atmospheric-pressure Solids Analysis Probe (ASAP)38 (Supplementary Figure S5), was found to exist essentially as the phenoxide form. Under APCI, HePI, and ASAP conditions, the p-hydroxybenzoic acid molecules are first transfer to the gas phase as neutral molecules, which only then undergo deprotonation by the gaseous negative ions generated by the source-plasma discharges. For multi-functional compounds, the ionization is predicted to take place preferentially at the most acidic site. In the gas phase, the hydroxyl group of p-hydroxybenzoic acid is more acidic than the carboxyl group.39 Thus, we envisaged that it is the contribution of the APCI mechanism that favors the phenoxide population as the capillary voltages is increased (Figure 2). Yamashita and Fenn reported that discharges are reduced by the presence of a strongelectron-affinity gas in the vicinity of the needle tip of the ion source.32 This is because gases such as O2 scavenge electrons and minimize the ionization by APCI-like mechanisms. To test our hypothesis that an increase in phenoxide population is correlated with the capillary voltage increase due to APCI discharges, we engulfed the ESI ion source with O2 gas. (Similar discharge suppression studies have been done with SF6.34,40). Figure 3 clearly shows that with the addition of O2 to the ion source, the relative abundance of the phenoxide decreases even at a capillary 12 ACS Paragon Plus Environment

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voltage of 2.5 kV. Our results demonstrate that there is some contribution from the APCI mechanism even at low capillary voltages. In general, as the capillary voltage increases, the impact due to discharges increases in turn, and consequently the relative contribution to the composite m/z 137 ion by the phenoxide tautomer also increases. Moreover, it can be seen from Figure 3 that the effect of the discharges can be greatly reduced by the engulfing ion source with O2.

Figure 3. Plots of the natural logarithm of average P−/C− peak intensity ratio versus capillary voltage (N = 3) prepared from ion-mobility separation data recorded for the m/z 137 ion in a closed source engulfed with oxygen at a flow rate of 400 L/Hr (red curve), or without oxygen (blue curve). For both experiments a sample of p-hydroxybenzoic acid in water containing 0.1% NH4OH was sprayed at a Vernier-probe-adjuster setting of 5.92 mm. Insets A, and B show arrival-time profiles recorded at a capillary voltage setting of 2.5 kV, whereas insets C and D depict profiles recorded at 4.5 kV. The peak at 4.2 ms represents the phenoxide ion. The sampling cone was set to 11 V, extraction cone 1.5 V, sample infusion flow rate 10 µL/min and desolvation-gas flow rate 370 L/Hr. The source and desolvation-gas temperatures were held at 80 and 100 °C, respectively. Effect of Desolvation-Gas Temperature



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The ambient temperature of the atmospheric-pressure ion source also influences the relative ratio of the tautomer population densities. In a Synapt G2 HDMS instrument the temperature of the enclosed spray chamber is controlled primarily by the desolvation-gas temperature.

Figure 4. A plot of the natural logarithm of average P−/C− peak intensity ratios against different desolvation gas temperatures settings (N = 3). The m/z 137 ion generated by ESI at a capillary voltage of 2.22 kV, from a sample of p-hydroxybenzoic acid in water containing 0.1% NH4OH was subjected to ion-mobility separation. The sampling cone was set to 11 V, extraction cone 1.5 V, sample infusion flow rate 10 µL/min, desolvationgas flow rate 370 L/Hr and Vernier-probe-adjuster position of 5.92 mm. The sourceblock temperature was held at 80 °C. The arrival times of the m/z 137 anion were recorded at different desolvation-gas temperatures. Insets A and B show arrival-time profiles recorded at desolvation-gas temperatures of 100 and 500 °C, respectively.

To demonstrate the temperature effect on the P−/C− intensity ratio, the mobility of the m/z 137 ions generated at different desolvation-gas temperatures by electrospraying (at a fixed probe position of 5.92 mm) a ca. 100-ppm aqueous solution of p-hydroxybenzoic acid containing 10-2 14 ACS Paragon Plus Environment

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M NH4OH was monitored. Although at lower desolvation-gas temperatures, the carboxylate predominates (Figure 4), as the desolvation-gas temperature is increased, a gradual increase of the phenoxide abundance was noted. At 500 ⁰C, the predominant species was the phenoxide. Schröder et al. briefly noted that the lowering the temperature in the ESI source leads to an increase of the carboxylate population. They presumed that an increase in temperature enables the carboxylate to surmount the energy barrier by a transformation mechanism mediated by protic solvent molecules to form the more stable phenoxide. Presumably, at higher temperatures the increase of the rate of collisions with residual water molecules leads to an increased conversion of the carboxylate to the phenoxide by the relay mechanism (Scheme 2). Although, the desolvation-gas temperature manifests a profound effect on the relative deprotomer populations of p-hydroxybenzoic acid, Janusson et al.14 reported that the relative peak intensities of signals recorded at two different temperatures from an equimolar mixture of tetramethylammoniun (TMA) and bis(triphenylphosphine)iminium (PPN) ions were not very different. In other words, the source temperature appears to have little direct effect on the extraction of ions from the liquid droplets. On the other hand, the temperature has a profound effect on transformations that take place after ions are extracted into the gas phase. Although the temperature of the desolvation gas is important, its flow rate does not appear to play a significant role on the tautomer ratio: no changes were observed in the arrivaltime profiles when the flow rate was gradually increased from 200 L/Hr to 400 L/Hr (Figure S6).

Effect of Sample Flow Rate Although Schröder and co-workers noted that the sample flow rate to bear some effect on the ratio of tautomer abundances, they considered this effect to be subtle, and did not pursue the matter in further detail.5 We conducted a series of experiments by ranging the infusion flow rate from 3 to 50 µL/min and noted that the phenoxide always dominates when the flow rates are under 20 µL/min (Figure 5). In contrast, the carboxylate prevails at flow rates between 30-50 µL/min, a change of the protonation site of p-(dimethylamino)chalcone (p-DMAC), from the carbonyl to the dimethylamino group has been noted when the sample infusion flow rate was increased under positive-ion-generating APCI conditions.11



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Figure 5. A plot of the natural logarithm of average P−/C− peak intensity ratios against different sample flow rate settings (N = 3). The m/z 137 ion generated by ESI at capillary voltage of 2.76 kV, from a sample of p-hydroxybenzoic acid in water containing 0.1% NH4OH was subjected to ion-mobility separation. The sampling cone was set to 7 V, extraction cone 1.3 V, desolvation-gas flow rate 300 L/Hr and Vernier-probe-adjuster position of 5.92 mm. The source and desolvation-gas temperatures were held at 80 and 200 °C, respectively. The arrival times of the m/z 137 anion were recorded at different sample flow rate settings. Insets A and B show arrival-time profiles recorded at sample flow rate settings of 3 and 50 µL/min, respectively. According to the equation proposed by Fernandez de la Mora and Locertales,41 when the liquid meniscus held at the exit of a metallic capillary tube charged to a high voltage forms a microjet, the radius of the droplets formed depends on the sample infusion flow rate (Equation 1, where R is the radius of droplets, and is the sample flow rate, is the permittivity of solvent and K is the conductivity of the solution). R ⁄

⁄

…………(Equation 1)

In other words, the droplet radius R is expected to increase with the flow rate.42 When the 16 ACS Paragon Plus Environment

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droplets are small, the solvent within the droplets evaporates rapidly due to the temperature of the hot ion source. However, when the radius of droplets increases, ion generation due to the charge-residue process is expected to be less efficient. In other words, ion generation from larger droplets is more congruent with the ion-evaporation model. As discussed in the probeposition section, the carboxylate is the more dominant product expected from the ion evaporation procedure. The relationship observed between the droplet size and the P−/C− ratio is congruent with the generalizations made by Dole.29 It is believed that the droplet size needed for the charge-residue model should be around 3 nm, whereas for the ion-evaporation model a droplet should be is about 10 nm in size.23,42

The Effect of Cone Voltage Gaseous ions generated by atmospheric-pressure ionization techniques must pass several intermediate vacuum regions before they enter the mass analyzer maintained under high vacuum. As the stream of atmospheric gases expands into the first low-pressure region, it forms a supersonic jet. For efficient ion transfer, different electrical potentials are applied to the entrance orifice and the skimmer (entrance to the high vacuum region). In this intermediate-pressure region, ions undergo acceleration, due to the potential difference applied between the entrance orifice and the skimmer, and collide with the background gases. This potential difference is often referred to as the cone voltage. As the cone voltage is increased, gaseous ions undergo acceleration in this region, collide with background gases, and experience fragmentation. 43-45 We found that the cone voltage also exerts a profound influence on the tautomer ratio. Our cone-voltage experiments show that the carboxylate is the dominant species when the voltage difference is relatively low (Figure 6, Inset A). However, when the cone voltage was gradually increased, the formation of phenoxide became more favored at about 35 V (Figure 6, Inset B). A similar observation had been noted by Schröder and his co-workers for the m/z 137 ion generated from acetonitrile solutions.5 However, they attributed the changes to the dissociation of the proton-bound dimer of p-hydroxybenzoic acid, which upon fragmentation only affords the phenoxide. We also noted that the P- tautomer is the dominant form when ions were generated from acetonitrile solutions (Supplementary Figure S7). However, it appears that the increase of phenoxide under high cone-voltage conditions in not due to the dissociation of the 17 ACS Paragon Plus Environment


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proton-bound dimer because an increase was observed even without the presence of the protonbound dimer in the ion source: MS1 spectra recorded from p-hydroxybenzoic acid under our experimental conditions, including low-to-high cone voltage settings, showed no observable peak (m/z 275) for the dimer (Supplementary Figure S8).

Figure 6. A plot of the natural logarithm of average P−/C− peak intensity ratios against different sampling cone voltage settings (N = 3). The m/z 137 ion generated by ESI at capillary voltage of 2.22 kV, from a sample of p-hydroxybenzoic acid in water containing 0.1% NH4OH, was subjected to ion-mobility separation. The ESI extraction cone 1.5 V, desolvation-gas flow rate 370 L/Hr, sample infusion flow rate 10 µL/min and Vernierprobe-adjuster position 5.92 mm. The source and desolvation-gas temperatures were held at 80 and 100 °C, respectively. The arrival times of the m/z 137 anion were recorded at different sampling cone voltage settings. Insets A and B show arrival-time profiles recorded at sampling voltage settings of 5 and 45 V, respectively.

4.

CONCLUSIONS


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Mass spectrometrists often adjust the position of the spray head, or reduce the capillary voltage to reduce the signal intensity when analyzing concentrated solutions. From the results presented here, it is evident that detuning an instrument is a poor practice because the relative intensities of distinct ions from different analytes, or even the same analyte can change discriminately as the probe position is adjusted. Attributing the gas-phase population distribution of carboxylate and phenoxide generated from p-hydroxybenzoic acid primarily to solvent effects, is an oversimplification. Herein, we have demonstrated that not only the relative populations of the two tautomers generated from the same solution change, but also that the predominant species can even be reversed by changing several source parameters. Evidently, the population ratio depends on factors such as probe position, capillary voltage, cone voltage, source temperature, flow rate, ambient gases present in the source region, concentration of the sample. Generally, these effects can be very subtle and further experimentations based on physicochemical principles are needed before conclusive generalization can be made. Our conclusions support the suggestions made by Janusson et al.14 that investigating of a certain response variation factor on population distributions of different ions without ensuring that all other parameters are held constant may not provide meaningful results, and may result in considerable confusion.

ASSOCIATED CONTENT

Supporting Information Supporting Information available free of charge on the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS



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This research was supported by funds provided by Stevens Institute of Technology (Hoboken, NJ). We thank Julius Pavlov for his comments on the manuscript, and suggesting the use of the word deprotomer.

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According to one definition, tautomers are isomers of a compound which differ only in the position of the protons and electrons. The carbon skeleton of the compound remains unchanged. A reaction which involves a simple proton transfer in an intramolecular fashion is called tautomerism (http://www.chem.ox.ac.uk/vrchemistry/nor/notes/tautomers.htm). Although often used in literature, sensu stricto, it is incorrect to refer to carboxylate (C-) and phenoxide (P-) forms as tautomers because their intramolecular conversion does not take place solely within themselves. see: Lalli, P. M.; Iglesias, B. A.; Toma, H. E.; de Sa, G. F.; Daroda, R. J.; Silva Filho, J. C.; Szulejko, J. E.; Araki, K.; Eberlin, M. N. J. Mass. Spectrom. 2012, 47, 712–719. We prefer to call such isomers, “deprotomers.”

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Figure captions Figure 1. A plot of the natural logarithm of average P−/C− peak intensity ratios against different Vernier-probe adjuster settings (N = 3). The m/z 137 ion generated by ESI at capillary voltage of 2.22 kV, from a sample of p-hydroxybenzoic acid in water containing 0.1% NH4OH was subjected to ion-mobility separation. The sampling cone was set to 11 V, extraction cone 1.5 V, sample infusion flow rate 10 µL/min and desolvation-gas flow rate 370 L/Hr. The source and desolvation-gas temperatures were held at 80 and 100 °C, respectively. The arrival times of the m/z 137 anions were recorded at different probe-position settings. Insets A and B show arrival-time profiles recorded at Vernier-probe-adjuster position of 4.92 or 9.92 mm, respectively (the small peak at 4.2 ms represents the phenoxide ion, a product of decarboxylation of m/z 137).

Figure 2. A plot of the natural logarithm of average P−/C− peak intensity ratios against different capillary voltage settings (N = 3). The m/z 137 ion generated by ESI at Vernier-probeadjuster setting of 5.92 mm, from a sample of p-hydroxybenzoic acid in water containing 0.1% NH4OH was subjected to ion-mobility separation. The sampling cone was set to 11 V, extraction cone 1.5 V, sample infusion flow rate 10 µL/min and desolvation-gas flow rate 370 L/Hr. The source and desolvation-gas temperatures were held at 80 and 100 °C, respectively. The arrival times of the m/z 137 anion were recorded at different capillary voltage settings. Insets A and B show arrival-time profiles recorded at capillary voltage settings of 2 and 3.75 kV, respectively.

Figure 3. Plots of the natural logarithm of average P−/C− peak intensity ratio versus capillary voltage (N = 3) prepared from ion-mobility separation data recorded for the m/z 137 ion in a closed source engulfed with oxygen at a flow rate of 400 L/Hr (red curve), or without oxygen (blue curve). For both experiments a sample of p-hydroxybenzoic acid in water containing 0.1% NH4OH was sprayed at a Vernier-probe-adjuster setting of 5.92 mm. Insets A, and B show arrival-time profiles recorded at a capillary voltage 23 ACS Paragon Plus Environment


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setting of 2.5 kV, whereas insets C and D depict profiles recorded at 4.5 kV. The peak at 4.2 ms represents the phenoxide ion. The sampling cone was set to 11 V, extraction cone 1.5 V, sample infusion flow rate 10 µL/min and desolvation-gas flow rate 370 L/Hr. The source and desolvation-gas temperatures were held at 80 and 100 °C, respectively.

Figure 4. A plot of the natural logarithm of average P−/C− peak intensity ratios against different desolvation gas temperatures settings (N = 3). The m/z 137 ion generated by ESI at a capillary voltage of 2.22 kV, from a sample of p-hydroxybenzoic acid in water containing 0.1% NH4OH was subjected to ion-mobility separation. The sampling cone was set to 11 V, extraction cone 1.5 V, sample infusion flow rate 10 µL/min, desolvation-gas flow rate 370 L/Hr and Vernier-probe-adjuster position of 5.92 mm. The source-block temperature was held at 80 °C. The arrival times of the m/z 137 anion were recorded at different desolvation-gas temperatures. Insets A and B show arrival-time profiles recorded at desolvation-gas temperatures of 100 and 500 °C, respectively.

Figure 5. A plot of the natural logarithm of average P−/C− peak intensity ratios against different sample flow rate settings (N = 3). The m/z 137 ion generated by ESI at capillary voltage of 2.76 kV, from a sample of p-hydroxybenzoic acid in water containing 0.1% NH4OH was subjected to ion-mobility separation. The sampling cone was set to 7 V, extraction cone 1.3 V, desolvation-gas flow rate 300 L/Hr and Vernier-probe-adjuster position of 5.92 mm. The source and desolvation-gas temperatures were held at 80 and 200 °C, respectively. The arrival times of the m/z 137 anion were recorded at different sample flow rate settings. Insets A and B show arrival-time profiles recorded at sample flow rate settings of 3 and 50 µL/min, respectively.

Figure 6. A plot of the natural logarithm of average P−/C− peak intensity ratios against different sampling cone voltage settings (N = 3). The m/z 137 ion generated by ESI at capillary 24 ACS Paragon Plus Environment

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voltage of 2.22 kV, from a sample of p-hydroxybenzoic acid in water containing 0.1% NH4OH, was subjected to ion-mobility separation. The ESI extraction cone 1.5 V, desolvation-gas flow rate 370 L/Hr, sample infusion flow rate 10 µL/min and Vernierprobe-adjuster position 5.92 mm. The source and desolvation-gas temperatures were held at 80 and 100 °C, respectively. The arrival times of the m/z 137 anion were recorded at different sampling cone voltage settings. Insets A and B show arrival-time profiles recorded at sampling voltage settings of 5 and 45 V, respectively.



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Graphical Abstract 26x15mm (600 x 600 DPI)

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Figure 1. A plot of the natural logarithm of average P−/C− peak intensity ratios against different Vernierprobe adjuster settings (N = 3). The m/z 137 ion generated by ESI at capillary voltage of 2.22 kV, from a sample of p-hydroxybenzoic acid in water containing 0.1% NH4OH was subjected to ion-mobility separation. The sampling cone was set to 11 V, extraction cone 1.5 V, sample infusion flow rate 10 µL/min and desolvation-gas flow rate 370 L/Hr. The source and desolvation-gas temperatures were held at 80 and 100 °C, respectively. The arrival times of the m/z 137 anions were recorded at different probe-position settings. Insets A and B show arrival-time profiles recorded at Vernier-probe-adjuster position of 4.92 or 9.92 mm, respectively (the small peak at 4.2 ms represents the phenoxide ion, a product of decarboxylation of m/z 137). 25x15mm (600 x 600 DPI)



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Figure 2. A plot of the natural logarithm of average P−/C− peak intensity ratios against different capillary voltage settings (N = 3). The m/z 137 ion generated by ESI at Vernier-probe-adjuster setting of 5.92 mm, from a sample of p-hydroxybenzoic acid in water containing 0.1% NH4OH was subjected to ion-mobility separation. The sampling cone was set to 11 V, extraction cone 1.5 V, sample infusion flow rate 10 µL/min and desolvation-gas flow rate 370 L/Hr. The source and desolvation-gas temperatures were held at 80 and 100 °C, respectively. The arrival times of the m/z 137 anion were recorded at different capillary voltage settings. Insets A and B show arrival-time profiles recorded at capillary voltage settings of 2 and 3.75 kV, respectively. 25x13mm (600 x 600 DPI)


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Figure 3. Plots of the natural logarithm of average P−/C− peak intensity ratio versus capillary voltage (N = 3) prepared from ion-mobility separation data recorded for the m/z 137 ion in a closed source engulfed with oxygen at a flow rate of 400 L/Hr (red curve), or without oxygen (blue curve). For both experiments a sample of p-hydroxybenzoic acid in water containing 0.1% NH4OH was sprayed at a Vernier-probe-adjuster setting of 5.92 mm. Insets A, and B show arrival-time profiles recorded at a capillary voltage setting of 2.5 kV, whereas insets C and D depict profiles recorded at 4.5 kV. The peak at 4.2 ms represents the phenoxide ion. The sampling cone was set to 11 V, extraction cone 1.5 V, sample infusion flow rate 10 µL/min and desolvation-gas flow rate 370 L/Hr. The source and desolvation-gas temperatures were held at 80 and 100 °C, respectively. 24x12mm (600 x 600 DPI)



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Figure 4. A plot of the natural logarithm of average P−/C− peak intensity ratios against different desolvation gas temperatures settings (N = 3). The m/z 137 ion generated by ESI at a capillary voltage of 2.22 kV, from a sample of p-hydroxybenzoic acid in water containing 0.1% NH4OH was subjected to ion-mobility separation. The sampling cone was set to 11 V, extraction cone 1.5 V, sample infusion flow rate 10 µL/min, desolvation-gas flow rate 370 L/Hr and Vernier-probe-adjuster position of 5.92 mm. The source-block temperature was held at 80 °C. The arrival times of the m/z 137 anion were recorded at different desolvation-gas temperatures. Insets A and B show arrival-time profiles recorded at desolvation-gas temperatures of 100 and 500 °C, respectively. 26x16mm (600 x 600 DPI)


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Figure 5. A plot of the natural logarithm of average P−/C− peak intensity ratios against different sample flow rate settings (N = 3). The m/z 137 ion generated by ESI at capillary voltage of 2.76 kV, from a sample of p-hydroxybenzoic acid in water containing 0.1% NH4OH was subjected to ion-mobility separation. The sampling cone was set to 7 V, extraction cone 1.3 V, desolvation-gas flow rate 300 L/Hr and Vernier-probeadjuster position of 5.92 mm. The source and desolvation-gas temperatures were held at 80 and 200 °C, respectively. The arrival times of the m/z 137 anion were recorded at different sample flow rate settings. Insets A and B show arrival-time profiles recorded at sample flow rate settings of 3 and 50 µL/min, respectively. 25x14mm (600 x 600 DPI)



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Figure 6. A plot of the natural logarithm of average P−/C− peak intensity ratios against different sampling cone voltage settings (N = 3). The m/z 137 ion generated by ESI at capillary voltage of 2.22 kV, from a sample of p-hydroxybenzoic acid in water containing 0.1% NH4OH, was subjected to ion-mobility separation. The ESI extraction cone 1.5 V, desolvation-gas flow rate 370 L/Hr, sample infusion flow rate 10 µL/min and Vernier-probe-adjuster position 5.92 mm. The source and desolvation-gas temperatures were held at 80 and 100 °C, respectively. The arrival times of the m/z 137 anion were recorded at different sampling cone voltage settings. Insets A and B show arrival-time profiles recorded at sampling voltage settings of 5 and 45 V, respectively. 24x12mm (600 x 600 DPI)


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Effect of Electrospray Ionization Source Conditions on the Tautomer

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