Nucleophilic Aromatic Substitution Between Halogenated Benzene Dopants and Nucleophiles in Atmospheric Pressure Photoionization
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In a preceding work with dopant assisted-atmospheric pressure photoionization (DA-APPI), an abundant ion at [M + 77]+ was observed in the spectra of pyridine and quinoline with chlorobenzene dopant. This contribution aims to reveal the identity and route of formation of this species, and to systematically investigate structurally related analytes and dopants. Compounds containing N-, O-, and S-lone pairs were investigated with APPI in the presence of fluoro-, chloro-, bromo-, and iodobenzene dopants. Computational calculations on a density functional theory (DFT) level were carried out to study the reaction mechanism for pyridine and the different halobenzenes. The experimental and computational results indicated that the [M + 77]+ ion was formed by nucleophilic aromatic ipso-substitution between the halobenzene radical cation and nucleophilic analytes. The reaction was most efficient for N-heteroaromatic compounds, and it was weakened by sterical effects and enhanced by resonance stabilization. The reaction was most efficient with chloro-, bromo-, and iodobenzenes, whereas with fluorobenzene the reaction was scarcely observed. The calculated Gibbs free energies for the reaction between pyridine and the halobenzenes were shown to increase in the order I < Br < Cl < F. The reaction was found endergonic for fluorobenzene due to the strong C–F bonding, and exergonic for the other halobenzenes. For fluoro- and chlorobenzenes the reaction was shown to proceed through an intermediate state corresponding to [M + dopant]+, which was highly stable for fluorobenzene. For the bulkier bromine and iodine, this intermediate did not exist, but the halogens were shown to detach already during the approach by the nucleophile.
KeywordsAtmospheric pressure photoionization Dopant Gas-phase ion/molecule reactions ipso-substitution Nucleophilic aromatic substitution
Atmospheric pressure photoionization (APPI) [1, 2] is one of the most important ionization techniques in liquid chromatography-mass spectrometry (LC-MS) [3, 4, 5]. Lately, it has found increasing use also in gas-chromatography-mass spectrometry (GC-MS) [6, 7, 8, 9, 10, 11]. In APPI, the analyte (and solvent in case of LC-MS) is vaporized using heat, after which the ionization is initiated by 10.0 and 10.6 eV photons emitted by a krypton discharge vacuum ultraviolet (VUV) lamp. In theory, the photons can ionize any compound with ionization energy (IE) below 10.6 eV, to generate the molecular ion (M+.). However, in many analytical applications, the presence of oxygen and evaporated solvent molecules with IEs > 10.6 eV significantly decreases the ionization efficiency in direct APPI. This is due to absorption of the photons by the matrix and subsequent energy conversion into neutral photo-dissociation processes. Consequently, an additional ionizable matrix constituent (IE < 10.6 eV), a so called dopant, is often added in great abundance to enhance the competing photon-ion conversion process. In subsequent ion–molecule reactions, the primary dopant ion population transfers the charge to the less abundant analytes. The most commonly used dopants include toluene, acetone, anisole, and chlorobenzene. The best known reactions in dopant-assisted APPI (DA-APPI) in positive ion mode include charge exchange and proton transfer, which lead to formation of M+. and protonated molecules ([M + H]+), respectively .
Our previous study compared the ionization mechanisms in direct and DA-APPI, as well as in direct and dopant-assisted atmospheric pressure laser ionization (APLI), both operated in an ultra-pure GC-MS system . Besides the expected M+. and [M + H]+ ions, very intense ions corresponding to [M + 77]+ were observed in pyridine and quinoline spectra in both DA-APPI and DA-APLI, when chlorobenzene was used as the dopant. According to high resolution mass spectra the mass 77 corresponded to C6H5. Two possible identities for the ion were suggested: a phenylium ion adduct, or a product of nucleophilic substitution reaction between chlorobenzene and pyridine/quinoline. Phenylium ion has been reported to be formed in the photodissociation of chlorobenzene , and a small ion (<1%) at m/z 77 was indeed observed in the pure chlorobenzene spectrum. The ion/molecule chemistry literature provides several reports on substitution reactions in the gas phase, often involving halobenzenes. Electrophilic ipso-substitution  was first reported by van Thuijl et al. in chemical ionization (CI) between protonated ammonia and methylamine reagent gases, and monosubstituted benzenes (including halobenzenes) [16, 17]. The reaction resulted in formation of aniline and N-methylaniline M+. ions, respectively. Later, Thölmann and Grützmacher reported a reaction between monosubstituted benzene radical cation and neutral ammonia in a Fourier transform-ion cyclotron resonance (FT-ICR) cell, again resulting in formation of aniline . Nucleophilic substitution between chloro- and nitrobenzene and small nucleophiles, such as ammonia and methylamine, has also been reported in atmospheric pressure chemical ionization (APCI), where the reaction was proven to take place between C6H5X+. and neutral ammonia . Later, Stone et al. showed that also [NH4]+ can act as the reagent ion . Ipso-substitution has also been shown to take place in clusters of aryl halides and ammonia after resonant multiphoton ionization [21, 22, 23, 24, 25, 26]. In 2010, Campbell observed the formation of [M + 77]+ ions from a reaction between pyridine and chloro-/bromobenzene M+. ions . He used a dual inlet APCI source, which he called a “dynamic reaction vessel” to point out the dominating ion chemistry. In collision induced experiments, Campbell proved the [M + 77]+ species to be a stable molecule, which he postulated to be the product of a gas-phase ipso-substitution. In APPI, substitution of the halogen with a methoxy group has been reported for halobenzene dopants in the presence of methanol . Several studies exist where density functional theory (DFT) has been utilized to explain the nucleophilic substitution reaction theoretically [29, 30].
This study aims at further investigations on the formation of the [M + 77]+ ion in DA-APPI. Besides chlorobenzene, the tendency of other halobenzene dopants to undergo the reaction is investigated, as well as a wider range of nucleophilic analytes. For a deeper understanding of the reaction mechanisms between pyridine and the different halobenzene dopants, computational calculations on a DFT level are presented.
Pyridine was purchased from Acros Organic (Fair Lawn, NJ, USA) and all other analytes from Sigma-Aldrich (Steinheim, Germany). Toluene (99.9%), bromobenzene (≥99.5%), iodobenzene (98%), and fluorobenzene (≥99.5%) were from Sigma-Aldrich, chlorobenzene (>99%) from Merck (Hohenbrunn, Germany), and hexane (HPLC grade) from VWR International (Leuven, Belgium).
Two mass spectrometers were used for the measurements: a Thermo Fisher Scientific (Bremen, Germany) Exactive Orbitrap and a Bruker Daltonik GmbH (Bremen, Germany) amaZon speed ETD trap. Custom-made, tightly sealed API interfaces for both instruments provided chemically and photo-physically inert matrices. The API interface for the Orbitrap has been described in detail before . Briefly, a commercial transfer line (Thermo Fisher Scientific) was used to guide the sample flow from the gas chromatograph (GC) column to an air-tight, conically-shaped ion source, which was heated to 250°C. A continuous make-up gas flow with high purity N2 was maintained at 850 mL/min. The capillary temperature was 250°C, and capillary, tube lens, and skimmer voltages were 25, 45, and 16 V, respectively. The mass range was set to m/z 50–1000. The source geometry of the ion source used with the amaZon speed ETD trap consisted of a conical ionization volume inlet, which lead into a cylindrical reaction tube . The length and the inner diameter of the reaction tube were 85 and 5 mm, respectively. The source was operated at elevated pressures of about 1200 mbar. N2 make-up gas was fed to the ionization chamber to match the inlet flow parameters of the mass spectrometer (760 mL/min) at operational source temperature of 300°C. In both setups, an additional dopant line was connected via a t-piece directly to the make-up gas entrance of the source enclosure. Headspace of the dopant was added to the make-up gas with a gas syringe and a syringe pump at a flow rate of 50–200 μL/min. A low-pressure Kr discharge lamp with a radio frequency (rf) driver from Syagen (Santa Ana, CA, USA) provided radiative output at 10.0 and 10.6 eV. All measurements were performed in positive ion mode. Comparative experiments were performed using an AB Sciex (Toronto, Canada) APPI source mounted on a API 300 triple quadrupole (AB Sciex) instrument. Continuous flow of 1 ppm pyridine in chlorobenzene was introduced at 20 μL/min flow rate. The ion spray voltage, and declustering, focusing, and entrance potentials were 2000, 34, 150, and 7 V, respectively.
For the Exactive Orbitrap setup the compounds were separated with a TRACE 1310 GC equipped with a TR-Dioxin 5MS column (30 m × 0.25 mm i.d. × 0.1 μm), both from Thermo Fisher Scientific. The helium carrier gas flow rate was set to 1.50 mL/min. The GC temperature program was as follows. Initial temperature was 50°C for 1 min, 30°C/min up to 150°C, 20°C/min up to 200°C, 30°C/min up to 300°C, and 20°C/min up to 320°C, hold time 5 min. The GC transfer line and injector temperature were both set to 250°C. Headspace samples of the pure standards (pyridine, 2-methylpyridine, 2,3-lutidine (2,3-dimethylpyridine), 2,6-lutidine (2,6-dimethylpyridine), pyridazine, pyrimidine, pyrrole, thiophene, furan, and benzofuran) were injected with a 0.5 μL syringe with a split ratio of 10,000. Acridine and quinoline were analyzed from a 10 μM solution, and pyrazine and thianaphthene were analyzed from a 1 μg/mL solution. Both solutions were prepared in hexane and injected in splitless injection mode.
With the amaZon speed ETD trap, an Agilent 7890 A GC system was used in combination with the same column type and carrier gas flow rate as for the Exactive Orbitrap setup. The GC temperature program was as follows. Initial temperature was 50°C for 1 min, 20°C/min up to 325°C, hold time 1 min. The injector as well as the transfer line temperature were maintained at 250°C. Headspace samples of the pure standards (piperidine, oxazole, isoxazole, 2-cyclohexen-1-one, cyclohexanone, N-methylpiperidine, morpholine, N-methylmorpholine) were injected with a 0.5 μL syringe with a split ratio of 200.
We expect the observed ion–molecule reactions to be in thermodynamic equilibrium, since the experimental conditions at atmospheric pressure provide high collision rates with fairly long reaction times in the ms up to s domain . Thus ∆RG is assumed to be the right quantity to picture the experimental results.
Results and Discussion
In order to test whether the observed reaction takes place also in a conventional LC-MS type APPI source, experiments were done using a commercially available APPI source, where a continuous flow of 1 ppm pyridine in chlorobenzene was introduced at 20 μL/min. The resulting spectrum showed both [M + H]+ and [M + 77]+ ions of pyridine at approximately same intensities (Figure S1 of the Supplementary material). Consequently, the reaction is possible under these conditions as well, but it is less efficient than in the ultra-pure APPI interface since the proportion of [M + 77]+ was significantly lower than in the measurements with the high purity APPI system. It is suggested that the reaction proceeds more efficiently in the closed, high-purity APPI interface. In conventional, non-tight APPI systems, the neutral background is more diverse, as also reflected in the elevated ion background of the spectrum, which consequently opens up additional and competing reaction pathways, such as the formation of [M + H]+.
Computational Calculations for the Reaction Between Pyridine and the Halobenzenes
DFT calculations were performed to elucidate feasible reaction mechanisms that would explain the experimental observations. Pyridine was chosen as an example compound, with the four halobenzenes as dopants. For the calculated geometries, see Tables S2–S5 in Supplementary Material. Calculations involving other compounds of this study are currently under work and will be reported in another publication.
In principle, there are two possible ways for a substitution reaction to proceed: either the halogen leaves the radical cation through a collision in a first reaction step creating a phenyl cation, which then would react with the analyte, or the analyte attacks the halobenzene radical cation and the halogen leaves the molecule because of this attack (Scheme 1). The latter mechanism is more likely since a phenyl cation was not observed in the MS experiments. As shown in Figure S2 (Supplementary Material), the C1 carbon of each halobenzene radical cation carries the most positive electrostatic potential. Consequently, the nitrogen lone pair of pyridine is expected to approach C1. However, with increasing weight, the positive charge distributes more and more to the halogen. Therefore, the configuration of the first approach in case of chlorine, bromine, or iodine is described by a weak bonding between the lone pair of the pyridine and the halogen atom. Interestingly, in the case of fluorobenzene, the dominance of the negative charge on the halogen leads to a hydrogen bond between the fluorine and the ortho-hydrogen of pyridine in the initial approach (see Figure S3 in Supplementary Material).
Calculated Overall Standard Gibbs Free Energies for the Nucleophilic Substitution Between Different Halobenzene Dopants and Pyridine
The [M + 77]+ ion observed in APPI is likely to have formed in a nucleophilic ipso-substitution between the nucleophilic analyte and the halobenzene dopant radical cation. Corresponding reactions have been reported in literature for (mainly) smaller amines but never before in APPI. Unlike in traditional APPI sources, the GC-APPI-MS interfaces used in this study utilize closed ionization volumes, where atmospheric gases and impurities are excluded. As a result, the number of possible ionization pathways is more limited than in a conventional APPI source. It is suggested that GC-APPI interfaces of this type could also be used to study other gas-phase ion/molecule reactions. Some reactions could perhaps be utilized for more efficient and selective ionization of particular compound classes that otherwise give low signals in APPI.
Financial support by the Academy of Finland (projects 218150, 255559, 268757, and 286014), Magnus Ehrnrooth Foundation, and iGenTrax is gratefully acknowledged. Thermo Scientific is acknowledged for supplying the Orbitrap, the GC, and the consumables, and Morpho Detection for supplying the APPI source. Professor Jari Yli-Kauhaluoma is acknowledged for his expert advice regarding the organic chemistry reactions.
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