Nucleophilic Aromatic Substitution Between Halogenated Benzene Dopants and Nucleophiles in Atmospheric Pressure Photoionization

  • Tiina J. Kauppila
  • Alexander Haack
  • Kai Kroll
  • Hendrik Kersten
  • Thorsten Benter
Research Article


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.

Graphical Abstract


Atmospheric 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 [12].

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 [13]. 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 [14], 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 [15] 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 [18]. 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 [19]. Later, Stone et al. showed that also [NH4]+ can act as the reagent ion [20]. 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 [27]. 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 [28]. 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).

Mass Spectrometry

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 [13]. 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 [31]. 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.

Gas Chromatography

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.

Computational Calculations

Theoretical support for the experimental data and proposed reaction mechanisms were provided by ab-initio calculations with the software package Gaussian09 [32] on a UNIX-based computer cluster, with 4 × 16-Core CPUs (6282SE AMD Opteron; Advanced Micro Devices GmbH, Dornach, Germany) and 32 × 16 GB memory. Input files were generated with the graphical user interface GaussView 4.1 [33], also used for data evaluation and graphical representation. Calculations were carried out on DFT level with the B3LYP [34, 35] hybrid functional and the implemented 6-311+G(3df,3p) basis set for the lighter elements of hydrogen to bromine. An additional basis set from Feller et al. [36, 37] was used for calculations with iodine-containing compounds. The basis set superposition error (BSSE) was not included; however, it should be negligible for a comparison between the intermediate structures, and its effect on the energetic comparison between intermediates and products/reactants should be marginal. For the presented reaction mechanism, the BSSE would not change the results in any case. With these conditions, the absolute errors of the computed ionization energies for fluoro-, chloro-, bromo-, and iodobenzene were within 3% compared with experimental values given by Fujisawa et al. [38]. Similarly, errors of the presented energy calculations are expected to be within the same range. Single steps and their corresponding Gibbs free energies along the reaction coordinates were obtained by the following computational procedure: (1) decrease of the analyte-dopant-distance along the nitrogen (N) and halogen (X) carrying carbon (C1) coordinate d(N-C1); (2) increase of the C1 and X distance along the d(C1-X) coordinate, with geometry optimizations for each point on these coordinates. The approximate minimum structures or transition states derived from these scans were then (3) fully optimized with tight convergence criteria to a minimum or first order saddle point, respectively; (4) subsequent frequency calculations for each optimized structure confirmed the presence of a transition state, by looking at the right number of imaginary frequencies along the reaction coordinate. Furthermore, they provided thermodynamic data to obtain the corrected Gibbs free energy Gcorr, which, in sum with the electronic energy ε 0 , yields the standard Gibbs free energy of a reaction step according to Equation 1) [39]:
$$ {\varDelta}_R{G}^{\varnothing }={\displaystyle \sum_{products}\left({\upvarepsilon}_0+{G}_{corr}\right)-{\displaystyle \sum_{\mathit{\mathsf{reactants}}}\left({\upvarepsilon}_0+{G}_{corr}\right)}} $$

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 [40]. Thus ∆RG is assumed to be the right quantity to picture the experimental results.

Results and Discussion

APPI Measurements

A large group of compounds containing nitrogen, oxygen, or sulfur lone pairs in their structure (Figure 1 and Supplementary Table S1) was analyzed by GC-APPI-MS with fluoro-, chloro-, bromo-, and iodobenzene dopants. For comparison purposes, the same compounds were also analyzed with direct APPI (without a dopant), and with toluene dopant. The ions formed in direct APPI and with different dopants were compared, and special attention was paid to the presence of the [M + 77]+ ion, which presumably results from of a gas-phase nucleophilic ipso-substitution reaction (Scheme 1) [15]. The compounds with vast and persistent [M + 77]+ are classified as Group 1, amongst which we found pyridine, quinoline, 2-methylpyridine, 2,3-lutidine, pyrimidine, pyrazine, pyridazine, and oxazole (see Figure 1). In combination with chloro-, bromo-, and iodobenzene dopants, Group 1 showed dominating [M + 77]+ formation and comparably low abundance of M+ or [M + H]+. A common structural feature of these compounds is a N-heteroaromatic ring, which in case of oxazole also contains oxygen. All the compounds in Group 1 are strong bases and have PAs between 885 and 977 kJ/mol (pyrimidine to pyrazine; see Table S1 in Supplementary Material) [41]. Group 2 in Figure 1 encompasses the investigated compounds with less reactivity regarding the ipso-substitution. In these cases, the [M + 77]+ ion was observed with at least one of the halobenzene dopants, but it was not the main ion. The last classification in Figure 1 is Group 3, which covers all the investigated compounds without any observed tendency for the ipso-substitution reaction. In these cases the [M + 77]+ ion signal was completely absent. Interestingly, part of this class are acridine and 2,6-lutidine, which are closely related to pyridine and quinoline, both Group 1 classified compounds. This result is attributed to sterical hindrance caused by the two methyl groups in the case of 2,6-lutidine and the two rings in the case of acridine, attached to the carbons adjacent to the pyridine-ring nitrogen. Instead of [M + 77]+, acridine and 2,6-lutidine showed [M + H]+ as the main ion. Comparative mass spectra for pyridine, quinoline, and acridine with chlorobenzene as the APPI dopant are shown in Figure 2.
Figure 1

Structures of the investigated compounds. Group (1): compounds that formed [M + 77]+ as the main ion with chloro-, bromo-, and iodobenzene dopants. Group (2): compounds that formed [M + 77]+ with one or several of the halobenzene dopants, but not as the main ion. Group (3): compounds that did not form [M + 77]+

Scheme 1

Nucleophilic ipso-substitution [15] between chlorobenzene radical cation and pyridine

Figure 2

GC-APPI-MS spectra of (a), (b) pyridine in the presence of chloro- and fluorobenzene dopants, respectively, and (c) quinoline and (d) acridine in the presence of chlorobenzene dopant (headspace 50 μL/min). D+. indicates the dopant molecular ion

Other N-containing compounds that showed [M + 77]+ ions in their spectra were isoxazole, piperidine, morpholine, and N-nitrosodimethylamine. For these Group 2 classified compounds the formation of [M + 77]+ was not as intense as for the N-heteroaromatic compounds in Group 1, and additional ions such as [M + H]+ or [M – H]+ were observed in pronounced abundance. In contrast, the substitution products for N-methylpiperidine and N-methylmorpholine were virtually absent, and the main ions observed with these compounds were [M + H]+ and [M – H]+ (see Figure 3a and b for piperidine and N-methylpiperidine, respectively). This is readily rationalized by sterical hindrance of the tertiary N, which reduces the nucleophilicity of the compounds, despite their high basicity (e.g., the PAs for piperidine and N-methylpiperidine are 954 and 971 kJ/mol, respectively). Furthermore, the absence of the substitution product for N-methylmorpholine suggests that the oxygen in morpholine and N-methylmorpholine is a weaker nucleophile than the nitrogen, and too weak to undergo the nucleophilic ipso-substitution. Besides nitrogen-containing compounds, the tendency for the nucleophilic substitution pathway of several oxygen- and sulfur-containing compounds, such as furan, 2,3-benzofuran, cyclohexanone, 2-cyclohexen-1-one, thiophene, and thianaphthene, was investigated. For those, the highest reaction yield was observed with 2-cyclohexen-1-one (see Figure 3d). The appearance of the ipso-substituted ion signal was not as persistent and vast; thus, 2-cyclohexen-1-one is classified as a Group 2 compound. Interestingly, the [M + 77]+ ion is absent from the cyclohexanone spectrum, shown in Figure 3c. Since the structures of cyclohexanone and 2-cyclohexen-1-one only differ by one double bond, it is suggested that a resonance stabilized end product is formed in the case of 2-cyclohexen-1-one, and this makes the nucleophilic substitution reaction favorable. Resonance stabilization may also explain the high efficiency of the nucleophilic substitution for the N-heteroaromatic compounds. Computational calculations to explain the possible effect of resonance stabilization in the nucleophilic substitution reaction are currently under study and will be subject to an upcoming publication. Furan and thiophene, which are heterocyclic compounds without nitrogen, were also classified as Group 2 compounds. For thiophene, a small [M + 77]+ ion signal was observed in the presence of bromobenzene dopant; however, its intensity did not exceed 1% of the main ion. 2,3-Benzofuran and thianaphthene showed no substitution reaction and, accordingly, belong to Group 3.
Figure 3

GC-APPI-MS spectra of (a) piperidine and (b) N-methylpiperidine in the presence of chlorobenzene dopant (headspace 200 μL/min) and background subtracted GC-APPI-MS spectra of (c) cyclohexanone and (d) 2-cyclohexen-1-one with bromobenzene dopant (headspace 200 μL/min)

In general, the formation of the [M + 77]+ ion was found to be highly dependent on the used halobenzene, as shown in Figure 4 for pyridine, 2-methylpyridine, and 2,3-lutidine. For the compounds of Group 1 (Figure 1), [M + 77]+ ions were observed in the presence of chloro-, bromo-, and iodobenzenes, but not with fluorobenzene, except for a low (~5%) signal in case of pyrimidine. A minor ion signal corresponding to [M + dopant]+ was, however, observed in the spectra of pyridine and pyrimidine in the presence of fluorobenzene (see Figure 2b for pyridine). For pyridine, the [M + 77]+ ion yield increased in the order Cl < Br < I, which is in good agreement with earlier reports of nucleophilic substitution between halobenzene radical cations and small amines in the gas phase [42, 43]. This reactivity order is reasonably explained by the oppositely directed order of C6H5-X bond strengths (I < Br < Cl < F [44]), and it is opposite to what has been reported for nucleophilic aromatic substitution involving neutral halo-, and nitrosubstituted benzenes in solution and in gas phase [45, 46]. For 2-methylpyridine the highest [M + 77]+ yield was observed with bromobenzene dopant. The lower signal with iodobenzene is attributed to the bulkier size of the halogen and to sterical hindrance by the methyl group adjacent to the nitrogen. Sterical hindrance of the nucleophilic attack in case of the bulky iodine molecule has also been reported in ammonia CI with halobenzenes [17]. Besides 2-methylpyridine, also 2,3-lutidine, quinoline, and pyrimidine showed decreased [M + 77]+ ion yield with iodobenzene; for 2,3-lutidine and quinoline sterical effects were observed also with bromobenzene, since the highest [M + 77]+ signal was observed with chlorobenzene (see Figure 4 for 2,3-lutidine). For compounds with lower reactivity (Group 2), the nucleophilic substitution reaction was even more selective, and in many cases only one of the halobenzenes, usually bromobenzene, led to the formation of the substituted product.
Figure 4

Proportions of different ions of pyridine, 2-methylpyridine, and 2,3-lutidine in direct APPI and dopant-assisted APPI with different halobenzene and toluene dopants

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 S2S5 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).

In each case, the initial approach subsequently leads to an intermediate state IS1 of lower energy (see Figure 5), where shared electron density between the lone pair and the π-system weakly bonds the pyridine to the C1 carbon of the halobenzene (shown for pyridine and fluorobenzene in Figure S4 of Supplementary Material). In addition, both ortho hydrogens of pyridine show bonding character; one towards the halogen and the other towards the meta and para carbons of the halobenzene. From this first intermediate state, the energy rises along the reaction coordinate to a transition state TS1, in which the nitrogen lone pair of pyridine attacks the C1 carbon of the halobenzene. The energy increase is mostly due to breaking the original π-system, which includes the halogen and C1, which is followed by the formation of a new π-system comprised of C2–C6. The Gibbs free energies of activation Δ R G ؇ for TS1 are calculated as 27.0, 28.3 and 34.8 kJ/mol for chloro-, bromo-, and iodobenzene, respectively. TS1 for fluorobenzene, calculated as Δ R G ؇ = 12.1 kJ/mol is significantly lower, which can be explained by strong localization of the positive charge at C1 due to the electronegativity of fluorine (see Figure S2 in Supplementary Material). The activation energy for this reaction step increases with the size of the halogen, which can be due to sterical effects, or due to the localized positive charge at the ipso-carbon of the smaller halobenzene radical cations [46]. Additionally, heavier halogens have to move out of the phenyl ring plane. With fluoro- and chlorobenzene TS1 leads to a second intermediate structure IS2, where the halogen and pyridine are both bonded to C1. This corresponds to a classical sp 3 configuration of the carbon. In case of fluorine, the planes of the two rings remain orthogonal and the H–F bond contributes to the 5-membered-ring-system that is formed between fluorobenzene and pyridine (see Figure S5 in Supplementary Material). For chlorobenzene, the pyridine ring plane is slightly distorted (D(Cl,C1,N,C) = −38.6°) and the hydrogen–chlorine bond is much weaker than the corresponding H–F bond in the case of fluorobenzene. The standard Gibbs free energies of the reaction for IS2 with respect to the first weakly bonded system IS1 are calculated as ∆RG = −5.1 kJ/mol and +18.0 kJ/mol for fluorine- and chlorobenzenes, respectively. Hence, in case of the chlorine-containing structure, this reaction step turns out to be endergonic, which is simply an overcompensation of the calculated negative standard reaction enthalpy of Δ R H Ø = −2.7 kJ/mol by the entropy contribution of reduced number of particles. An increase of the C1–X bond length causes the electronic energy to rise to the next transition state TS2. This one is fairly high in case of fluorobenzene (Δ R G ؇ = +85.0 kJ/mol) and quite low for chlorobenzene (Δ R G ؇ = +3.6 kJ/mol), which indicates that the C1–F bond is very strong, as one would expect, and the C1–Cl bond is much weaker. The latter is partially attributed to sterical hindering of the bulkier halogen in the less favorable intermediate IS2. Following the reaction path, the transition state TS2 leads to a third intermediate IS3, where the halogen is not attached to the C1, but instead to the C2 carbon of the halobenzene. Interestingly, for bromo- and iodobenzene, computational analysis shows that the intermediate IS3 is formed directly after the first transition state TS1 (Figure S6 in Supplementary Material). In these cases, the C1–N bond forms with a simultaneous break of the C1–X bond, without formation of the second intermediate structure IS2, in which N and X are both attached to C1. Computation of the last step to the formation of the finally separated products (i.e., the N-phenyl-pyridine cation [M + 77]+ and the halogen radical) shows a monotone increase of the electronic energy towards the products. Consequently, no further transition state is expected. Despite this increase of ε0 the last step is exergonic for chlorine, bromine, and iodine by ∆RG = −1.7, −13.8, and −16.3 kJ/mol, owing to the compensating entropy contribution. The situation is quite different for the lightest halogen, in which ε0 for the partition of the fluorine and the N-phenyl-pyridine cation cannot be compensated by the entropy contribution, which thus renders this step strongly endergonic by ∆RG = +113 kJ/mol. These results are in good agreement with the experimental data. The N-phenyl-pyridine cation [M + 77]+ was observed with the three heavier halobenzenes but not with fluorobenzene; on the other hand, with fluorobenzene, an ion with m/z corresponding to the calculated overall minimum IS2 along the reaction pathway was observed ([M + D]+ in Figure 2b). Of course, the mass spectral information alone does not reveal the bonding situation; however, the high activation barrier TS2, which moves the fluorine to another carbon, strongly indicates that this reaction pathway stops at IS2 and simply competes with other ionization pathways. For the other halogens, the activation barriers along the reaction pathway are low enough to be overcome with thermal energy so that these systems rush through to the final products according to the overall Gibbs free energies of the nucleophilic substitution reaction given in Table 1. Optimum geometry for the N-phenyl-pyridine cation [M + 77]+ is shown in Figure S7 of the Supplementary Material.
Figure 5

ΔRG0 plotted against every reaction intermediate for pyridine and each halobenzene. Similar structures are aligned vertically. The difference between two steps always equals the standard Gibbs free energy of reaction

Table 1

Calculated Overall Standard Gibbs Free Energies for the Nucleophilic Substitution Between Different Halobenzene Dopants and Pyridine


ΔRG0 [kJ/mol]










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.

Supplementary material

13361_2015_1315_MOESM1_ESM.pdf (660 kb)
ESM 1 (PDF 660 kb)


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Copyright information

© American Society for Mass Spectrometry 2015

Authors and Affiliations

  • Tiina J. Kauppila
    • 1
  • Alexander Haack
    • 2
  • Kai Kroll
    • 2
  • Hendrik Kersten
    • 2
  • Thorsten Benter
    • 2
  1. 1.Faculty of PharmacyUniversity of HelsinkiHelsinkiFinland
  2. 2.Department of Physical and Theoretical ChemistryUniversity of WuppertalWuppertalGermany

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