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Nebulization prior to ionization for mechanistic studies of chemical reactions
Analytica Chimica Acta xxx (xxxx) xxx
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Nebulization prior to ionization for mechanistic studies of chemical reactions Hong Zhang a, b, 1, Lina Qiao b, 1, Wenxin Wang a, b, Jing He a, b, Kai Yu a, b, Miao Yang a, b, Hong You a, b, Jie Jiang a, b, * a b
School of Marine Science and Technology, Harbin Institute of Technology at Weihai, Weihai, 264209, China State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin, 150090, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A nebulization method followed by on-line ionization was developed. The method enables on-line separation of ionic and neutral intermediates. Isolating ionic and neutrals intermediates in two reactions was investigated. The carbocation and its isomer lactone products were readily isolated and identified.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 January 2020 Received in revised form 12 February 2020 Accepted 14 February 2020 Available online xxx
Many important chemical transformations proceed by way of ionic and/or neutral intermediates. Great effort has been expended to understand the mechanism, with only minimum attention given to separate associated ionic and neutral intermediates. Herein, we present a nebulization method followed by online ionization to isolate and characterize the ionic and neutral intermediates. The separation of nebulization and ionization and electrical deflection of ionic species guarantee that only neutrals undergo the subsequent on-line ionization. We present data that show the formation of neutral intermediates and iminium ions with short lifetime in Eschweiler-Clarke methylation of di-n-butylamine, as well as data that provide evidence for the formation of carbocation and its isomer lactone products resulting from copper-mediated oxidative cyclization of 4-phenylbutyric acid. Experiments in which dissociation behavior of these two isomers varied at the same collision energy confirmed the carbocation during the cyclization. The nature of this process, which online isolates the ionic and neutral intermediates prior to ionization, greatly advances in mechanistic studies. © 2020 Elsevier B.V. All rights reserved.
Keywords: Mass spectrometry Nebulization Ionic/neutral intermediate di-n-butylamine 4-Phenylbutyric acid
1. Introduction
* Corresponding author. School of Marine Science and Technology, Harbin Institute of Technology at Weihai, Weihai, 264209, China. E-mail address: [email protected] (J. Jiang). 1 These authors contributed equally to this work.
The reaction mechanism is the step-by-step description of a reaction process in which reactive intermediates formed in reacting solution are prime importance to mechanistic studies [1]. Reactive intermediates can be indirectly identified by physical organic methods, or directly for example by spectroscopic methods (e.g. UV/vis [2], IR [3], and NMR [4]). Alternatively, mass spectrometry
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Please cite this article as: H. Zhang et al., Nebulization prior to ionization for mechanistic studies of chemical reactions, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.02.031
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(MS) monitoring of chemical reactions complements these methods in tracking reaction progress and detecting reactive intermediates, and can interrogate mechanisms via cleverly designed experiments. The underlying applications for MS monitoring benefit from its ionization methods, taking the form of plasma [5], and especially of electrospray ionization (ESI) coupled with microreactors [6,7] and other methods [8,9]. Reactions varied from seconds to hours can be monitored online with these massspectrometry-informed methods, providing high-quality timedependent spectra, a topic that has been reviewed [10,11]. Given these established capacities, the use of ESI-MS, for example, to identify reaction intermediates without affecting the ongoing chemical process, includes complex multicomponent reactions, organometallic transformations [12], and even oxygen- and moisture-sensitive reaction mixtures [13]. The reaction intermediates in the field of pharmaceuticals, medicines and synthetic chemistry include ionic species [14] such as carbocations, carbanions, carbenes and radical cations, and neutral intermediates/products. In a typical electrospray-based experiment, the ionic and neutral species are simultaneously sampled in the charged-microdroplets generated by applying a high voltage to the reacting solution. Since the neutrals are ionized and expressed at the form of charged state (e.g., [MþH]þ, [MþNa]þ) in this scenario, the differentiation of ionic and neutral intermediates poses an experimental challenge and to some extent relies on the creativity and experience of chemists. This restricts MS applications in reaction mechanism studies. Moreover, the ionic intermediates and ionized products in some cases are isomer and thus cannot be distinguished on the basis of mass-to-charge alone [15]. This represents an additional concern in capturing and detecting reactive intermediates experiments. The commonly used electrospray-based technologies such as desorption electrospray ionization (DESI) [16,17], extractive electrospray ionization (EESI) [18], sonic spray ionization (SSI) [19], have also been applied to identify the reaction intermediates in chemical reactions. However, the concern on differentiating the reaction intermediates at ionic or neutral forms using these ionization strategies is yet challenging. Herein, we provide a new strategy that the reaction solution undergoes a solution-phase nebulization prior to ionization to achieve ionic and neutral intermediates separation and characterization. This takes inspiration from the nebulization where the ionic intermediates transferred to the gas phase can be electrically deflected. The reaction mixture was transferred to a homemade flow injection sprayer to generate an electrically neutral spray. After gentle desolvation in a straight heated tube, the ionic species in drying gas stream were electrically deflected using an ion deflector and the remaining neutral species were exclusively ionized for MS analysis without the interference of ionic species. The ionization of gas-phase neutral species in this way has been widely used in atmospheric pressure ionization techniques such as neutralization/re-ionization experiments [20e22]. We also performed the other case that in absence of ion deflection the ionic and neutral species from the heated tube were simultaneously ionized and detected by MS. This in fact is similar to previous electrospraybased methods [23e25] where the ionic and neutral species were simultaneously presented in the electrospray plume. Owing to the removal of ionic species by ion deflection, the differences in abundances in these two cases are assigned as ionic intermediates and the neutral molecules which are regardless of the ion deflection and have the same abundance. To evaluate this performance, two reactions, EschweilereClarke reaction and copper-catalyzed CeH oxidative cyclization of carboxylic acids, were demonstrated. The ionic and neutral intermediates were separated and characterized, which gave new insights into the mechanism studies.
Fig. 1. (a) Experimental setup for isolating and characterizing ionic and neutral species. The trajectories of ionic and neutral species when the ion deflector operated at (b) ON mode, and (c) OFF mode. For ON mode, the ionic species from nebulization are removed and only neutral species are sequentially ionized for MS analysis; for OFF mode, both the ionic and neutral species pass through the ion deflector and are simultaneously ionized and detect-ed by MS. The reaction mixture introduced methods: (d) direct injection for EschweilereClarke methylation of di-n-butylamine; (e) pressurized sampling injection for copper-mediated oxidative cyclization of 4phenylbutyric acid.
2. Materials and methods 2.1. Reagents Methanol and acetonitrile at MS grade were purchased from Sigma-Aldrich (Darmstadt, Germany). Potassium persulfate (K2S2O8), tris(p-bromophenyl)aminium hexachloroantimonate, dichloromethane, 4-aminophenol, 4-phenylbutyric acid, di-nbutylamine, acetic acid, and formaldehyde (36.5e38% in H2O) were purchased from Sigma-Aldrich (St. Louis, MO). Formic acid and copper (II) acetate (Cu(OAc)2$H2O) were purchased from Laiyang Fine Chemical Factory (Laiyang, China). Ultrapure H2O was obtained from a water purification system (Milli-Q, Milford, MA).
2.2. Experiment apparatus As shown in Fig. 1a, the apparatus used for nebulization and isolation of the ionic and neutral species consisted of a home-built flow injection sprayer (id., 0.10 mm, od., 0.30 mm; id., 0.45 mm, od., 0.75 mm), a stainless steel tube (id., 5.8 mm; od., 7.4 m; length 10 cm), and an ion deflector. For the sprayer, nitrogen was used as nebulization gas and a gas pressure of 20 psi was used. To maximize collection of the electrically neutral microdroplets, the tip of the sprayer was inserted into the heated tube. The transmission tube was heated using a coiled heating wire (0.3 mm) controlled by temperature regulator (Zhonghuan Temperature instrument Co., LTD, Tianjing, China) coupled with a thermocouple, with the range of 25 Ce400 C. The drying gas stream emerging from the heated tube (~150 C) passed through an ion deflector to remove the ionic species in the gas stream. The dimension of the ion deflector is shown in Fig. S1. The ion deflector consisted of a pair of parallel metal plates (10 10 mm2) and was integrated into a box made with polyetheretherketone (PEEK). The distance between the two plates was
Please cite this article as: H. Zhang et al., Nebulization prior to ionization for mechanistic studies of chemical reactions, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.02.031
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10 mm. The holder was placed in line with the MS inlet and the distance to the MS inlet was ca. 1 cm. One plate was grounded while the other was supplied with a potential to deflect ions. The deflection voltage increased with the increase of gas pressure for nebulization. It was found that a bias of 2 kV could completely remove all ionic species as monitored by MS. After deflection, the separated neutral species were then ionized by ESI (id., 0.10 mm, od., 0.30 mm). The distance between the tip of the ESI sprayer and the MS inlet was ca. 0.4 cm, and an angle formed between the spray tip and the inlet was ca. 30 . The heated tube and ion deflector were integrated to minimize turbulence of gas stream at the interface and maximize the transfer efficiency of ionic/neutral species at atmosphere pressure. For ESI, the spray solvents for EschweilereClarke reaction and copper-catalyzed CeH oxidative cyclization were methanol and methanol/water/acetic acid mixture (90:10:0.1), respectively. The details for experimental procedures were given in Supporting Information. 2.3. Mass spectrometry experiments All the experiments were implemented on an LTQ/Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Full-scan positive ion mass spectra were recorded over an m/z range from 100 to 400 with a resolution of 60000. Typical instrumental parameters included 1 microscans, 500 ms maximum ion injection time, 110 V tube lens voltage, 35 V capillary voltage, and 275 C capillary temperature. MS/MS data were recorded using the default values except that the isolation window and nominal collision energy were adjusted to improve spectral quality. A standard LTQ XL ESI positive ion calibration solution was rigorously used for the positive mass calibration before each experiment. All experiments were conducted three times or more. Without special notes, the mass spectra given were the average of three scans. 3. Results 3.1. Performance of the heated tube and ion defector The aim of the ion deflector was to remove the ionic species from nebulization. There were two operation modes of the ion deflector, OFF mode and ON mode. At OFF mode, the ionic species and neutrals from nebulization both passed through the ion deflector and were then ionized for MS analysis. In this case, the ionic and neutral species were simultaneously ionized, which is similar to previous studies where the reaction mixture was ionized by ionization sources such as ESI. At ON mode, the ionic species from nebulization were removed by the ion deflector with a bias voltage of 2 kV, and the neutral species passed through the ion deflector and underwent ionization for MS analysis. In this scenario, the neutrals were analyzed without the interference of ionic species, and thus only neutrals involved in the gas stream were ionized and then detected by MS. The straight heated tube, similar to sample transfer capillary, was grounded to merely function as gentle desolvation and simultaneously minimize any possible interference with heating within the tube. The performance of desolvation was evaluated. Evidence for complete drying of the electrically neutral microdroplets is seen in the fact that free gas-phase cation radicals (m/z 480.8496) were detected by MS when nebulization of tris(pbromophenyl)aminium hexachloroantimonate and dried by being passed through the straight heated tube (Fig. S2a). The attempted removal of the ionic species employed an ion deflector. The ionic species from nebulization were removed by the ion deflector with a bias voltage of 2 kV. No signal of cation radical was observed (Fig. S2b). The success of the approach in detecting neutral species
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is clear from the representative mass spectrum of 4-aminophenol in aqueous methanol. No signal of 4-aminophenol (m/z 110.0603) was observed after a gentle desolvation in the straight heated tube (Fig. S3a), but a peak of m/z 110.0603 was dominated after deflection (2 kV) and then ionization using ESI (Fig. S3c). This sharp contrast between organic salts and 4-aminophenol in detection behavior emphasizes the fact that the radical cations are from the organic salt solution and that the neutral 4-aminophenol well lies in the ionization source. 3.2. EschweilereClarke reaction With the success of separation and detection of ionic and neutral species, we turned attention to investigate the ionic and neutral intermediates involved in EschweilereClarke [26], in which a primary (or secondary) amine is methylated into the hemiaminal with formaldehyde and formic acid in excess. Di-n-butylamine was selected as a model secondary amine to examine this process, and the proposed mechanism is shown in Scheme 1. Di-n-butylamine reacting with formaldehyde leads to the transient 2 via the hemiaminal 1. In presence of formic acids in excess acting as a source of hydride, 2 is reduced to afford tertiary amine 3 as the final product. Previous MS studies have reported these reaction intermediates on the basis of ionization where a high voltage is applied to the reaction solution [27]. A significant question is that the ionic and neutral reaction intermediates are simultaneously ionized and analyzed, resulting in that the existing forms of these intermediates during the transform of primary (or secondary) amine to tertiary amine cannot be confirmed. Formaldehyde (0.1%), formic acid (1.0%) and di-n-butylamine (1.0%) were mixed in aqueous methanol (1/3: v/v) and then directly injected into the flow injection sprayer for nebulization. When the species emerging from the heated tube were directly ionized using ESI with methanol as solvent, peaks were observed at m/z 130.1593, 142.1594, 144.1751 and 160.1699 (Fig. 2a). Then, we used an ion deflector to remove the ionic species in the gas stream before ionization for MS analysis, and no signals were observed with the ion deflector applied (Fig. S4). After ionization, the application of the ion deflector showed no detectable change in the abundance of m/z 130.1593, 144.1751 and 160.1699. When no ion deflector was applied (Fig. 2a), signal of m/z 130.1593 was dominant and the relative intensities of m/z 144.1751 and 160.1699 were 0.11 and 0.06; when ion deflector was used (Fig. 2c), signal associated with m/z 130.1593 was still dominant and the relative intensities of m/z 144.1751 and 160.1699 were 0.12 and 0.06. Polarity switch of the ion deflector and deflection voltage had no influence on these peaks (Figs. S5 and S6). The time-dependent information also showed that the deflection voltage had no influence on these species (Fig. S7). These results establish the independence of m/z 130.1593, 144.1751 and 160.1699 on any ions formed during nebulization and its dependence on the ESI. MS/MS experiments for these peaks were also performed (Fig. S8) consistent with previous studies [27]. Therefore, peaks of m/z 130.1593, 144.1751 and 160.1699 represented neutral molecules in the course of Eschweiler-Clarke methylation and correspond to protonated di-nbutylamine, 3 and 1, respectively. When deflection was applied prior to ionization (Fig. 2c), the resulting peak of m/z 142.1594 was quite different from that recorded when no ion deflector was applied (Fig. 2a). The signal recorded at m/z 142.1594 was absent. The time-dependent data further supported that signal of m/z 142.1594 was related to the deflection voltage (Fig. S7). This suggests the fact that the response of m/z 142.1594 depends on the deflection voltage applied rather than ionization. The signal of m/z 142.1594 was assigned as the iminium ion 2 and was further supported by the tandem mass
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Scheme 1. Proposed EschweilereClarke methylation of di-n-butylamine leading to the tertiary amine.
Scheme 2. Proposed mechanism for copper-mediated oxidative cyclization of 4phenylbutyric acid to lactone.
Fig. 2. EschweilereClarke methylation of di-n-butylamine. (a) Mass spectra showing nebulization and ionization of reaction mixture without ion deflection, and (b) expansion showing the spectrum from m/z 135 to 165. (c) Mass spectra showing nebulization of reaction mixture and ionization of the neutral species passing through the ion deflector (2 kV), and (d) expansion showing the spectrum form m/z 135 to 165. Data were shown at reaction time of 1 min.
spectrometry experiment (Fig. S8d). It has been reported that the lifetime of iminium ions in aqueous solution is quite short and the reaction of an iminium ion with a source of hydride is very rapid under acidic conditions [27,28]. These observations suggest that this setup is capable of online separation, monitoring and identification of ionic/neutral species and permits the detection of transient intermediates. 3.3. Copper-catalyzed CeH oxidative cyclization of carboxylic acids Encouraged by these results, we turned to the conversion of CeH to CeO bonds in complex molecules, which remains an outstanding challenge in synthetic chemistry [29,30]. The coppercatalyzed CeH oxidative cyclization of carboxylic acids affording lactone [31] was investigated. As shown in Scheme 2, benzylic hydrogen abstraction of 4-phenylbutyric acid by SO$4 generates a carbon radical, which is then oxidized by Cu(II) catalyst to a resonancestabilized carbocation 4. The 4 can cyclize directly to give lactone 5 or be trapped by water to furnish alcohol 6, which is further converted to 5 or terminates with the formation of ketone 7. The oxidative cyclization with the formation of carbocation 4 differs from related investigations that proposed the intermediacy of a carboxylate radical [32,33]. The proposed mechanism has been investigated by DESI-MS combined with physical organic experiments [15]. However, the carbocation 4 and lactone 5 are not isolated and characterized by MS on basis of m/z alone because the
carbocation 4 and protonated 5 have the same m/z. We use the setup described herein coupling with a heated reactor, similar to that used in pressurized sample infusion [9,34], to online isolation and characterization of the carbocation and the neutral products including its isomer. The experiment details are given in the Supporting Information. The reaction solution was transferred to the flow injection sprayer for nebulization through a sample capillary at a gas pressure of 4 psi. When no ion deflection was applied, the ionic and neutral species were directly ionized in presence of a plume of positive-electrosprayed droplets created using methanol/water/acetic acid mixture (90:10:0.1). The mass spectrum is dominated with signal of m/z 165.0917 (Fig. 3a). The mass spectrum also shows signals of m/z 179.0705, 180.0785, 201.0315 and 217.0266. With ion deflection (2 kV), no signals were observed (Fig. S9), indicating that the ionic species are completely removed. Then, the remaining neutral species underwent the subsequent on-line ionization. Peaks associated with m/z 165.0917, 179.0705, 201.0315 and 217.0266 were observed with the corresponding abundances to no ion deflection applied (Fig. 3a and b). The time-dependent information further supports that the deflection voltage had no influence on these species (Fig. S10). This indicates that these species are neutral molecules in the oxidative cyclization of 4-phenylbutyric acid. Peaks of m/z 165.0917 corresponded to protonated 4-phenylbutyric acid. Signals of m/z 179.0705 and 217.0266 were assigned as [7 þ H]þ and [7 þ K]þ, respectively. As shown in Fig. 3b, the resulting abundance for 163.0761 peak with ion deflection applied is quite different from that recorded when nebulizing the mixed solution followed no ion deflection (Fig. 3a). Only relative abundance of 46% was observed, which significantly decreased compared to the case in absence of ion deflection where the relative abundance of 163.0761 peak was 82%. Polarity switching of the ion deflector showed no detectable change in the abundance of this peak (Fig. S11). The relative abundance of 163.0761 peak decreased with the increase of deflection voltage from 0 to 2 kV (Fig. S12). The time-dependent results with ion deflector also showed the decrease of relative abundance of 163.0761 peak compared to no ion deflector applied
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Fig. 4. MS/MS spectra of m/z 163.0761 obtained from (a) deflection then ionization, (b) nebulization alone, and (c) standard 5. Isolation window is m/z 1; CID energy is 21 (arbitrary units). Different dissociation features were obtained.
Fig. 3. Copper-mediated oxidative cyclization of 4-phenylbutyric acid to lactone. Mass spectra showing (a) nebulization then ionization without ion deflector, and (b) nebulization with ion deflection (2 kV) then ionization. (c) Mass spectrum showing nebulization of reaction solution without deflection and ionization. Data were obtained at reaction time of 2.5 min.
(Fig. S10). Together these experiments suggest that peaks of m/z 163.0761 in Fig. 3a and b represent different species. Since the ionic species were completely removed using an ion deflector, the 163.0761 peak in presence of ion deflection was assigned to lactone 5 (Fig. 3b); and the 163.0761 peak in absence of ion deflection was a mixture of carbocation 4 and protonated lactone 5 (Fig. 3a). The case where only nebulization was also performed. We observed different mass spectrum feature; the mass spectrum is shown in Fig. 3c and dominated with peak of m/z 163.0761 associated with carbocation 4. The above assignment of 163.0765 peak was further supported by collision induced dissociation (CID) experiment, as now discussed. This experiment was carried out by selecting the m/z 163.0765 from deflection then ionization (Fig. 3b) and nebulization alone (Fig. 3c) and dissociating under various collision energies. As shown in Fig. 4a and b, fragmentations from these two cases generate two similar fragments, one at m/z 145 associated with loss of H2O and the other one with m/z 117 associated with loss of CH2O2 (Fig. S13). However, the resulting dissociation behavior respect to collision energy for these two cases was quite different. CID on m/z 163.0765 from deflection then ionization established 80% dissociation of this species in the gas phase at normal collision energy (NCE) of 21 (arbitrary units). This is consistent well with standard lactone 5 that undergoes 80% dissociation with NCE of 21 (Fig. 4c). When the same NCE of 21 was applied in the case of merely nebulizing the reaction mixture, 99% dissociation was observed (Fig. 4b). To obtain the same dissociation, the NCE used was 15 (Fig. S14). Together, these results suggest that the intrinsic reactivity of carbocation 4 requires less energy to undergo the same
extent dissociation as compared to lactone 5. The sharp contrast for these two species behaviors in fragmentation supports the formation of carbocation 4 in the oxidative cyclization of 4-phenylbutyric acid, which was not previously isolated and characterized [31]. In contrast to experiments associated with prolonging the reaction time to obtain pure lactone [15], detection of the lactone 5 can be directly achieved respect to the separation of nebulization and ionization and electrical deflection of carbocation 4. 4. Conclusions In summary, we have demonstrated a nebulization method followed by on-line ionization in which the ionic and neutral intermediates in reaction solution can be separated for characterization without any interference. Nebulization then deflection guarantees that only neutral intermediates undergo the subsequent on-line ionization. The ionic and neural intermediates resulting from EschweilereClarke methylation of di-n-butylamine were successfully captured and analyzed by MS. Response to ion deflection provides direct evidence for the formation of short-lived iminium ions in the reaction progress rather than ionization. The isomer (carbocation, lactone product) generated in Cu(II)-catalyzed oxidative cyclization of 4-phenylbutyric acid was separated. Provided that the individual dissociation feature of the isomer with normal collision energies, the carbocation intermediates showed high reactivity compared to the formed lactone. The two steps coupled with tandem mass spectrometry give this approach high specificity. This platform was also compatible with a wide range of analytical needs such as time-dependent reaction information and different reaction conditions (e.g., temperature, reaction solvent). This on-line mass spectrometric technique respect to nebulization probes the existence form of reaction intermediates (e.g., ionic, neutral), which would advance mechanistic information for many solution reactions. Declaration of competing interest The authors declare that they have no known competing
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financial interests or personal relationships that could have appeared to influence the work reported in this paper. CRediT authorship contribution statement Hong Zhang: Conceptualization, Writing - original draft, Methodology, Investigation, Formal analysis. Lina Qiao: Conceptualization, Writing - original draft, Methodology, Investigation, Formal analysis. Wenxin Wang: Investigation, Validation. Jing He: Investigation, Validation. Kai Yu: Writing - review & editing, Data curation. Miao Yang: Validation, Data curation. Hong You: Writing - review & editing. Jie Jiang: Supervision, Conceptualization, Funding acquisition, Project administration. Acknowledgments This work was supported by the supported by the Natural Science Foundation of China (No.21904029), Key R&D program of Shandong (NO.2016YYST013), fundamental research funds for the central universities (No.HIT.NSRIF.2020087) and Weihai science and technology development program (No. 2019KYCXJJYB03). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca.2020.02.031. References [1] L.A.B. Moraes, M.N. Eberlin, Ketalization of gaseous acylium ions, J. Am. Soc. Mass Spectrom. 12 (2001) 150e162. [2] J.M. Beames, F. Liu, L. Lu, M.I. Lester, Ultraviolet spectrum and photochemistry of the simplest criegee intermediate CH2OO, J. Am. Chem. Soc. 134 (2012) 20045e20048. [3] O. Zandi, T.W. Hamann, Determination of photoelectrochemical water oxidation intermediates on haematite electrode surfaces using operando infrared spectroscopy, Nat. Chem. 8 (2016) 778e783. [4] R.M. Bain, S. Sathyamoorthi, R.N. Zare, "On-droplet" chemistry: the cycloaddition of diethyl azodicarboxylate and quadricyclane, Angew. Chem. Int. Ed. 56 (2017) 15083e15087. [5] X.X. Ma, S.C. Zhang, Z.Q. Lin, Y.Y. Liu, Z. Xing, C.D. Yang, X.R. Zhang, Real-time monitoring of chemical reactions by mass spectrometry utilizing a lowtemperature plasma probe, Analyst 134 (2009) 1863e1867. [6] L.S. Santos, J.O. Metzger, Study of homogeneously catalyzed Ziegler-Natta polymerization of ethene by ESI-MS, Angew. Chem. Int. Ed. 45 (2006) 977e981. [7] J. Griep-Raming, S. Meyer, T. Bruhn, J.O. Metzger, Investigation of reactive intermediates of chemical reactions in solution by electrospray ionization mass spectrometry: radical chain reactions, Angew. Chem. Int. Ed. 41 (2002) 2738e2742. [8] H. Zhang, X. Li, K. Yu, N. Li, J. He, H. You, J. Jiang, On-line monitoring of photolysis reactions using electrospray ionization mass spectrometry coupled with pressurized photoreactor, Anal. Chim. Acta 1013 (2018) 36e42. [9] X. Yan, E. Sokol, X. Li, G.T. Li, S.Q. Xu, R.G. Cooks, On-line reaction monitoring and mechanistic studies by mass spectrometry: negishi cross-coupling, hydrogenolysis, and reductive amination, Angew. Chem. Int. Ed. 53 (2014) 5931e5935. [10] A. Ray, T. Bristow, C. Whitmore, J. Mosely, On-line reaction monitoring by mass spectrometry, modern approaches for the analysis of chemical reactions, Mass Spectrom. Rev. 37 (2018) 565e579.
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Please cite this article as: H. Zhang et al., Nebulization prior to ionization for mechanistic studies of chemical reactions, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.02.031
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Nebulization prior to ionization for mechanistic studies of chemical reactions Hong Zhang a, b, 1, Lina Qiao b, 1, Wenxin Wang a, b, Jing He a, b, Kai Yu a, b, Miao Yang a, b, Hong You a, b, Jie Jiang a, b, * a b
School of Marine Science and Technology, Harbin Institute of Technology at Weihai, Weihai, 264209, China State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin, 150090, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A nebulization method followed by on-line ionization was developed. The method enables on-line separation of ionic and neutral intermediates. Isolating ionic and neutrals intermediates in two reactions was investigated. The carbocation and its isomer lactone products were readily isolated and identified.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 January 2020 Received in revised form 12 February 2020 Accepted 14 February 2020 Available online xxx
Many important chemical transformations proceed by way of ionic and/or neutral intermediates. Great effort has been expended to understand the mechanism, with only minimum attention given to separate associated ionic and neutral intermediates. Herein, we present a nebulization method followed by online ionization to isolate and characterize the ionic and neutral intermediates. The separation of nebulization and ionization and electrical deflection of ionic species guarantee that only neutrals undergo the subsequent on-line ionization. We present data that show the formation of neutral intermediates and iminium ions with short lifetime in Eschweiler-Clarke methylation of di-n-butylamine, as well as data that provide evidence for the formation of carbocation and its isomer lactone products resulting from copper-mediated oxidative cyclization of 4-phenylbutyric acid. Experiments in which dissociation behavior of these two isomers varied at the same collision energy confirmed the carbocation during the cyclization. The nature of this process, which online isolates the ionic and neutral intermediates prior to ionization, greatly advances in mechanistic studies. © 2020 Elsevier B.V. All rights reserved.
Keywords: Mass spectrometry Nebulization Ionic/neutral intermediate di-n-butylamine 4-Phenylbutyric acid
1. Introduction
* Corresponding author. School of Marine Science and Technology, Harbin Institute of Technology at Weihai, Weihai, 264209, China. E-mail address: [email protected] (J. Jiang). 1 These authors contributed equally to this work.
The reaction mechanism is the step-by-step description of a reaction process in which reactive intermediates formed in reacting solution are prime importance to mechanistic studies [1]. Reactive intermediates can be indirectly identified by physical organic methods, or directly for example by spectroscopic methods (e.g. UV/vis [2], IR [3], and NMR [4]). Alternatively, mass spectrometry
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Please cite this article as: H. Zhang et al., Nebulization prior to ionization for mechanistic studies of chemical reactions, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.02.031
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(MS) monitoring of chemical reactions complements these methods in tracking reaction progress and detecting reactive intermediates, and can interrogate mechanisms via cleverly designed experiments. The underlying applications for MS monitoring benefit from its ionization methods, taking the form of plasma [5], and especially of electrospray ionization (ESI) coupled with microreactors [6,7] and other methods [8,9]. Reactions varied from seconds to hours can be monitored online with these massspectrometry-informed methods, providing high-quality timedependent spectra, a topic that has been reviewed [10,11]. Given these established capacities, the use of ESI-MS, for example, to identify reaction intermediates without affecting the ongoing chemical process, includes complex multicomponent reactions, organometallic transformations [12], and even oxygen- and moisture-sensitive reaction mixtures [13]. The reaction intermediates in the field of pharmaceuticals, medicines and synthetic chemistry include ionic species [14] such as carbocations, carbanions, carbenes and radical cations, and neutral intermediates/products. In a typical electrospray-based experiment, the ionic and neutral species are simultaneously sampled in the charged-microdroplets generated by applying a high voltage to the reacting solution. Since the neutrals are ionized and expressed at the form of charged state (e.g., [MþH]þ, [MþNa]þ) in this scenario, the differentiation of ionic and neutral intermediates poses an experimental challenge and to some extent relies on the creativity and experience of chemists. This restricts MS applications in reaction mechanism studies. Moreover, the ionic intermediates and ionized products in some cases are isomer and thus cannot be distinguished on the basis of mass-to-charge alone [15]. This represents an additional concern in capturing and detecting reactive intermediates experiments. The commonly used electrospray-based technologies such as desorption electrospray ionization (DESI) [16,17], extractive electrospray ionization (EESI) [18], sonic spray ionization (SSI) [19], have also been applied to identify the reaction intermediates in chemical reactions. However, the concern on differentiating the reaction intermediates at ionic or neutral forms using these ionization strategies is yet challenging. Herein, we provide a new strategy that the reaction solution undergoes a solution-phase nebulization prior to ionization to achieve ionic and neutral intermediates separation and characterization. This takes inspiration from the nebulization where the ionic intermediates transferred to the gas phase can be electrically deflected. The reaction mixture was transferred to a homemade flow injection sprayer to generate an electrically neutral spray. After gentle desolvation in a straight heated tube, the ionic species in drying gas stream were electrically deflected using an ion deflector and the remaining neutral species were exclusively ionized for MS analysis without the interference of ionic species. The ionization of gas-phase neutral species in this way has been widely used in atmospheric pressure ionization techniques such as neutralization/re-ionization experiments [20e22]. We also performed the other case that in absence of ion deflection the ionic and neutral species from the heated tube were simultaneously ionized and detected by MS. This in fact is similar to previous electrospraybased methods [23e25] where the ionic and neutral species were simultaneously presented in the electrospray plume. Owing to the removal of ionic species by ion deflection, the differences in abundances in these two cases are assigned as ionic intermediates and the neutral molecules which are regardless of the ion deflection and have the same abundance. To evaluate this performance, two reactions, EschweilereClarke reaction and copper-catalyzed CeH oxidative cyclization of carboxylic acids, were demonstrated. The ionic and neutral intermediates were separated and characterized, which gave new insights into the mechanism studies.
Fig. 1. (a) Experimental setup for isolating and characterizing ionic and neutral species. The trajectories of ionic and neutral species when the ion deflector operated at (b) ON mode, and (c) OFF mode. For ON mode, the ionic species from nebulization are removed and only neutral species are sequentially ionized for MS analysis; for OFF mode, both the ionic and neutral species pass through the ion deflector and are simultaneously ionized and detect-ed by MS. The reaction mixture introduced methods: (d) direct injection for EschweilereClarke methylation of di-n-butylamine; (e) pressurized sampling injection for copper-mediated oxidative cyclization of 4phenylbutyric acid.
2. Materials and methods 2.1. Reagents Methanol and acetonitrile at MS grade were purchased from Sigma-Aldrich (Darmstadt, Germany). Potassium persulfate (K2S2O8), tris(p-bromophenyl)aminium hexachloroantimonate, dichloromethane, 4-aminophenol, 4-phenylbutyric acid, di-nbutylamine, acetic acid, and formaldehyde (36.5e38% in H2O) were purchased from Sigma-Aldrich (St. Louis, MO). Formic acid and copper (II) acetate (Cu(OAc)2$H2O) were purchased from Laiyang Fine Chemical Factory (Laiyang, China). Ultrapure H2O was obtained from a water purification system (Milli-Q, Milford, MA).
2.2. Experiment apparatus As shown in Fig. 1a, the apparatus used for nebulization and isolation of the ionic and neutral species consisted of a home-built flow injection sprayer (id., 0.10 mm, od., 0.30 mm; id., 0.45 mm, od., 0.75 mm), a stainless steel tube (id., 5.8 mm; od., 7.4 m; length 10 cm), and an ion deflector. For the sprayer, nitrogen was used as nebulization gas and a gas pressure of 20 psi was used. To maximize collection of the electrically neutral microdroplets, the tip of the sprayer was inserted into the heated tube. The transmission tube was heated using a coiled heating wire (0.3 mm) controlled by temperature regulator (Zhonghuan Temperature instrument Co., LTD, Tianjing, China) coupled with a thermocouple, with the range of 25 Ce400 C. The drying gas stream emerging from the heated tube (~150 C) passed through an ion deflector to remove the ionic species in the gas stream. The dimension of the ion deflector is shown in Fig. S1. The ion deflector consisted of a pair of parallel metal plates (10 10 mm2) and was integrated into a box made with polyetheretherketone (PEEK). The distance between the two plates was
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10 mm. The holder was placed in line with the MS inlet and the distance to the MS inlet was ca. 1 cm. One plate was grounded while the other was supplied with a potential to deflect ions. The deflection voltage increased with the increase of gas pressure for nebulization. It was found that a bias of 2 kV could completely remove all ionic species as monitored by MS. After deflection, the separated neutral species were then ionized by ESI (id., 0.10 mm, od., 0.30 mm). The distance between the tip of the ESI sprayer and the MS inlet was ca. 0.4 cm, and an angle formed between the spray tip and the inlet was ca. 30 . The heated tube and ion deflector were integrated to minimize turbulence of gas stream at the interface and maximize the transfer efficiency of ionic/neutral species at atmosphere pressure. For ESI, the spray solvents for EschweilereClarke reaction and copper-catalyzed CeH oxidative cyclization were methanol and methanol/water/acetic acid mixture (90:10:0.1), respectively. The details for experimental procedures were given in Supporting Information. 2.3. Mass spectrometry experiments All the experiments were implemented on an LTQ/Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Full-scan positive ion mass spectra were recorded over an m/z range from 100 to 400 with a resolution of 60000. Typical instrumental parameters included 1 microscans, 500 ms maximum ion injection time, 110 V tube lens voltage, 35 V capillary voltage, and 275 C capillary temperature. MS/MS data were recorded using the default values except that the isolation window and nominal collision energy were adjusted to improve spectral quality. A standard LTQ XL ESI positive ion calibration solution was rigorously used for the positive mass calibration before each experiment. All experiments were conducted three times or more. Without special notes, the mass spectra given were the average of three scans. 3. Results 3.1. Performance of the heated tube and ion defector The aim of the ion deflector was to remove the ionic species from nebulization. There were two operation modes of the ion deflector, OFF mode and ON mode. At OFF mode, the ionic species and neutrals from nebulization both passed through the ion deflector and were then ionized for MS analysis. In this case, the ionic and neutral species were simultaneously ionized, which is similar to previous studies where the reaction mixture was ionized by ionization sources such as ESI. At ON mode, the ionic species from nebulization were removed by the ion deflector with a bias voltage of 2 kV, and the neutral species passed through the ion deflector and underwent ionization for MS analysis. In this scenario, the neutrals were analyzed without the interference of ionic species, and thus only neutrals involved in the gas stream were ionized and then detected by MS. The straight heated tube, similar to sample transfer capillary, was grounded to merely function as gentle desolvation and simultaneously minimize any possible interference with heating within the tube. The performance of desolvation was evaluated. Evidence for complete drying of the electrically neutral microdroplets is seen in the fact that free gas-phase cation radicals (m/z 480.8496) were detected by MS when nebulization of tris(pbromophenyl)aminium hexachloroantimonate and dried by being passed through the straight heated tube (Fig. S2a). The attempted removal of the ionic species employed an ion deflector. The ionic species from nebulization were removed by the ion deflector with a bias voltage of 2 kV. No signal of cation radical was observed (Fig. S2b). The success of the approach in detecting neutral species
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is clear from the representative mass spectrum of 4-aminophenol in aqueous methanol. No signal of 4-aminophenol (m/z 110.0603) was observed after a gentle desolvation in the straight heated tube (Fig. S3a), but a peak of m/z 110.0603 was dominated after deflection (2 kV) and then ionization using ESI (Fig. S3c). This sharp contrast between organic salts and 4-aminophenol in detection behavior emphasizes the fact that the radical cations are from the organic salt solution and that the neutral 4-aminophenol well lies in the ionization source. 3.2. EschweilereClarke reaction With the success of separation and detection of ionic and neutral species, we turned attention to investigate the ionic and neutral intermediates involved in EschweilereClarke [26], in which a primary (or secondary) amine is methylated into the hemiaminal with formaldehyde and formic acid in excess. Di-n-butylamine was selected as a model secondary amine to examine this process, and the proposed mechanism is shown in Scheme 1. Di-n-butylamine reacting with formaldehyde leads to the transient 2 via the hemiaminal 1. In presence of formic acids in excess acting as a source of hydride, 2 is reduced to afford tertiary amine 3 as the final product. Previous MS studies have reported these reaction intermediates on the basis of ionization where a high voltage is applied to the reaction solution [27]. A significant question is that the ionic and neutral reaction intermediates are simultaneously ionized and analyzed, resulting in that the existing forms of these intermediates during the transform of primary (or secondary) amine to tertiary amine cannot be confirmed. Formaldehyde (0.1%), formic acid (1.0%) and di-n-butylamine (1.0%) were mixed in aqueous methanol (1/3: v/v) and then directly injected into the flow injection sprayer for nebulization. When the species emerging from the heated tube were directly ionized using ESI with methanol as solvent, peaks were observed at m/z 130.1593, 142.1594, 144.1751 and 160.1699 (Fig. 2a). Then, we used an ion deflector to remove the ionic species in the gas stream before ionization for MS analysis, and no signals were observed with the ion deflector applied (Fig. S4). After ionization, the application of the ion deflector showed no detectable change in the abundance of m/z 130.1593, 144.1751 and 160.1699. When no ion deflector was applied (Fig. 2a), signal of m/z 130.1593 was dominant and the relative intensities of m/z 144.1751 and 160.1699 were 0.11 and 0.06; when ion deflector was used (Fig. 2c), signal associated with m/z 130.1593 was still dominant and the relative intensities of m/z 144.1751 and 160.1699 were 0.12 and 0.06. Polarity switch of the ion deflector and deflection voltage had no influence on these peaks (Figs. S5 and S6). The time-dependent information also showed that the deflection voltage had no influence on these species (Fig. S7). These results establish the independence of m/z 130.1593, 144.1751 and 160.1699 on any ions formed during nebulization and its dependence on the ESI. MS/MS experiments for these peaks were also performed (Fig. S8) consistent with previous studies [27]. Therefore, peaks of m/z 130.1593, 144.1751 and 160.1699 represented neutral molecules in the course of Eschweiler-Clarke methylation and correspond to protonated di-nbutylamine, 3 and 1, respectively. When deflection was applied prior to ionization (Fig. 2c), the resulting peak of m/z 142.1594 was quite different from that recorded when no ion deflector was applied (Fig. 2a). The signal recorded at m/z 142.1594 was absent. The time-dependent data further supported that signal of m/z 142.1594 was related to the deflection voltage (Fig. S7). This suggests the fact that the response of m/z 142.1594 depends on the deflection voltage applied rather than ionization. The signal of m/z 142.1594 was assigned as the iminium ion 2 and was further supported by the tandem mass
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Scheme 1. Proposed EschweilereClarke methylation of di-n-butylamine leading to the tertiary amine.
Scheme 2. Proposed mechanism for copper-mediated oxidative cyclization of 4phenylbutyric acid to lactone.
Fig. 2. EschweilereClarke methylation of di-n-butylamine. (a) Mass spectra showing nebulization and ionization of reaction mixture without ion deflection, and (b) expansion showing the spectrum from m/z 135 to 165. (c) Mass spectra showing nebulization of reaction mixture and ionization of the neutral species passing through the ion deflector (2 kV), and (d) expansion showing the spectrum form m/z 135 to 165. Data were shown at reaction time of 1 min.
spectrometry experiment (Fig. S8d). It has been reported that the lifetime of iminium ions in aqueous solution is quite short and the reaction of an iminium ion with a source of hydride is very rapid under acidic conditions [27,28]. These observations suggest that this setup is capable of online separation, monitoring and identification of ionic/neutral species and permits the detection of transient intermediates. 3.3. Copper-catalyzed CeH oxidative cyclization of carboxylic acids Encouraged by these results, we turned to the conversion of CeH to CeO bonds in complex molecules, which remains an outstanding challenge in synthetic chemistry [29,30]. The coppercatalyzed CeH oxidative cyclization of carboxylic acids affording lactone [31] was investigated. As shown in Scheme 2, benzylic hydrogen abstraction of 4-phenylbutyric acid by SO$4 generates a carbon radical, which is then oxidized by Cu(II) catalyst to a resonancestabilized carbocation 4. The 4 can cyclize directly to give lactone 5 or be trapped by water to furnish alcohol 6, which is further converted to 5 or terminates with the formation of ketone 7. The oxidative cyclization with the formation of carbocation 4 differs from related investigations that proposed the intermediacy of a carboxylate radical [32,33]. The proposed mechanism has been investigated by DESI-MS combined with physical organic experiments [15]. However, the carbocation 4 and lactone 5 are not isolated and characterized by MS on basis of m/z alone because the
carbocation 4 and protonated 5 have the same m/z. We use the setup described herein coupling with a heated reactor, similar to that used in pressurized sample infusion [9,34], to online isolation and characterization of the carbocation and the neutral products including its isomer. The experiment details are given in the Supporting Information. The reaction solution was transferred to the flow injection sprayer for nebulization through a sample capillary at a gas pressure of 4 psi. When no ion deflection was applied, the ionic and neutral species were directly ionized in presence of a plume of positive-electrosprayed droplets created using methanol/water/acetic acid mixture (90:10:0.1). The mass spectrum is dominated with signal of m/z 165.0917 (Fig. 3a). The mass spectrum also shows signals of m/z 179.0705, 180.0785, 201.0315 and 217.0266. With ion deflection (2 kV), no signals were observed (Fig. S9), indicating that the ionic species are completely removed. Then, the remaining neutral species underwent the subsequent on-line ionization. Peaks associated with m/z 165.0917, 179.0705, 201.0315 and 217.0266 were observed with the corresponding abundances to no ion deflection applied (Fig. 3a and b). The time-dependent information further supports that the deflection voltage had no influence on these species (Fig. S10). This indicates that these species are neutral molecules in the oxidative cyclization of 4-phenylbutyric acid. Peaks of m/z 165.0917 corresponded to protonated 4-phenylbutyric acid. Signals of m/z 179.0705 and 217.0266 were assigned as [7 þ H]þ and [7 þ K]þ, respectively. As shown in Fig. 3b, the resulting abundance for 163.0761 peak with ion deflection applied is quite different from that recorded when nebulizing the mixed solution followed no ion deflection (Fig. 3a). Only relative abundance of 46% was observed, which significantly decreased compared to the case in absence of ion deflection where the relative abundance of 163.0761 peak was 82%. Polarity switching of the ion deflector showed no detectable change in the abundance of this peak (Fig. S11). The relative abundance of 163.0761 peak decreased with the increase of deflection voltage from 0 to 2 kV (Fig. S12). The time-dependent results with ion deflector also showed the decrease of relative abundance of 163.0761 peak compared to no ion deflector applied
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Fig. 4. MS/MS spectra of m/z 163.0761 obtained from (a) deflection then ionization, (b) nebulization alone, and (c) standard 5. Isolation window is m/z 1; CID energy is 21 (arbitrary units). Different dissociation features were obtained.
Fig. 3. Copper-mediated oxidative cyclization of 4-phenylbutyric acid to lactone. Mass spectra showing (a) nebulization then ionization without ion deflector, and (b) nebulization with ion deflection (2 kV) then ionization. (c) Mass spectrum showing nebulization of reaction solution without deflection and ionization. Data were obtained at reaction time of 2.5 min.
(Fig. S10). Together these experiments suggest that peaks of m/z 163.0761 in Fig. 3a and b represent different species. Since the ionic species were completely removed using an ion deflector, the 163.0761 peak in presence of ion deflection was assigned to lactone 5 (Fig. 3b); and the 163.0761 peak in absence of ion deflection was a mixture of carbocation 4 and protonated lactone 5 (Fig. 3a). The case where only nebulization was also performed. We observed different mass spectrum feature; the mass spectrum is shown in Fig. 3c and dominated with peak of m/z 163.0761 associated with carbocation 4. The above assignment of 163.0765 peak was further supported by collision induced dissociation (CID) experiment, as now discussed. This experiment was carried out by selecting the m/z 163.0765 from deflection then ionization (Fig. 3b) and nebulization alone (Fig. 3c) and dissociating under various collision energies. As shown in Fig. 4a and b, fragmentations from these two cases generate two similar fragments, one at m/z 145 associated with loss of H2O and the other one with m/z 117 associated with loss of CH2O2 (Fig. S13). However, the resulting dissociation behavior respect to collision energy for these two cases was quite different. CID on m/z 163.0765 from deflection then ionization established 80% dissociation of this species in the gas phase at normal collision energy (NCE) of 21 (arbitrary units). This is consistent well with standard lactone 5 that undergoes 80% dissociation with NCE of 21 (Fig. 4c). When the same NCE of 21 was applied in the case of merely nebulizing the reaction mixture, 99% dissociation was observed (Fig. 4b). To obtain the same dissociation, the NCE used was 15 (Fig. S14). Together, these results suggest that the intrinsic reactivity of carbocation 4 requires less energy to undergo the same
extent dissociation as compared to lactone 5. The sharp contrast for these two species behaviors in fragmentation supports the formation of carbocation 4 in the oxidative cyclization of 4-phenylbutyric acid, which was not previously isolated and characterized [31]. In contrast to experiments associated with prolonging the reaction time to obtain pure lactone [15], detection of the lactone 5 can be directly achieved respect to the separation of nebulization and ionization and electrical deflection of carbocation 4. 4. Conclusions In summary, we have demonstrated a nebulization method followed by on-line ionization in which the ionic and neutral intermediates in reaction solution can be separated for characterization without any interference. Nebulization then deflection guarantees that only neutral intermediates undergo the subsequent on-line ionization. The ionic and neural intermediates resulting from EschweilereClarke methylation of di-n-butylamine were successfully captured and analyzed by MS. Response to ion deflection provides direct evidence for the formation of short-lived iminium ions in the reaction progress rather than ionization. The isomer (carbocation, lactone product) generated in Cu(II)-catalyzed oxidative cyclization of 4-phenylbutyric acid was separated. Provided that the individual dissociation feature of the isomer with normal collision energies, the carbocation intermediates showed high reactivity compared to the formed lactone. The two steps coupled with tandem mass spectrometry give this approach high specificity. This platform was also compatible with a wide range of analytical needs such as time-dependent reaction information and different reaction conditions (e.g., temperature, reaction solvent). This on-line mass spectrometric technique respect to nebulization probes the existence form of reaction intermediates (e.g., ionic, neutral), which would advance mechanistic information for many solution reactions. Declaration of competing interest The authors declare that they have no known competing
Please cite this article as: H. Zhang et al., Nebulization prior to ionization for mechanistic studies of chemical reactions, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.02.031
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financial interests or personal relationships that could have appeared to influence the work reported in this paper. CRediT authorship contribution statement Hong Zhang: Conceptualization, Writing - original draft, Methodology, Investigation, Formal analysis. Lina Qiao: Conceptualization, Writing - original draft, Methodology, Investigation, Formal analysis. Wenxin Wang: Investigation, Validation. Jing He: Investigation, Validation. Kai Yu: Writing - review & editing, Data curation. Miao Yang: Validation, Data curation. Hong You: Writing - review & editing. Jie Jiang: Supervision, Conceptualization, Funding acquisition, Project administration. Acknowledgments This work was supported by the supported by the Natural Science Foundation of China (No.21904029), Key R&D program of Shandong (NO.2016YYST013), fundamental research funds for the central universities (No.HIT.NSRIF.2020087) and Weihai science and technology development program (No. 2019KYCXJJYB03). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca.2020.02.031. References [1] L.A.B. Moraes, M.N. Eberlin, Ketalization of gaseous acylium ions, J. Am. Soc. Mass Spectrom. 12 (2001) 150e162. [2] J.M. Beames, F. Liu, L. Lu, M.I. Lester, Ultraviolet spectrum and photochemistry of the simplest criegee intermediate CH2OO, J. Am. Chem. Soc. 134 (2012) 20045e20048. [3] O. Zandi, T.W. Hamann, Determination of photoelectrochemical water oxidation intermediates on haematite electrode surfaces using operando infrared spectroscopy, Nat. Chem. 8 (2016) 778e783. [4] R.M. Bain, S. Sathyamoorthi, R.N. Zare, "On-droplet" chemistry: the cycloaddition of diethyl azodicarboxylate and quadricyclane, Angew. Chem. Int. Ed. 56 (2017) 15083e15087. [5] X.X. Ma, S.C. Zhang, Z.Q. Lin, Y.Y. Liu, Z. Xing, C.D. Yang, X.R. Zhang, Real-time monitoring of chemical reactions by mass spectrometry utilizing a lowtemperature plasma probe, Analyst 134 (2009) 1863e1867. [6] L.S. Santos, J.O. Metzger, Study of homogeneously catalyzed Ziegler-Natta polymerization of ethene by ESI-MS, Angew. Chem. Int. Ed. 45 (2006) 977e981. [7] J. Griep-Raming, S. Meyer, T. Bruhn, J.O. Metzger, Investigation of reactive intermediates of chemical reactions in solution by electrospray ionization mass spectrometry: radical chain reactions, Angew. Chem. Int. Ed. 41 (2002) 2738e2742. [8] H. Zhang, X. Li, K. Yu, N. Li, J. He, H. You, J. Jiang, On-line monitoring of photolysis reactions using electrospray ionization mass spectrometry coupled with pressurized photoreactor, Anal. Chim. Acta 1013 (2018) 36e42. [9] X. Yan, E. Sokol, X. Li, G.T. Li, S.Q. Xu, R.G. Cooks, On-line reaction monitoring and mechanistic studies by mass spectrometry: negishi cross-coupling, hydrogenolysis, and reductive amination, Angew. Chem. Int. Ed. 53 (2014) 5931e5935. [10] A. Ray, T. Bristow, C. Whitmore, J. Mosely, On-line reaction monitoring by mass spectrometry, modern approaches for the analysis of chemical reactions, Mass Spectrom. Rev. 37 (2018) 565e579.
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Please cite this article as: H. Zhang et al., Nebulization prior to ionization for mechanistic studies of chemical reactions, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2020.02.031