B American Society for Mass Spectrometry, 2014

J. Am. Soc. Mass Spectrom. (2014) 25:1870Y1881 DOI: 10.1007/s13361-014-0988-7

RESEARCH ARTICLE

The Ionization Mechanisms in Direct and Dopant-Assisted Atmospheric Pressure Photoionization and Atmospheric Pressure Laser Ionization Tiina J. Kauppila,1 Hendrik Kersten,2 Thorsten Benter2 1

Faculty of Pharmacy, University of Helsinki, Helsinki 00014 Finland Department of Physical and Theoretical Chemistry, University of Wuppertal, 42119 Wuppertal( Germany

2

Abstract. A novel, gas-tight API interface for gas chromatography–mass spectrometry was used to study the ionization mechanism in direct and dopant-assisted atmospheric pressure photoionization (APPI) and atmospheric pressure laser ionization (APLI). Eight analytes (ethylbenzene, bromobenzene, naphthalene, anthracene, benzaldehyde, pyridine, quinolone, and acridine) with varying ionization energies (IEs) and proton affinities (PAs), and four common APPI dopants (toluene, acetone, anisole, and chlorobenzene) were chosen. All the studied compounds were ionized by direct APPI, forming mainly molecular ions. Addition of dopants suppressed the signal of the analytes with IEs above the IE of the dopant. For compounds with suitable IEs or Pas, the dopants increased the ionization efficiency as the analytes could be ionized through dopant-mediated gas-phase reactions, such as charge exchange, proton + transfer, and other rather unexpected reactions, such as formation of [M+77] in the presence of chlorobenzene. Experiments with deuterated toluene as the dopant verified that in case of proton transfer, the proton originated from the dopant instead of proton-bound solvent clusters, as in conventional open or non-tight APPI sources. In direct APLI using a 266 nm laser, a narrower range of compounds was ionized than in direct APPI, because of exceedingly high IEs or unfavorable two-photon absorption cross-sections. Introduction of dopants in the APLI system changed the ionization mechanism to similar dopant-mediated gas-phase reactions with the dopant as in APPI, which produced mainly ions of the same form as in APPI, and ionized a wider range of analytes than direct APLI. Key words: Atmospheric pressure photoionization, Atmospheric pressure laser ionization, Gas chromatography– mass spectrometry, Ionization mechanism, Resonance-enhanced multiphoton ionization Received: 3 July 2014/Revised: 19 August 2014/Accepted: 19 August 2014/Published online: 24 September 2014

Introduction

A

tmospheric pressure photoionization (APPI) is an important ionization method, which after its introduction in 2000 has significantly broadened the range of low polarity compounds amenable to LC-MS [1, 2]. Although electrospray ionization (ESI) is still the most popular LC-MS ionization technique for the analysis of polar compounds, APPI is often the method of choice, when neutral or nonpolar analytes are analyzed. APPI has found wide use in pharmaceutical, biological, environmental, and food analysis areas, to mention just a few [3–5]. As the name suggests, the initial process in APPI is photoionization. Typically, APPI uses a krypton discharge lamp,

Correspondence to: Tiina J. Kauppila; e-mail: [email protected]

which provides vacuum ultraviolet (VUV) radiation with energies at 10.0 and 10.6 eV. In theory, any species with ionization energy (IE) below the energy of the photons can be ionized. Accordingly, in non-tight atmospheric pressure ionization sources, the abundantly present atmospheric gases do not contribute to the overall charge generation. This way APPI is a more selective method to generate ions of common analytical interest, compared with for instance atmospheric pressure chemical ionization (APCI). However, in the provided wavelength range, several species such as water and oxygen photodissociate with significant absorption cross-sections [6]. Consequently, open systems are prone to extensive neutral radical-induced ion transformation processes and radiative loss into non-charged species. This results in poor performance for direct photoionization. To enhance the ionization efficiency in APPI, a solvent with an IE below the energy of the photons, called dopant, is

T. J. Kauppila et al.: APPI and APLI Mechanism

typically used in APPI to enhance the ionization efficiency. The idea is to beat competing photodissociation processes, and quantitatively convert VUV photons into a primary charged species, mostly molecular ions. These can form proton-bound clusters with the neutral analogues, with water or other solvents present in the ion source [7]. The neutral analytes can react with these clusters in fast ligand-switching or association reactions, and contribute to thermodynamically well equilibrated cluster distribution. Further downstream, the clusters dissociate in the low pressure region of the mass spectrometer mainly by collision induced dissociation, where protonated analyte molecules will be formed, if the analyte has a higher proton affinity (PAs) than the other species in the cluster. The most important ionization route for nonpolar compounds is charge exchange, which can take place in case the IE of the analyte is below the IE of the dopant. In LC-MS, the LC solvents have been shown to play a major role in the ionization reactions: high PA LC solvents, such as methanol and acetonitrile, can completely neutralize the dopant molecular ions and thus prevent the ionization of analytes through charge exchange [8]. Although APPI was originally developed as an interface for LC-MS, some gas chromatography–mass spectrometry (GCMS) applications utilizing APPI have also emerged [9–18]. A general advantage of GC is its overwhelming separation efficiency compared with LC, and in particular for APPI the absence of LC solvents significantly enhances the charge exchange process between the dopant molecular ions and the neutral analyte. However, water clusters and atmospheric gases can still affect the ionization process in non-tight GC-APPI sources. Recently, a highly sensitive atmospheric pressure ionization (API) interface for GC-MS has been introduced [19]. In this design, the crucial parameters for efficient direct ionization and chromatographic performance were carefully considered. The key part of this interface is a small, airtight, conically shaped ionization unit. Inside, a vortex is maintained by a highly purified (99.999999%) nitrogen flow, to the eye of which the minor GC gas stream is placed. Optical access is provided by a MgF2 window, on which the photoionization lamp is directly sited. All materials were carefully selected, in particular issuing outgassing and thermal expansion coefficients. The interface has been shown to give reasonable sensitivity for trace analysis, with LODs in the femtogram range [20]. Besides APPI, the interface can also be used for atmospheric pressure laser ionization (APLI), simply by replacing the VUV lamp with a suitable laser. APLI has been introduced as an alternative ionization method for LC-MS and GC-MS [21, 22]. In APLI, the ionization is based on resonanceenhanced two-photon ionization. It is highly selective towards compounds exhibiting long-living resonant states, such as polycyclic aromatic hydrocarbons (PAHs). APLI is not as widely spread as APPI, and thus less explored. Nevertheless, its outstanding performance in the analysis of nonpolar aromatic compounds in crude oil, petroleum, and environmental samples has been demonstrated in several publications [23–25]. A comprehensive study on the addition of different dopants in APLI has thus far not been done.

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Due to the purity of the matrix and, thus, well-defined conditions, this novel GC-API interface offers a unique opportunity for studying ionization mechanisms in direct and dopant-assisted APPI and APLI, without interference from LC solvents, water-clusters, or atmospheric gases. Therefore, a group of compounds with varying IEs and PAs was chosen for the study, and their ionization behavior in direct APPI and APLI, as well as in the presence of four most typical APPI dopants, toluene, acetone, anisole, and chlorobenzene, was investigated.

Experimental Chemicals Toluene (99.9%), toluene-d8 (99%), acetone (99.9%), naphthalene, benzaldehyde, quinoline, acridine, ethylbenzene, and bromobenzene were purchased from Sigma-Aldrich (Steinheim, Germany), hexane (HPLC grade) from VWR International (Leuven, Belgium), chlorobenzene (999%) and anisole (999%) from Merck (Hohenbrunn, Germany), pyridine from Acros Organic (Fair Lawn, NJ, USA), and anthracene from Fluka (Buchs, Germany).

Sample Preparation Stock solutions, 1–10 mM of quinoline, acridine, anthracene, and naphthalene were prepared in toluene (acridine) or hexane (all others). The stock solutions were used to prepare a 1 μM mixture of the compounds in hexane, which was used for the GC-MS analyses. Pyridine, ethylbenzene, bromobenzene, and benzaldehyde were analyzed by headspace injection.

Instrumentation Gas Chromatography The samples were separated using a Thermo Scientific 450 Series gas chromatograph and a TR-Dioxin 5MS column (30 m×0.25 mm i.d. × 0.1 μ; Milan, Italy). 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, 20 °C/min up to 320°C, hold time 5 min. Helium with 99.999% purity (Messer Industriegase GmbH, Bad Soden, Germany) at a constant flow rate of 1.50 mL/min was used as the carrier gas. The GC transfer line and injector temperatures were both set to 250°C. Splitless injection of 0.5 μL was used for the anthracene, naphthalene, acridine, and quinoline mixture (corresponding to 500 fmol on column/compound). Split injection of 0.5 μL with a split ratio of 10,000 was used for bromobenzene, ethylbenzene, and benzaldehyde headspace. Split injection of 0.05 μL with a split ratio of 10,000 was used for pyridine headspace.

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Mass Spectrometry The mass spectrometer was a Thermo Scientific (Bremen, Germany) Exactive Orbitrap, equipped with a custom-made API interface. As shown in Figure 1 (left), a commercial transfer line (Thermo Scientific) was used to guide the sample flow from the GC column to the ion source enclosure. The column and the ion source enclosure were gastightly connected using a standard GC ferrule. The original MS transfer capillary was replaced by a stainless steel capillary (Klaus Ziemer GmbH, Langerwehe, Germany) modified with a gas-tight adapter to the ion source. Optical access for the UV laser or VUV lamp radiation was provided with a cemented MgF window (Edmund Optics Inc., Barrington, NJ, USA) in 2 the circular depression of the source enclosure (cf. Figure 1 right). The ion source enclosure was made of invar36 (Enpar Sonderwerkstoffe GmbH, Gummersbach, Germany) to adapt to the thermal expansion coefficient of MgF and the cement (OMEGA CC High Temperature Cement2 from OMEGA Engineering Inc., Stamford, CT, USA). The ionization volume was of conical shape with the aperture to the MS located at the tip, parallel to the GC-column entrance. As shown in Figure 1 (right), the column and the make-up gas entrance were located directly below the window, the latter to the far left of the cone and perpendicular to the column. In this way, the main flow maintained a vortex, to the eye of which the minor GC flow was directed. Two heater cartridges (HORST GmbH, Lorsch, Germany) with 200 W heat output each kept the interface at a constant temperature of up to 350°C. Electrical isolation at certain points in the setup ensured that the capillary voltage in the first differential pumping stage was still applicable. In the present experiments, the interface was held at 250°C. For the make-up gas, N2 from compressed gas cylinders (Messer Industriegase GmbH, Bad Soden, Germany) was further purified with a Vici Metronics (Poulsbo, WA, USA) N2 purifier to sub ppbV levels of impurities. The gas flow was adjusted to 850 mL/ min with a MKS mass flow controller and multigas controller 647C from MKS Instruments Deutschland (München, Germany). The capillary temperature was 250°C, and capillary, tube lens, and skimmer voltages were 5, 43, and 14 V, respectively. The

T. J. Kauppila et al.: APPI and APLI Mechanism

mass range was 50–1000 (pyridine), 100–1000 (ethylbenzene and benzaldehyde), or 120–1000 m/z (the rest of the compounds). All measurements were performed in positive ion mode. For the dopant experiments, an additional line was connected via a tpiece directly to the make-up gas entrance of the source enclosure. Headspace of the dopant (toluene, toluene-d8, acetone, anisole, and chlorobenzene) was added to the make-up gas with a gas syringe and a syringe pump at a flow rate of 100 μL/min. For the APPI measurements, a low-pressure Kr discharge lamp with a rf driver from Syagen (Santa Ana, CA, USA) provided radiative output of 10.0 and 10.6 eV. The entire ionization volume was irradiated. For the APLI measurements, an OEM laser device from CryLaS (Berlin, Germany) was used. The laser was a frequency-quadrupled diode-pumped solid-state (DPPS) Nd:YAG laser with 266 nm (4.66 eV) wavelength, a pulse duration of 0.9 ns, a max repetition rate of 60 Hz, a spot size of 0.5 mm, a pulse energy of 200 μJ, and a power density of 108 W/cm2. The laser beam pointed straight down the cone, sweeping past the exit of the GC-column.

Results and Discussion For this study, eight compounds (naphthalene, quinoline, anthracene, acridine, benzaldehyde, ethylbenzene, pyridine, and bromobenzene) with ionization energies (IEs) ranging from 7.4 to 9.5 eV and proton affinities (PAs) from 754.1 to 972.6 were chosen (Figure 2). The compounds were analyzed with direct and dopant-assisted APPI and direct and dopant-assisted APLI. Toluene, anisole, chlorobenzene, and acetone were used as dopants. The IEs and PAs of the studied compounds and dopants are listed in Table 1.

Direct APPI and APLI Table 2 summarizes the observed ions for the analytes studied with direct APPI and APLI. As expected, all the compounds were ionized in APPI, since their IEs are well below the provided photon energies of 10.0 and 10.6 eV (cf. Table 1). Mainly

Figure 1. Schematic of the entire GC-API interface (left) and close up of the ion source enclosure with indicated volume flows and radiation entrance (right)

T. J. Kauppila et al.: APPI and APLI Mechanism

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Figure 2. Structures and molecular weights (MW) of the studied compounds

molecular ions (M+.) were formed (Scheme 1, Reaction 1), except in case of benzaldehyde, for which the formation of [M – H]+ was observed, and pyridine, which formed both M+. and protonated molecule (MH+). The [M – H]+ ion of benzaldehyde can be either a hydride abstraction product, a fragment of MH+ formed by loss of H2, or a fragment of M+. formed by loss of H. (Scheme 1, Reactions 2, 3, and 4, respectively). In case of ethylbenzene, the M+. appeared to be the most abundant species; however, several minor intensity ions were observed as well. Formation of MH+ of pyridine was thought to be due to self-protonation (Scheme 1, Reaction 5), which is supported by the observation that the proportion of MH+ in the pyridine spectrum increased with higher injection volume or smaller split ratio. In direct APLI, bromobenzene, naphthalene, and anthracene formed exclusively M+. ions, similarly to APPI. Ethylbenzene showed a similar diverse ion distribution as in APPI with M+. and other minor ions (Table 2). However, unlike in APPI, benzaldehyde, pyridine, quinoline, and acridine were not observed in APLI at all. The underlying process of resonanceenhanced multi-photon ionization (REMPI) readily explains this [21]. Typical laser power densities in APLI are in the range of 107 to 108 W/cm2, which restrict the REMPI process to two photons. The first photon excites the molecule into a resonant state of a certain lifetime (Scheme 1, Reaction 6), typically in the ns–μs range, in which the second photon provides the residual energy for ionization (Scheme 1, Reaction 7). The summed photon energy has to exceed the IE of the compound. The laser in this study operated at 266 nm wavelength, corresponding to 4.66 eV/photon and, hence, the maximum energy provided for the ionization process was 9.32 eV. Consequently, the present experimental laser setup simply could not ionize benzaldehyde with an IE of 9.5 eV in direct APLI. Pyridine, quinoline, and acridine have IEs below 9.32 eV (9.26, 8.63 and 7.80, respectively) and, therefore, the IE condition in their case is fulfilled. However, the lifetime of the excited transition state of pyridine has been reported to be particularly short [26, 27], with the competing deactivation process of the resonant state

into an electronically lower-lying state (Scheme 1, Reaction 8) faster than the absorption process of the second photon, which explains why pyridine could not be ionized in the current setup. Since quinoline and acridine share a similar N-heterocyclic ring structure, it is likely that the lifetimes of their transition states were also too short for the current laser setup to obtain appreciable ionization rates. On the other hand, the one-color limit of pyridine has been reported to lie beneath the S0 ➔ S1 transition, which makes 1+1 REMPI of pyridine impossible using this transition [28]. However, ionization using higher vibrational levels of the S1 state is possible with lower wavelength laser. Also, the ionization of quinoline has been reported to proceed via higher vibrational levels.

Dopant-Assisted APPI and APLI Effect of Dopants on the Background The introduction of dopant increased the amount of background ions in both APPI and APLI, but in APPI the increase was much more pronounced than in APLI (Figure 3). The elevated background was mostly due to dopant-derived ions: M+. formed by photoionization of the dopant in cases of toluene, anisole, and chlorobenzene (Scheme 1, Reaction 9), and MH+ in case of acetone. Ions that are typically observed in the dopant spectra in APPI, such as oxidation products at m/z 107–109 in toluene spectra [8, 29], were not observed in the present study. In both APPI and APLI the background was highest with chlorobenzene, toluene, and anisole. With chlorobenzene also the amount of other ions in the background increased. In APPI, acetone increased the background as well, although less than the other dopants, but in APLI the introduction of acetone caused no changes in the spectrum since the IE of acetone is 9.70 eV, which is above the two-photon energy of the used laser (9.32 eV). The higher signal intensities of the dopantderived ions in APPI indicate that the used APPI setup ionizes the dopants more efficiently than the APLI setup.

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T. J. Kauppila et al.: APPI and APLI Mechanism

Table 1. Ionization energies (IE) and proton affinities (PA) of the studied compounds and dopants [49], and the enthalpy changes (ΔH) for charge exchange and proton transfer reactions between the analytes and toluene, acetone and anisole dopants

ΔH for charge exchange (eV)

ΔH for proton transfer (eV) methoxy

IE

PA

PA

(eV)

(kJ/mol)

(eV)

Analytes

benzyl toluene

acetone

anisole

acetone

phenol

radical radical

Anthracene

7.44

877.4

9.09

-1.39

-2.26

-0.76

-0.47

-0.67

-0.34

Naphthalene

8.14

802.9

8.32

-0.68

-1.56

-0.06

0.30

0.10

0.43

Acridine

7.80

972.6

10.08

-1.03

-1.90

-0.40

-1.46

-1.66

-1.33

Quinoline

8.63

953.2

9.88

-0.20

-1.07

0.43

-1.26

-1.46

-1.13

Pyridine

9.26

930.1

9.64

0.43

-0.44

1.06

-1.02

-1.22

-0.89

Benzaldehyde

9.50

834.0

8.64

0.67

-0.20

1.30

-0.02

-0.22

0.11

Bromobenzene

9.00

754.1

7.82

0.17

-0.70

0.80

0.80

0.60

0.93

Ethylbenzene

8.77

788.0

8.17

-0.06

-0.93

0.57

0.45

0.25

0.58

IE

PA

PA

(eV)

(kJ/mol)

(eV)

Dopants Green: Observed, exothermic reaction Toluene

8.83

Chlorobenzene

9.07

Anisole

8.20

Acetone

9.70

Magenta: Reaction not observed, although it was the most exothermic

Benzyl radical

812.0

8.42

831.4

8.62

844.0

8.75

Methoxyphenol radical

Table 2. The analyte ions observed with direct APPI and APLI Compound

Ethylbenzene Bromobenzene Naphthalene Anthracene Benzaldehyde Pyridine Quinoline Acridine

APPI

APLI m/z (%) identity

m/z (%) identity

106 (100) M+., 123 (36) [M+17]+, 105 (25) [M–H]+, 104 (20) [M–H2]+, 103 (5) [M–H–H2]+ 156, 158 (100) M+. 128 (100) M+. 178 (100) M+. 105 (100) [M-H]+ 79 (100) M+., 80 (79) MH+ 129 (100) M+. 179 (100) M+.

106 (M+., 100), 119 (28) [M+13]+, 105 (22) [M–H]+ 156, 158 (100) M+. 128 (100) M+. 178 (100) M+. -

T. J. Kauppila et al.: APPI and APLI Mechanism

M+.

M +h M MH+ M

(1) +

+.

[M H] + H

(2)

[M H]+ + H2

(3)

+

.

[M H] + H

(4)

M+. + M

MH+ + [M H].

(5)

M +h

M*

(6)

+.

M* + h

M

M*

M

D+h

D+.

+.

(7) (8) (9) +.

D +M

D+M

D+. + D

DH++ [D H].

+

(10) (11)

+

DH + M

D + MH

D+. + M

[D H]. + MH+ +.

M + C6H5Cl

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assisted APPI and APLI. [Note that absolute signals rather than signal-to-noise ratios are discussed here. This approach was chosen to make possible the direct comparison of the total amount of ions formed in different conditions. For comparing the sensitivities of direct and dopant-assisted methods, the effect of the dopant on the background should be considered.] The ionization of each compound in dopant-assisted APPI and APLI is discussed individually below. For discussions about the exothermicity/endothermicity of the charge exchange and proton transfer reactions between the analytes and the toluene, acetone, and anisole dopants, see the calculated enthalpy changes (ΔH) in Table 1.

(12)

+

(13) .

[M C6H5] + Cl

(14)

Scheme 1. The main ionization reactions observed with APPI and APLI. M=analyte, D=dopant

There are several reasons for this dissimilarity, such as the significant restriction of the ionization volume with the small spot size of the DPSS laser beam. Also, different dependencies on the power densities (first order in APPI and up to second order in APLI) and compound-dependent properties (lifetime of the resonant state, UV versus VUV absorption cross-section) contribute to the observed differing ionization rates. The higher ionization efficiency of the dopants in APPI is likely to affect also the ionization of the analytes, since more dopant-derived ions are available for gas-phase ion/molecule reactions in APPI than in APLI. This will be discussed in more detail below, together with the results obtained with the analytes.

Effect of the Dopants on the Ionization of Analytes in APPI and APLI Figures 4 and 5 show the heights of different ions formed from the studied compounds with direct and dopant-

Ethylbenzene The ionization behavior of ethylbenzene was very similar in APPI and APLI, as can be seen in Figures 4 and 5. Besides direct APPI and APLI, ethylbenzene was ionized with chlorobenzene and acetone dopants. Chlorobenzene enhanced the ionization efficiency of ethylbenzene by almost an order of magnitude in APPI, and 4-fold in case of APLI. In all cases, the main ion formed was M+.. The signal enhancement in the presence of chlorobenzene is likely due to charge exchange between M+. of chlorobenzene and ethylbenzene (Scheme 1, Reaction 10). In both APPI and APLI, the introduction of anisole suppressed the signal of ethylbenzene, attributable to the lower IE of anisole, which makes the charge exchange from the M+. of anisole to neutral ethylbenzene thermodynamically impossible. The introduction of anisole also seems to suppress the direct photoionization of ethylbenzene, very likely due to the markedly competing photon absorption and neutralizing reactions between the dopant and the analyte. In toluene, a major ion, which is likely to be ethylbenzene or xylene present as a solvent impurity [30] [note, however, the possibility that the ion at m/z 106 resides from gasphase chemistry could not be excluded] was observed at the same mass as ethylbenzene M+. and, therefore, the

Figure 3. Height of the dopant main ion and naphthalene M+. in APPI and APLI

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T. J. Kauppila et al.: APPI and APLI Mechanism

Figure 4. Ionization of the studied compounds with direct and dopant-assisted APPI. *In toluene the ionization of ethylbenzene could not be verified due to high background signal at m/z 106

ionization of ethylbenzene in toluene could not be verified. However, since the IE of ethylbenzene is below that of toluene (8.77 and 8.83 eV, respectively), the charge exchange reaction between toluene M+. and ethylbenzene is theoretically possible.

Bromobenzene Besides direct APPI and APLI, bromobenzene was ionized with chlorobenzene and acetone dopants (Figures 4 and 5) as well. In all cases, bromobenzene formed M+.. In APPI, the introduction of dopants did not cause significant changes in the ion signal,

Figure 5. Ionization of the studied compounds with direct and dopant-assisted APLI. *Ionization of ethylbenzene could not be verified due to high background signal at m/z 106

T. J. Kauppila et al.: APPI and APLI Mechanism

but in APLI over two orders of magnitude increase in the bromobenzene M +. was observed upon introduction of chlorobenzene. The IE of bromobenzene is below that of chlorobenzene and acetone (9.00, 9.07, and 9.70 eV, respectively); therefore, charge exchange in the presence of these dopants is thermodynamically possible. However, acetone generally lacks the ability for charge exchange, since rapid self-protonation between M+. and the abundant neutral acetone is the predominant reaction channel (Scheme 1, Reaction 11) [31]. Hence, the observed formation of the M +. of bromobenzene in the presence of acetone is most likely due to direct photoionization (Scheme 1, Reaction 1). As expected from the IE criteria, the use of toluene and anisole did not lead to ionization of bromobenzene (cf. Table 1). Although a much more significant change was observed in the bromobenzene signal in the presence of chlorobenzene in APLI than in APPI, the resulting signal was still an order of magnitude lower in APLI than in APPI. The obvious difference in the ionization efficiencies in APLI between bromobenzene and chlorobenzene is readily explained by the nature of the resonant state. In both compounds, the excited transition state upon excitation with 266 nm radiation is pre-dissociative, but with significantly differing lifetimes of 36 and 1000 ps for bromobenzene and chlorobenzene, respectively [32]. This means that in twophoton absorption process the probability for a second photon to excite the molecule from the resonant state into an ionized state (Scheme 1, Reaction 8) is far lower in case of bromobenzene. However, once formed, the chlorobenzene M+. can efficiently ionize the neutral bromobenzene by charge exchange (Scheme 1, Reaction 10).

Naphthalene As shown in Figures 4 and 5 for naphthalene, only the M+. ion was observed in all cases. In APPI, the introduction of toluene and chlorobenzene increased the naphthalene signal 7- to 8-fold because of increased ionization by charge exchange (Scheme 1, Reaction 10). The addition of anisole, on the other hand, had only little effect on signal enhancement compared with direct APPI, probably because of the low exothermicity of the charge exchange reaction (0.06 eV, cf. Table 1). With acetone, the naphthalene signal decreased to one-fifth of the signal in direct APPI, indicating signal suppression by the less transparent matrix for VUV radiation and thus reduced photon flux. Additionally, as mentioned above for acetone, charge exchange with the analyte is not likely. Instead, acetone can efficiently promote formation of protonated analytes through proton transfer (Scheme 1, Reaction 12), in case the PA of the analytes is higher than the PA of acetone (812.0 kJ/mol). The PA of naphthalene, however, is 802.9 kJ/ mol and, therefore, proton transfer from acetone MH+ to naphthalene is not thermodynamically possible. In APLI, the addition of dopants caused no significant change in the naphthalene signal (cf. Figure 5). First, a smaller amount of dopant-derived ions is available for gas-phase ionization reactions as compared to APPI (Figure 3). Second, the appreciable ionization efficiency of naphthalene in direct APLI

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simply exceeds any noticeable effect of additional chemical ionization reactions. The two-photon absorption cross-section is composed of the product of the UV absorption cross-section from the electronic ground state to the resonant excited state, the lifetime of the resonant state, and the UV absorption cross section from the excited state to the ionized state. In case of naphthalene, the fluorescence lifetime upon 266 nm excitation was reported by Laor et al. as 43 ns [33], which is sufficiently long to explain the observed ionization rate. The signal heights obtained for naphthalene and the other compounds of the study in direct APLI can be compared from the graphs in Figure 5, where it shows that the signal for naphthalene is the highest. Figure 3 gives a direct comparison between the APPI and APLI obtained signals for naphthalene. For both direct methods the signals were of similar intensity, although the highest signal was obtained with dopant-assisted APPI. High ionization efficiency for naphthalene in (1+1) REMPI has also been reported in previous publications [25, 34, 35]. Anthracene Anthracene showed solely M+. with direct and dopant-assisted APPI, except in case of acetone, where it showed both M+. and MH+ ions (Figure 4). Toluene, anisole, and chlorobenzene increased the anthracene signal, the latter even 4-fold. With acetone, both M+. and MH+ ions were formed in a ratio of approximately 2:3. Anthracene has a higher PA than acetone (877.4 and 812.0 kJ/mol, respectively) and, therefore, proton transfer from protonated acetone to anthracene is thermodynamically possible. Nevertheless, the signal obtained in the presence of acetone was only roughly one-third of the signal obtained with direct APPI, indicating suppression effects. In studies using desorption atmospheric pressure photoionization (DAPPI), the presence of acetone has repeatedly been observed to suppress the ionization of anthracene completely [36–38]. In the open DAPPI source the protonation of analytes is dependent on their ability to enter proton-bound acetone-water clusters, which must be difficult for the nonpolar anthracene. However, in the current setup the protonated molecules are likely to be formed in a direct bimolecular gas-phase proton transfer process between the dopant and the analyte (Scheme 1, Reaction 12). Therefore, some protonation of anthracene is observed, although the reaction is obviously not very efficient. In APLI, anthracene formed M+. ions with all the dopants (Figure 5). The proton transfer with acetone is not possible in APLI, since acetone is not amenable for the (1+1) REMPI process at 266 nm. Introduction of chlorobenzene doubled the anthracene signal, whereas the other dopants did not have any effect on the signal intensity. This is different from APPI, where the anthracene signal also increased with toluene and anisole (Figure 4). Similarly to naphthalene, also anthracene is efficiently ionized by (1+1) REMPI at 266 nm. Benzaldehyde Similarly to direct APPI, benzaldehyde gave a [M – H]+ with anisole dopant, but with acetone, both MH+ and [M – H]+ were observed (in ratio of approx. 3:2, respectively,

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Figure 3). The overall signal with acetone dopant was over three orders of magnitude higher than with direct APPI. With anisole dopant the signal was lower than with direct APPI, whereas with toluene and chlorobenzene no signal for benzaldehyde was observed. Benzaldehyde has a fairly high IE at 9.5 eV, which is higher than the IEs of toluene, chlorobenzene, and anisole and, therefore, charge exchange (Scheme 1, Reaction 10) with these dopants is not possible. Formation of M+. ions through direct photoionization (Scheme 1, Reaction 1) should, however, be thermodynamically possible, but still M+. ions of benzaldehyde were not observed under any circumstance. A more important ionization route for benzaldehyde seems to be the formation of [M – H]+, either through hydride abstraction or fragmentation of M+. or MH+. Hydride abstraction is a typical reaction for compounds with aliphatic alkane chains [39], but benzaldehyde does not have such functionality. However, in electron ionization, fragmentation of M+. of benzaldehyde to a resonance-stabilized benzoylium ion at m/z 105 has been reported to be a major pathway [40]. It is, therefore, possible that also here, a M+. ion is formed, which rapidly fragments to the more stable benzoylium ion (Scheme 1, Reaction 4). However, the high intensity of both MH+ and [M-H]+ ions in the presence of acetone suggests that protonation and a subsequent loss of H2 is a much more favorable ionization route for benzaldehyde (Scheme 1, Reactions 12 and 3, respectively), and at least in the presence of acetone the [M – H]+ ion is more likely to be a fragment of MH+. The proton transfer from acetone to benzaldehyde is thermodynamically possible, since the PA of benzaldehyde (834.0 kJ/mol) is above the PA of acetone (812.0 kJ/ mol). The resulting ion is still likely to be the benzoylium ion, as in direct APPI and anisole dopant, although it is formed via a different route. As it appears from Table 1, the PA of benzyl radical (deprotonated toluene M+.) is slightly lower than the PA of benzaldehyde (831.4 and 834.0 kJ/mol, respectively) and, therefore, also the proton transfer from M+. of toluene to benzaldehyde is energetically possible. However, this reaction was not observed, which could be due to the very low exothermicity of the reaction. In chlorobenzene-assisted APLI, benzaldehyde showed MH+ as the main ion and [M+77]+ and [M+93]+ ions (according to the exact mass calculator of Xcalibur, corresponding to additions of C6H5 and C6H5O, respectively) with lower intensity (Figure 5). With the other dopants (and direct APLI) no signal was observed. This is very different from APPI, where benzaldehyde was not ionized by chlorobenzene dopant at all, but instead formed an intense MH + with acetone. Chlorobenzene is more prone to react through charge exchange than proton transfer because it has electron-donating groups, which have a stabilizing effect on the M+. [41] and, therefore, it is surprising that such a high signal for MH+ of benzaldehyde was observed in the presence of chlorobenzene. However, chlorobenzene gave high background ions also at other m/z besides chlorobenzene M+., either due to gas-phase chemistry or impurities, and the proton could therefore also originate from other species besides the chlorobenzene dopant. Similar [M+ 77]+ ions as here were also observed with pyridine and

T. J. Kauppila et al.: APPI and APLI Mechanism

quinoline, and they are likely to have formed through a similar mechanism (see discussion below), although with benzaldehyde the signal for this ion was much lower, indicating a lower efficiency for the reaction. The reason why these ions were not observed for benzaldehyde with chlorobenzene-assisted APPI is unknown.

Pyridine Unlike with direct APPI, where pyridine formed both M+. and MH+ ions, with toluene, acetone, and anisole dopants only MH+ ions of pyridine were observed (Figure 4). The addition of these dopants increased the pyridine signal 2.5- to 10-fold (most with toluene), compared with the signal with direct APPI, indicating that more favorable conditions for the formation of MH+ were achieved in the presence of dopants. Pyridine has a PA of 930.1 kJ/mol, which is higher than the PAs of acetone, and benzyl and methoxyphenol radicals (deprotonated anisole M+., PAs 812, 831.4, and 844.0 kJ/mol, respectively), and, therefore, the proton transfer from these species is thermodynamically possible (Table 1, Scheme 1, Reaction 13). On the other hand, the IE of pyridine (9.26 eV) is higher than the IEs of toluene, anisole, and chlorobenzene (8.83, 8.20, and 9.07 eV, respectively), which makes the charge exchange with these species unlikely. In LC-MS conditions, formation of protonated molecules is known to be mediated by proton-bound water and solvent clusters, which also act as proton donors [7]. Proton-bound water clusters are likely to be the proton donors also in case of open, non-tight GC-APPI-MS and DAPPI setups, which do not use LC solvents, but is that the case in the current setup as well? According to the assumption of a water-free setup, the only possible origin for the proton is the dopant (Scheme 1, Reaction 13). Self-protonation (Scheme 1, Reaction 5) is likely in case of direct APPI of pyridine, but since the signals increased significantly in the presence of the dopants, some dopantmediation was involved. In order to test this hypothesis, the analytes were ionized with APPI using deuterated toluene as the dopant. Figure 6 shows the spectrum obtained from the analysis of pyridine with toluene-d8. Traces of ordinary toluene were left in the syringe and/or tubing from the previous experiment and, therefore, also a toluene M+. peak is observed at m/z

Figure 6. Pyridine APPI spectrum in the presence of toluene and toluene-d8

T. J. Kauppila et al.: APPI and APLI Mechanism

92, in addition to the M+. of toluene-d8 at m/z 100. In the pyridine spectrum, the ratios of m/z 80 (MH+) to m/z 81 (MD+) are nearly the same as the ratios of M+. of toluene and toluene-d8, indicating that the proton indeed originates from the toluene M+.. It is assumed that also in cases of anisole and acetone the protons originate from the dopant, although this was not tested with deuterated solvents. This is a major difference between the current setup and previous designs of open or non-tight APPI sources, where the protons have been shown to originate from atmospheric water [7]. With chlorobenzene as the dopant, MH+ of pyridine was observed (approximately twice the signal compared with direct APPI), but the main ion was [M+77]+ at m/z 156 (Figure 4). The signal of [M+77]+ was over two orders of magnitude higher than the overall signal obtained for pyridine in direct APPI, and also much higher than the signals obtained with other dopants. The mass 77 may correspond to phenylium ion, which has been reported to be formed in the photodissociation of chlorobenzene [32]. A minor signal at m/z 77 was observed in the chlorobenzene spectrum and, therefore, the ion at [M+77]+ could be a phenylium ion adduct. Another possibility is that it could be the product of a nucleophilic attack by chlorobenzene to the nitrogen lone pair of electrons (Scheme 1, Reaction 14), as has previously been reported for chlorobenzene and ammonia to form aniline [42]. The identity of the ion could not be clarified with certainty, but will be addressed in an upcoming investigation. The same ion was observed in chlorobenzene-assisted APLI (Figure 5). However, in APLI the highest signal for pyridine was the MH+ in the presence of toluene.

Quinoline In direct APPI, only the molecular ion of quinoline was observed, whereas in toluene and acetone-assisted APPI the protonated form was the most abundant species; up to five times more intense than in the direct method (Figure 4). The IE of quinoline is 8.63 eV, thus well below the IEs of toluene, acetone, and chlorobenzene (cf. Table 1) and, therefore, charge exchange in the presence of these dopants is possible. On the other hand, the PA of quinoline exceeds the PAs of acetone, and benzyl and methoxyphenol radicals, which paves the reaction pathway for proton transfer as well. As it appears from Table 1, the proton transfer in case of toluene, acetone, and anisole dopants is more exothermic than the charge exchange reaction. This fact might tentatively explain the predominant formation of MH+ from a purely thermodynamic point of view. With chlorobenzeneassisted APPI, the main ion of quinoline was observed at m/z 206, corresponding to [M+77]+, similarly to the behavior of pyridine (see above). There seems to be a reaction pattern involving the N-heterogenic ring structure, which is present in both pyridine and quinoline (see Figure 2). Furthermore, this reaction seems to be very efficient. The presence of chlorobenzene caused a manifold increase in the ion intensity for both compounds, in case of quinoline even by a factor of 30, compared with direct APPI. In APLI, quinoline could only be ionized in the presence of toluene and chlorobenzene (Figure 5). Similarly to APPI, with toluene and chlorobenzene, MH+ and [M+77]+, respectively,

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were shown. With anisole and acetone-assisted APLI, no signal for quinoline was observed.

Acridine In APPI, acridine formed M+. ions with all dopants, except acetone, with which it formed an MH+ (Figure 4). The addition of dopants increased the acridine signal in all cases, most with chlorobenzene (over 5-fold). The IE of acridine is sufficiently low (7.80 eV) to allow charge exchange with all the dopants, but also the PA of acridine is high enough (972.6 kJ/ mol) for proton transfer with toluene, acetone, and anisole. In LC-APPI-MS conditions, acridine has typically been observed as an MH+ ion [30, 43–45]. As it appears from the ΔH values in Table 1, proton transfer for acridine is more exothermic than charge exchange in cases of acetone, toluene, and anisole, however, MH+ ions were only observed with acetone. This behavior cannot be simply explained by plain thermodynamic arguments; instead a deeper insight into the crucial mechanism is needed. It has been shown earlier that proton transfer requires more reaction time than the formation of M+. ions [46, 47], but one would expect the reaction rate for acridine protonation to be at least as high as it is for quinoline and pyridine with lower PAs (Table 2). The difference between acridine and pyridine and quinoline may be the lower IE of acridine, which makes the charge exchange in all cases exothermic. Only proton transfer is possible for pyridine in the presence of toluene and anisole, and for quinoline in the presence of anisole, respectively. In the presence of acetone, charge exchange is unlikely for all analytes and proton transfer prevails for any compounds with sufficiently high PA. Acridine ions were not observed in direct APLI, but the addition of chlorobenzene, toluene, and anisole dopants enabled the formation of M+. ions of acridine through the charge exchange process (Figure 5). However, the signal intensity was almost an order of magnitude lower than in APPI with the same dopants. Formation of [M+77]+ ions in the presence of chlorobenzene, observed with pyridine and quinoline, did not occur for acridine. This is interesting, since acridine has a similar N-heterocyclic ring in its structure as well as pyridine and quinolone, and one would expect a similar reaction behavior. However, it is possible that the two aromatic rings on both sides of the N-heterocyclic ring of acridine sterically hinder the formation of the [M+77]+ ion.

Conclusions Direct-APPI is a very efficient ionization method, when operated (1) in a chemically and photo-physically inert matrix, (2) with optimized overlap between photon flux and analyte density, and (3) with longest possible irradiation time of the analyte. In case of GC-APPI, chromatographic constraints and the need for a major additional gas flow require a well-balanced system concerning irradiation time, convectional/diffusive peak broadening, and radiation overlap with the eluent. In general, a broader range of compounds, with significantly lower background, is amenable to direct APPI than to any dopant-assisted method.

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Dopant-assisted GC-APPI is a very efficient and useful ionization method as well, when (1) the chemical system is well known, and (2) no other chemically active constituents than the dopant and the analyte are present. As presented, the combination of certain dopants with certain analyte compound classes significantly enhanced analyte attributable mass signals. Noticeable suppression of the direct photoionization process demonstrated that the addition of dopants significantly decreased the VUV transparency of the matrix and /or added neutralizing reactions between the dopant and the analyte ions, and, consequently, analytes with higher IEs than the dopants were not observed anymore. However, analytes with suitable IEs or PAs were ionized through dopant-mediated gas-phase ion/molecule reactions, such as charge exchange and proton transfer. In many cases, the signal of the analytes increased as a result of a more preferred ionization pathway than direct photoionization. In the event of several possible pathways, the ionization mainly tended to proceed towards the direction of highest exothermicity, although for some cases thermodynamics did not explain the observed ionization pathway. Following thermodynamic considerations, with acridine, the formation of protonated molecules was expected with several dopants, but only the charge exchange process was observed. This behavior is different from previous experiments under LC-MS conditions. Direct APLI was shown to be more selective than direct APPI. First of all, the laser system in the present experimental setup provided a lower photon energy for the ionization process, in total 9.32 eV compared with 10.0 and 10.6 eV in APPI. Additionally, the efficiency of the two-photon laser ionization process depends more on molecular properties than the efficiency of the single-photon ionization process in APPI. The presence of (1+1) REMPI suitable dopants at 266 nm increased the number of compounds amenable to APLI, due to gas-phase chemical ionization processes. In most cases, similar reactions were observed in dopant-assisted APPI and APLI. The dopant acetone was not ionized with the laser and, consequently, acetone-mediated reactions were not observed in APLI. The ionization efficiency for direct and dopant-assisted APLI was generally lower than the ionization efficiency in APPI, but this was due to the low intensity of the laser used in this work. Higher signals and an extended analyte range are expected with high intensity lasers, such as a KrF-excimer laser, operating with up to 10 mJ/pulse and a two-photon energy of 10.0 eV [48]. This is currently under investigation and will be issued in an upcoming publication. Besides formation of molecular ions or protonated molecules, also unexpected reactions were observed in both APPI and APLI, such as the formation of [M+77]+ ions of benzaldehyde, pyridine, and quinoline in the presence of chlorobenzene. This species gave a signal that was several orders of magnitude higher than the signals obtained in direct APPI or APLI, and could, therefore, possibly be useful in trace analysis. The mechanism for the formation of this ion is not yet clarified, but will be investigated together with possible compound types amenable to this reaction.

T. J. Kauppila et al.: APPI and APLI Mechanism

Acknowledgments The authors acknowledge financial support by the Academy of Finland (projects 218150, 255559, and 268757), and Magnus Ehrnrooth Foundation and iGenTrax. Thermo Scientific is acknowledged for supplying the Orbitrap, the GC, and the consumables, and Morpho Detection for supplying the APPI source.

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