Infrared spectrum of a protonated fluorescence dye: Acridine orange

Journal of Molecular Spectroscopy 268 (2011) 66–77

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Infrared spectrum of a protonated fluorescence dye: Acridine orange Anita Lagutschenkov, Otto Dopfer ⇑ Institut für Optik und Atomare Physik, Technische Universität Berlin, Hardenbergstraße 36, 10623 Berlin, Germany

a r t i c l e

i n f o

Article history: Available online 2 April 2011 Special issue dedicated to A.R.W. McKellar and P.R. Bunker Keywords: Acridine orange Protonation IR spectroscopy Quantum chemical calculations Photodissociation Dye

a b s t r a c t The infrared (IR) spectrum of protonated acridine orange (AOH+) has been measured in the fingerprint range (600–1740 cm 1) by means of IR multiple photon dissociation (IRMPD) spectroscopy. The IRMPD spectrum of mass-selected AOH+ ions was recorded in a Fourier transform ion cyclotron resonance mass spectrometer equipped with an electrospray ionization source using an IR free electron laser. Quantum chemical calculations at the B3LYP and RI-MP2 levels of theory using the cc-pVDZ basis set were employed to guide the isomer and vibrational assignment of the measured IR spectrum. Protonation at the nitrogen atom of the central ring (N10) was predicted to be by far the most stable protonation site. Good agreement is observed between the IRMPD spectrum and the linear IR absorption spectrum of the N10 isomer calculated at the B3LYP level. The IRMPD spectrum exhibits 14 bands in the spectral range investigated, which are assigned to individual normal modes of N10. The fragmentation process of AOH+ upon IR activation in the ground electronic state is analyzed in some detail, revealing that elimination of CH4 is thermodynamically favored over loss of CH3NCH2. The effects of protonation on the geometric and electronic structure are revealed by comparison with neutral AO. Astrophysical implications of the IR spectrum of AOH+ are briefly discussed in the context of the unidentified IR emission bands. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction 3,6-Bis(dimethylamino)acridine, C17H19N3, also known as acridine orange (AO, Fig. 1), is a dark orange to brownish solid and exhibits bright orange color in aqueous solution. AO belongs to the family of acridines, the nitrogen-containing analogues of anthracene. Several of these planar heterocyclic aromatic compounds are used as fluorescence dyes in molecular biology, biochemistry, toxicology, and supramolecular chemistry [1–4]. Depending on the pH value, this metachromic dye can appear in two prototropic forms, AO and AOH+, and the solution shows a sharp change in color arising from different absorption maxima of the two species [1]. Under physiologic conditions (pH = 7.4) essentially all AO molecules exist in their protonated form [1,5]. The strong ion–dipole interaction between this cationic dye molecule and the polar environment is responsible for the wide application of AOH+ in, for example, biochemical and biomedical processes, supramolecular host–guest chemistry, and material science. Within the acridine family, 9-aminoacridine, proflavin, and AO are often used as intercalating agents. For example, as the three-membered ring of acridines has roughly the same size as DNA and RNA base pairs, they intercalate easily between the double strand [6]. The spectral shifts in fluorescence of AO(H+) sensi⇑ Corresponding author. Fax: +49 30 31423018. E-mail address: [email protected] (O. Dopfer). 0022-2852/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jms.2011.03.024

tively depend on the host molecule and have thus often been employed to discriminate between DNA and RNA. In addition, crystals of AOH+ have been investigated as promising organic material exhibiting electroluminescence and photoconductivity [2]. Apart from the common use of acridines as organic dyes in life and material science, they are also of general interest as heterocyclic aromatic compounds in organic and interstellar chemistry. In addition to bare protonated polycyclic aromatic hydrocarbons (H+PAH), their nitrogen derivatives are suggested as possible carriers for spectra of the interstellar medium, including the unidentified IR emission (UIR) bands and the diffuse interstellar bands [7–12]. As an example from the acridine family, proflavin and related dye molecules have been considered to contribute to the astronomical spectra in the optical range [13], whereas protonated heterocyclic aromatic molecules, such as protonated acridine and related nitrogen-substituted aromatic compounds, are invoked as potential carriers of the UIR spectrum [7,9,14]. The geometric, vibrational, and electronic structure of AOH+ in the condensed phase has been characterized by NMR, IR, Raman, and optical spectroscopy, as well as X-ray crystallography [1– 4,15–19]. NMR [3,15] and X-ray [16,17] studies demonstrate that protonation of AO in aqueous solution and in ionic AOH+Y crystals occurs at the acridine N10 atom (Fig. 1). While the planar acridine nucleus of AOH+ in AOH+Y salts has perfect C2v symmetry, the C atoms of the methyl groups are slightly out-of-plane due to steric hindrance and packing effects. The vibrational structure in the

A. Lagutschenkov, O. Dopfer / Journal of Molecular Spectroscopy 268 (2011) 66–77

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Fig. 1. Calculated structures of AO and AOH+. Most stable structures of neutral (C2) and protonated acridine orange (C2v) calculated at the B3LYP/cc-pVDZ level along with the atom labeling.

ground electronic state of AOH+ has been characterized by IR and Raman spectroscopy in the condensed phase [2,19], and a detailed vibrational assignment has been reported on the basis of semiempirical PM3 calculations assuming C2v symmetry [19]. Very little experimental and theoretical information is available for the geometric, vibrational, and electronic structure of isolated neutral AO [20,21]. As no spectroscopic data are at hand, the knowledge of the effects of protonation of AO relies completely on theoretical characterization [20,21]. Low level quantum chemical calculations at the B3LYP/6-31G⁄ level suggest that the structure of AO in the ground electronic state has a planar acridine core with pyramidal dimethylamino groups (Fig. 1) [20]. All condensed-phase studies reveal substantial effects of solvation and counter ions on the geometric, vibrational, electronic, and optical properties of AOH+. These observations make this fluorescing dye indeed a sensitive probe of the local environment because of the strong ionic interactions with the nearby counter ions, polar solvent molecules, biomolecules, or host molecules. These effects have often been analyzed using semi-empirical or low level ab initio and density functional theory calculations [4,15,19,22,23]. Spectroscopic studies of AOH+ isolated in the gas phase are lacking so far, although such studies are required to separate the intrinsic molecular properties of this fundamental cation from the strong solvation effects. Similarly, laboratory spectra of isolated AOH+ are desired for direct comparison with the astronomical UIR spectrum. To this end, the present work reports a detailed analysis of the IR spectrum of isolated AOH+ in the fingerprint range (600–1740 cm 1) using quantum chemical calculations at the B3LYP and RI-MP2 levels of theory. These quantum chemical efforts are more sophisticated than previous

calculations, which are at most at the B3LYP/6-31+G(d) level [22]. The first spectroscopic data for isolated AOH+ allow for an unambiguous determination of the protonation site of the isolated molecule and provide detailed information about its geometric and vibrational properties. Spectroscopic characterization of protonated aromatic ions (AH+) and their clusters in the gas phase is a challenging task, because even sophisticated direct absorption techniques, which are frequently applied to neutral and ionic transient molecules and clusters [24–26], are usually not sensitive enough to overcome the problem of low AH+ ion concentrations [27–29]. Thus, most spectroscopic data for AH+ have been obtained via photodissociation. Early pioneering electronic photodissociation spectra of fundamental isolated AH+ ions were recorded by Freiser and Beauchamp in room temperature ion traps [30,31], and these studies have recently been extended to molecular beam experiments [10,32,33]. The major experimental strategies for recording IR spectra of AH+ ions and their clusters have been reviewed recently [34]. One technique involves single-photon IR photodissociation (IRPD) of mass-selected weakly bound clusters of AH+ (messenger technique [35–37]) using optical parametric oscillator lasers [12,32,38–45]. In this approach the absorption of an IR photon is signaled by evaporation of the weakly bound ligand. In rare cases, this method can also be applied to weakly bound AH+ ions with low fragmentation thresholds [46–48]. In most cases, however, the usually strong chemical bonds in AH+ ions require the resonant absorption of multiple IR photons to drive the dissociation process (IRMPD) [11,49–56]. Details of the IRMPD mechanism were described in recent reviews [57,58]. Briefly, IRMPD relies on resonant noncoherent sequential absorption of

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multiple IR photons, heating up the molecular ion to energies above the lowest dissociation threshold of the ground electronic state. In order to avoid the anharmonicity bottleneck, fast intramolecular vibrational energy redistribution has to occur in between each absorption step of the first few photons. The high IR laser intensities necessary to drive the IRMPD process are often provided by IR free electron lasers, and their application for IRMPD spectroscopy of AH+ ions and their clusters has been reviewed recently [34,58–62]. Despite the multiple photonic nature of the resonant noncoherent absorption process, the IRMPD cross section approximately reflects the absorption of the first IR photon, which justifies the comparison of IRMPD spectra with calculated linear one-photon IR absorption spectra [57,58]. Deviations of IRMPD from linear IR absorption spectra include minor but noticeable frequency shifts (usually to lower frequency) and modulation of IR intensities due to the multiple photon process (vide infra). In the present work, the IRMPD spectrum of mass-selected AOH+ ions is obtained in the fingerprint range (600–1740 cm 1) using the Free Electron Laser for Infrared eXperiments (FELIX) in the Netherlands. The analysis of the IRMPD spectrum is accomplished by quantum chemical calculations, which provide detailed information about the protonation site, and the geometric, vibrational, and electronic properties of AOH+ in the ground electronic state. Comparison with the corresponding properties of AO demonstrates the effects of protonation on these parameters. Moreover, comparison of acridineH+ [9] with AOH+ reveals the impact of the substitution of the two dimethylamino groups on its IR spectrum, which is discussed in the context of the astronomical UIR spectrum. Finally, IRMPD of AOH+ provides valuable insight into the fragmentation process of this molecular ion in its ground electronic state.

2. Experimental and theoretical techniques The IRMPD spectrum of AOH+ in Fig. 2b was obtained in the fingerprint range (600–1740 cm 1) in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer equipped with an electrospray ionization (ESI) source [63]. The mass spectrometer is coupled to the IR beamline of the free electron laser FELIX [64]. AO was purchased from Sigma–Aldrich as 3,6-bis(dimethylamino)acridine hydrochloride zinc chloride double salt with a dye content of 90% (product number A6014) and used without further purification. AOH+ ions were produced by spraying a 4  10 5 molar solution in water/methanol (1:4) at a flow rate of 10 lL/min. After accumulation in a hexapole trap for 4 s, the ions were transferred into the ICR via an octopole guide. Subsequently, AOH+ ions with m/z = 266 were mass selected in the ICR trap and irradiated for 2 s with 10 macropulses from FELIX operating at a repetition rate of 5 Hz. Each macropulse is about 5 ms long and composed of 5000 micropulses of 1 ps length. The average macropulse energy was measured as 45 mJ corresponding to micropulse energies of 10 mJ. The bandwidth of the FELIX radiation is specified as 0.5% of the central wavelength (FWHM), which corresponds to 5 cm 1 at 1000 cm 1. Calibration of the wavelength was achieved using a grating spectrometer with an accuracy of ±0.02 lm, which corresponds to ±0.5 and ±8 cm 1 at frequencies of 500 and 2000 cm 1, respectively. Depending on the laser frequency, the step size varied between 3 and 7 cm 1. Major fragmentation channels observed upon IRMPD of AOH+ were m/z = 250, 234, and 223, which correspond to formal loss of CH4, 2  CH4, and CH3NCH2, respectively. Four additional fragments with lower intensities were identified at m/z = 207, 206, 180, and 179 (Fig. 2c). The parent and all fragment ion intensities were monitored as a function of the laser frequency, and the IRMPD yield was then calculated as the integrated intensity of the fragment

(a)

(b)

(c)

Fig. 2. IR action spectra of AOH+. (a) Ion current of AOH+ (m/z = 266) and IR laser intensity (dashed line) as a function of the laser frequency. (b) IRMPD yield of AOH+ linearly normalized for variations in the IR laser intensity. The positions and vibrational and isomer assignments of the bands observed (A–O) are given in Table 1. (c) Photodissociation spectra recorded in the seven most intense fragment channels (m/z = 250, 234, 223, 207, 206, 180, 179) and used to evaluate the IRMPD yield. No other fragmentation channels were observed. The spectra are not normalized for laser intensity and vertically expanded by factors up to 19 to bring them roughly to the same scale.

ions divided by the sum of parent and fragment ion intensities. The IRMPD yield was linearly normalized for variations in the laser intensity shown in Fig. 2a. The widths of bands assigned to individual transitions vary between 10 and 30 cm 1 and arise from a combination of the finite laser band width, unresolved rotational structure and sequence hot bands of the ions in the ICR cell held at room temperature, and the multiple photon absorption process resulting in heating of the ions during the 2 s irradiation time [58]. Rotational band contour simulations of the vibrational transitions using the calculated rotational constants (A = 1.01277 GHz, B = 0.14103 GHz, C = 0.12419 GHz) and assuming T = 300 K suggest that 5 cm 1 of the widths of the observed bands results from unresolved rotational fine structure. The most narrow band at 694 cm 1 (A) has a width of 10 cm 1, consistent with additional broadening mainly arising from the limited laser bandwidth at this wavelength (4 cm 1). Quantum chemical calculations at the B3LYP and RI-MP2 levels of theory using the cc-pVDZ basis set were performed for AO(H+) and fragment ions in order to identify various low lying isomers on their potential energy surfaces and to evaluate their structural, energetic, electronic, and IR spectral properties. Reported energies include harmonic zero point energy corrections, scaled with a factor of 0.975 (B3LYP, vide infra) and 0.97 (MP2) [56], respectively. For all stationary points, frequency analysis ensures their nature as minimum or transition state on the potential energy surface. B3LYP calculations were performed with GAUSSIAN03 [65], whereas for RI-MP2 calculations TURBOMOLE

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6.1 was employed [66]. Initial test calculations at the B3LYP and (RI-)MP2 levels revealed that the (RI-)MP2 method fails to reproduce the frequencies and IR intensities of the AOH+ spectrum. This observation was previously noted for related protonated aromatic molecules [67–69], and has been related to the failure of MP2 calculations of properly describing the structures of benzene and related arene molecules [70]. However, the B3LYP spectra are in good agreement with the experimental IRMPD spectrum when using correlation-consistent basis sets [67–69]. In contrast, energetic properties are often reproduced with higher accuracy by correlated methods. Therefore, some of the energetic results at the RI-MP2 level are also reported in the present work for comparison with the B3LYP data. Theoretical IR stick spectra are convoluted with a width (FWHM) of 30 cm 1 to facilitate convenient comparison with the experimental spectrum. A natural bond orbital (NBO) analysis was performed to evaluate charge distributions in AOH+ and AO.

3. Results and discussion 3.1. IR spectrum and structure of AOH+ The IRMPD spectrum of AOH+ in Fig. 2b is highly structured and exhibits 14 discernible features labeled A–O in the spectral range between 600 and 1740 cm 1 (Table 1). IR action spectra monitored in the fragment channels with m/z = 250, 234, 223, 207, 206, 180, and 179 are compared to the depletion spectrum of the parent ion with m/z = 266 in Fig. 2. The depletion of the parent ion signal exceeds 50% at intense resonances indicating efficient IRMPD. As the IRMPD yield is normalized for variations of the parent ion production in the ESI source, it displays a better signal-to-noise ratio than the parent depletion signal. Consequently, the IRMPD yield will be compared to the calculated spectra. Quantum chemical calculations at the B3LYP and RI-MP2 levels were performed to establish the vibrational and isomer assignment

Table 1 Experimental vibrational frequencies of AOH+ (IRMPD spectrum, Fig. 2) compared to frequencies of the N10 isomer of AOH+ and neutral AO (C2 isomer) calculated at the B3LYP/ccpVDZ level. AOH+a

AOH+ (C2v)b

AO (C2)b

Vibrationc

AOH+d crystal

PM3d,e

1613 (O)f 1600 (N)

1652 (a1,94) 1637 (b2,1758) 1605 (b2,305)

1648 (a,51) 1614 (b,1253) 1600 (b,3)

rCC (8b of ring 2/8a of ring 1) rCN (8b of ring 1/8b of ring 2) rCC (8b of ring 1/8b of ring 2)

1603 m

1595 (b2)

1554 (M)

1550 (b2,237) 1529 (a1,123)

1507 (b,6) 1506 (a,68)

rCC (8a of ring 2/8b of ring 1/bNH) rCC (8a of ring 1/8a/b of ring 2)

1508 (L)

1506 (b2,758)

1474 (b,469)

rCC (8b of ring 2)/rCN

1502 m

1519 (b2)

1448 (K)

1467 1460 1444 1441

(a1,17) (b2,54) (b2,4) (a1,0)

1460 1461 1448 1440

(a,21) (b,98) (b,0) (a,24)

bCH3(asym) bCH3(asym) bCH3(asym) bCH3(asym)

1444 w

1437 (a1)

1425 (I)

1437 1431 1428 1427 1401 1396

(a1,9) (b1,34) (b2,96) (b2,68) (a1,34) (b2,43)

1439 1425 1423 – 1401 1396

(a,0) (a,13) (b,187)

bCH3(sym) bCH3(asym) bCH3(sym) bNH rCN/bCH3(sym) bCH3(sym)/bNH

1409 w

1410 (b2)

1380 (H)

1387 (b2,9) 1375 (b2,565) 1367 (a1,15)

1389 (b,0) 1349 (b,43) 1353 (a,44)

rCC (19a of ring 1)/bNH rCC (19b of ring 1)/rCN/bNH/CH3(sym) rCC (19b of ring 2/19a of ring 1)

1383 m

1372 (a1)

1345 (G)

1344 (b2,532) 1316 (a1,5)

1326 (b,514) 1309 (a,0)

rCN/bCH rCC (19a of ring 1)

1359 m

1357

1290 (F)

1279 (b2,18) 1273 (a1,7)

1308 (b,48) 1254 (a,0)

bCH/bNH bCH (3 of ring 1)

1295 w

1292 (b2)

1235 (E)

1242 (b2,89) 1222 (a1,23) 1216 (b2,9)

1243 (b,50) 1220 (a,11) 1203 (b,25)

bCH (9a of ring 2) bCH (14 of ring 2) bCH (3 of ring 2)

1250 m 1231 m

1250 (a1) 1231

1168 (D)

1167 (b2,370) 1149 (b2,28) 1132 (b2,40)

1152 (b,26) 1142 (b,122) 1129 (b,7)

bCH (14 of ring 2)

1137 m

1138 (b2)

1045 (a1,33) 1045 (b2,8) 974 (b1,4)

1048 (a,34) 1048 (b,7) 949 (b,4)

cCH3 cCH3 cCH

965 s

936 (b2)

937 (b1,8) 911 (b2,43)

908 (b,11) 921 (b,25)

cCH

922 vs

913 (b1)

810 (B)

813 (b1,89) 801 (b1,13)

860 (b,34) 794 (b,24)

cCH (11 of ring 2) cCH (10a of ring 2)

824 vs 790 s

809 (b1) 754 (b1)

694 (A)

695 (b2,53) 670 (b1,34)

691 (b,5) –

ring (6a of ring 2)

699 vs

669 (b2)

908 (C)

a

(a,34) (b,0)

cCH3 bCH

ring (12 of ring 2)

cNH

Peak positions are taken from the IRMPD spectrum (Fig. 2b). Vibrational symmetries and IR intensities in km/mol are given in parentheses. Only transitions with I > 3 km/mol are listed. c The notation r, c, b, and s refers to stretch, out-of-plane bend or rock, in-plane bend, and torsional modes, respectively; rings 1 and 2 refer to the pyridine and benzene moieties, respectively. The notation for normal modes mi is adapted from Wilson [73]. d Ref. [19]. e Some of the assignments in Ref. [19] are in conflict with those given in the present work with respect to both frequency, IR intensity, and vibrational symmetry. f In the m/z = 250 and 223 primary fragments, this band occurs at 1626 cm 1. b

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Table 2 Relative energies (kJ/mol) of various isomers of AOH+ calculated at the B3LYP and RI-MP2 levels.

a

B3LYP/cc-pVDZ RI-MP2/cc-pVDZb MP2/cc-pVDZa a b c

C1

C2

C4

C9

C11

N10

N15a

N15b

203.0 195.1

132.9 146.7

76.4 75.0

204.6 196.2

196.2 206.5

0 0 0

120.1 76.1

121.6 78.2 76.6 (TS)c

Using GAUSSIAN03. Using TURBOMOLE. Transition state.

of the transitions observed in the IRMPD spectrum. Eight isomers were found with protonation occurring at different C or N atoms within the molecule (Table 2). Only r complexes of AOH+ were considered, as p complexes are saddle points on the potential energy surface [39]. The isomer lowest in energy at both levels of theory is the N10 isomer with C2v symmetry obtained by N protonation of the central ring (Fig. 1). The proton affinity (PA) is calculated as 1088 and 1047 kJ/mol at the B3LYP and RI-MP2 levels, respectively. No experimental value seems to be available for comparison in order to decide which of the two values is more reliable. Protonation at the C4 atom is about 75 kJ/mol less exothermic than at N10 at both theoretical levels, and protonation at other C atoms are far less favorable (Table 2). In particular, protonation at C3, C12, C16, and C17 does not yield stable minima. On the other hand, protonation is feasible at the amino nitrogen atoms, leading to two isomers N15a/b with similar energies and cis/trans orientation of the planar H+AN15AC3AC4 unit. While for all other isomers the agreement between the relative energies evaluated at the B3LYP and RI-MP2 levels is within 15 kJ/mol, the difference is rather substantial for the N15 isomers (44 kJ/mol). At the moment, the reason for the latter, rather large discrepancy is unclear. Test calculations for dimethylaniline and dimethylamine demonstrate that both the B3LYP and the MP2 level yield PAs for N protonation in good agreement with experiment. The deviations are less than 10 kJ/mol, suggesting that protonation of the aliphatic N atom is described properly by both theoretical levels (Table T1 in Supplementary information). On the other hand, the PA for N protonation of acridine calculated at the RI-MP2 level (966 kJ/mol) is much closer to the experimental value (973.9 kJ/mol [71]) than the PA derived at the B3LYP level (992 kJ/mol). This observation may be taken as evidence that energies for protonation at the aromatic ring are somewhat overestimated at the B3LYP level (by 20–40 kJ/mol) and that the PA value of 1047 kJ/mol for the N10 isomer of AOH+ obtained at the RI-MP2 level is more reliable than the B3LYP value (1088 kJ/mol). In any case, thermodynamic considerations predict a single isomer of AOH+ to be populated at thermal equilibrium at room temperature, namely N10. This isomer is also expected to be predominant in solution and thus readily produced in the ESI source. The IRMPD spectrum is compared in Fig. 3 with the linear IR absorption spectra of all relevant isomers of AOH+ calculated at the B3LYP level. As the IR spectra show much higher activity in the range above 1100 cm 1, the spectra below 1100 cm 1 are shown vertically expanded by a factor 20. As demonstrated in Fig. F1 in Supplementary information, the IR spectrum evaluated at the (RI-)MP2 level substantially deviates from that obtained at the B3LYP level in the fingerprint range with respect to both frequencies and IR intensities. Moreover, the spectra at the MP2 and RI-MP2 levels are very similar, demonstrating that the resolution-of-identity (RI) approximation has little impact on the IR spectrum. Previous work has shown that IR spectra of aromatic molecules obtained at the B3LYP level are consistent with the experimental spectra, whereas those at the MP2 level are often not reliable [67–70]. The B3LYP spectrum calculated for N10 demonstrates satisfactory agreement with the measured IRMPD spectrum. For the other

isomers, the agreement is far less favorable, so that the assignment of the IRMPD spectrum to N10 is preferred both energetically and spectroscopically. The C4 spectrum obviously lacks intensity near 1600 cm 1 (band N/O), whereas it exhibits its highest IR activity near 1530 cm 1 in disagreement with experiment. Isomers N15a/b do not reproduce the IRMPD spectrum well in the spectral range below 1100 cm 1. In particular, the transition predicted near 950 cm 1 is not observed in the IRMPD spectrum. Also, the pronounced IRMPD band D at 1165 cm 1 is not predicted for N15a/b. A similar mismatch between calculated and measured IR spectra is observed for the even higher-lying carbenium isomers C1, C2, C9, and C11. For example, none of these have significant predicted intensity in the vicinity of band D. In summary, comparison of the IR spectra in Fig. 3 clearly demonstrates that N10 is the predominant carrier of the IRMPD spectrum and contributions from the higher-energy isomers are not significant. After identification of N10 as the major carrier of the IRMPD spectrum, we consider the effects of protonation on the geometric and vibrational properties of this isomer (Fig. 1). The geometry of N10 has C2v symmetry, i.e. the aromatic acridine ring is strictly planar, and only two of the three protons of the methyl rotors are located above and below the plane. The barriers calculated for internal rotation of the CH3 groups are rather high (5.8 and 4.1 kJ/mol for rotation of C17H3 and C16H3 at the B3YLP level, respectively), suggesting that the molecular ion is relatively rigid. On the other hand, neutral AO has lower symmetry with a nearly planar aromatic ring system and slightly pyramidal dimethylamino groups [20,21], yielding essentially isoenergetic minima with either C2 or Cs symmetry, depending on whether the two N atoms of the dimethylamino groups are on the same or on different sides of the aromatic plane. The dihedral angle C2AC3AN15AC17 amounts to 173.5°. The low barriers for simultaneous inversion of both amino groups (<0.5 kJ/mol) imply, however, that this molecule is probably quasi-planar with effective C2v symmetry [72]. The IR spectra of all three stationary points of AO (C2, Cs, C2v) are very similar in the considered fingerprint range. Similar to AOH+, the barriers for internal CH3 rotation are rather high for AO (6.5 and 3.4 kJ/mol for rotation of C17H3 and C16H3). The NBO analysis of the charge distribution indicates that proton attachment to the nonbonding lone pair of N10 leads to some charge reorganization. Although most of the positive charge is redistributed over the whole molecule, +0.42 e still resides on the excess proton (see Table T2 and Fig. F2 in Supplementary information for atomic NBO charges). The impact of protonation on the structural parameters is detailed in Table 3 (B3LYP) and Fig. F3 in Supplementary information. As expected, protonation at N10 has the largest influence on the bond lengths and angles in the vicinity of the heteroatom in the pyridine ring, with alternating affects as a function of the distance of the protonation site [19]. In particular, the bonds in the heavyatom backbone between the two dimethylamino groups are strongly affected. Specifically, the N10AC12 bond elongates by 28 mÅ, and as a consequence the neighboring C11AC12 and C4AC12 bonds contract by 12 and 29 mÅ. The adjacent C3AC4 bond is stretched by 22 mÅ and as a consequence the C3AN15

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IRMPD

B

C

D

A

NO

G E

F

H

I K

L M

N10

C4

N15a

x2 N15b

x2 C2

x2 C11

x2 C1

x2 C9

x2 600

700

800

900

1000

1100

ν / cm-1

1100

1200

1300

1400

ν / cm

1500

1600

1700

-1

Fig. 3. Comparison between IRMPD spectrum of AOH+ and calculated spectra. IRMPD spectrum of AOH+ and linear IR absorption spectra of a variety of isomers of AOH+ calculated at the B3LYP/cc-pVDZ level. The stick spectra are sorted in energetic order of the isomers, scaled by 0.975, and convoluted with a Gaussian profile with FWHM = 30 cm 1. All calculated spectra are drawn to the same intensity scale when taking into account the given vertical expansion factors. All spectra in the left panel are vertically expanded by a factor of 20.

bond to the amino side chain shrinks by 29 mÅ. This in turn leads to stretching of the aliphatic N15AC16 and N15AC17 bonds by 10 and 8 mÅ, respectively. All other bond lengths between heavy

atoms change less than 2 mÅ. Notable changes in bond angles include a drastic increase in the C12AN10AC13 angle by 7°, whereas the adjacent N10AC12AC11 angle decreases by 5°. All other bond

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Table 3 Bond lengths (Å) and angles (degrees) of neutral and protonated AO evaluated at the B3LYP/cc-pVDZ level.

a

Bond

AO

AOH+

C1AC2 C2AC3 C3–C4 C4AC12 C11AC12 C1AC11 C9AC11 N10AC12 C3–N15 N15AC16 N15AC17 N10AH

1.3694 1.4443 1.3931 1.4238 1.4467 1.4268 1.3979 1.3482 1.3879 1.4529 1.4514

1.3675 1.4431 1.4150 1.3953 1.4351 1.4266 1.3992 1.3757 1.3588 1.4632 1.4594 1.0156

Da 0.0019 0.0012 0.0219 0.0285 0.0116 0.0002 0.0013 0.0275 0.0291 0.0103 0.0080

Angle

AO

AOH+

C1AC2AC3 C2AC3AC4 C3AC4AC12 C4AC12AC11 C12–C11AC1 C11AC1AC2 C11AC12AN10 C12AN10AC13 C13AC14AC9 C14AC9AC11 C2AC3AN15 C4AC3AN15 C3AN15AC16 C3AN15AC17 C12AN10AH

121.1 118.2 122.0 119.0 118.2 121.6 122.8 118.5 118.0 119.9 119.9 122.0 120.4 118.8

121.1 118.1 120.5 121.5 117.2 121.7 117.5 125.1 119.0 121.9 120.4 121.5 121.0 120.2 117.4

Da 0.0 0.1 1.5 2.5 1.0 0.1 5.3 6.6 1.0 2.0 0.5 0.5 0.6 1.4

Difference between AOH+ and AO.

angles change less than 2.5°. Significantly, the present B3LYP/ccpVDZ structure is close to the one obtained from the X-ray data [16,17] and previous B3LYP/6-31+G(d) calculations [22] but differs substantially from that obtained at the semi-empirical PM3 level [19]. As a consequence, the detailed vibrational assignments of the IR and Raman spectrum of AOH+ in Ref. [19] based on the PM3 calculations are unreliable (vide infra). The structural changes induced by protonation translate directly into frequency shifts of the corresponding normal modes. Moreover, protonation has also a profound impact on the IR intensities on several vibrational fundamentals. Table 1 and Fig. 4 compare the IR spectral properties of AO and the N10 isomer of AOH+ in the fingerprint range and suggest a vibrational assignment of the bands detected in the IRMPD spectrum. N10 has 114 normal modes, which can be classified in C2V symmetry in Cvib = 37a1 + 19a2 + 22b1 + 36b2. Most vibrations observed in the fingerprint range are in-plane modes (a1, b2). Only vibrations of N10 with IR intensities larger than 3 km/mol are considered in Table 3. This approach is justified in view of the achieved signal-to-noise ratio (only modes with I > 40 km/mol are detected experimentally) and

the most intense vibration of N10 in the fingerprint range with I = 1758 km/mol. For the nomenclature of the vibrational modes, we refer to the notation of Wilson used for benzene derivatives [73]. Most of the transitions in the IRMPD spectrum can be attributed to a single isolated vibrational mode of the N10 isomer of AOH+, providing detailed information about the force field of this ion. Band A at 694 cm 1 is assigned to an out-of-phase combination of the two m6a ring modes of the benzene moieties (denoted ring 2). This transition has a narrow width of only 10 cm 1. Band B at 810 cm 1 is due to an out-of-plane CAH bending mode (cCH), in which all aromatic protons vibrate in phase against the aromatic skeleton (m11). Such transitions are typical for IR spectra of (protonated) polycyclic aromatic hydrocarbon molecules [7,11,57,67]. This mode has a substantial predicted red shift of 47 cm 1 from the frequency of neutral AO, indicating that protonation softens the potential for out-of-plane bending motions of the aromatic ring system. Band C at 908 cm 1 is assigned to an out-of-phase combination of the two m12 ring modes of the benzene rings slightly coupled to the symmetric CAN stretch of the amino groups. The

Fig. 4. Effect of protonation on the IR spectrum of AO. IRMPD spectrum of AOH+ compared to linear IR absorption spectra of the N10 isomer of AOH+ and neutral AO.

A. Lagutschenkov, O. Dopfer / Journal of Molecular Spectroscopy 268 (2011) 66–77

intense and broad transition D at 1168 cm 1 with a width of 30 cm 1 is ascribed to an in-plane CAH bending mode (bCH), arising from an in-phase combination of the m14 modes of the benzene rings along with a large component of the NAH bend (bNH) of the excess proton. The latter implies substantial motion of the positive charge, which explains the strong enhancement in the IR intensity of this mode upon protonation (from 26 to 370 km/mol). The weak band E at 1235 cm 1 is mainly attributed to a bCH bend, obtained by coupling the m9a modes of the benzene rings with bNH. Similarly, the weak band F at 1290 cm 1 is due to a bCH bend coupled with bNH. The intense band G at 1345 cm 1 is ascribed to a bCH bend coupled to aliphatic CAN stretch modes (rCN) of the amino groups. Its blue shift of 18 cm 1 from neutral AO is partly due to the substantial protonation-induced contraction of the C3AN15 bond. Band H appears at 1380 cm 1 in the blue shoulder of band G and is assigned to a similar normal mode as G. It also has large components of the aliphatic CAN stretch, coupled to bCH and bNH. The latter component may again be responsible for the large intensity enhancement of this transition upon protonation (from 43 to 565 km/mol), whereas the rCN component induces again part of the blue shift of 26 cm 1. The weak band I at 1425 cm 1 is due to a variety of overlapping rocking modes of the methyl groups (cCH3) and the bNH mode, which does not exist in neutral AO. However, bNH is largely coupled to CAH bending motions so that its IR intensity is moderate (68 km/mol). Transition K at 1448 cm 1 is produced by an asymmetric cCH3 mode. As the methyl groups are not much affected upon protonation, this mode displays almost no frequency shift and change in IR intensity. The intense band L at 1508 cm 1 is attributed to an aliphatic rCN stretch coupled to aromatic rCC stretch motion. The rCN contribution is again mainly responsible for the large protonation-induced blue shift of 32 cm 1. Band M at 1554 cm 1 is generated by an aromatic rCC mode obtained by coupling of m8a of the benzene rings with m8b of the pyridine ring. It also contains substantial part of bNH, which may explain some of the intensity enhancement from 6 to 237 km/mol. The blue shift of 43 cm 1 upon protonation is consistent with the geometrical changes of the aromatic carbon skeleton. The signal between bands L and M at 1530 cm 1 is produced by an aromatic rCC mode (mainly m8a of pyridine) coupled to aliphatic rCN, which also exhibits a blue shift of 22 cm 1 upon protonation. The most intense feature in the IRMPD spectrum near 1600 cm 1 has a large width of 60 cm 1, consistent with an assignment to an unresolved doublet N and O. The first component N at 1600 cm 1 arises from a coupled aromatic rCC mode (m8b of pyridine coupled to m8b of benzene). A substantial bNH component enhances its IR intensity from 3 to 305 km/mol. The second component O at 1613 cm 1 is assigned to the mode with highest IR intensity in the fingerprint range (1758 km/mol), which also arises from coupling of m8b of pyridine with m8b of benzene. It has a large asymmetric aromatic rCN component strongly coupled to bNH, which leads to an overall intensity enhancement and frequency blue shift of 24 cm 1. In general, there is satisfactory agreement between the measured IRMPD spectrum and the linear absorption spectrum calculated for N10. There are, however, several notable discrepancies, most of which can be attributed to the multiple photonic nature of the IRMPD process [57,58]. First, although the IRMPD technique reflects mainly the absorption of the first photon and thus justifies in zero-order direct comparison with a linear absorption spectrum, a certain threshold intensity Ith of the vibrational transition is required to allow for its detection. In the present case, Ith lies near 40 km/mol as indicated in Fig. 4. For example, the ring mode calculated at 911 cm 1 with I = 43 km/mol can clearly be detected (band C), whereas the CH3 rocking mode predicted at cCH3 = 1132 cm 1 with I = 40 km/mol is absent in the IRMPD spectrum. The observed value for Ith sensitively depends on several parameters of the laser

73

(intensity, pulse shape and chirp, irradiation time) and the molecular ion (temperature, dissociation energy, size, density of states, rigidity, symmetry). In general, low Ith values are observed for molecules with low dissociation thresholds and fast rates for intramolecular vibrational energy redistribution (IVR). Efficient IVR is a basic requirement for the sequential noncoherent absorption of IR photons with the same frequency. IVR rates are higher for larger molecular ions featuring more degrees of freedom and also for more flexible molecules exhibiting more efficient anharmonic vibrational couplings [72]. Interestingly, IRMPD for azuleneH+ was observed for bands with I > Ith = 10 km/mol under very similar experimental conditions, such as laser intensity and irradiation time [67]. The lower Ith value for azuleneH+ is somewhat surprising, because it has a much higher dissociation energy than AOH+ (327 vs. 54 kJ/mol) and a much lower density of states due to the smaller molecular size and the lack of the four methyl rotors, which is supposed to suppress the IVR rates and thus the IRMPD efficiency. A second effect of the IRMPD process on the IR spectrum of a molecule are red shifts of the transitions due to anharmonic couplings [57]. Depending on the vibrational mode of the aromatic molecule, this red shift typically varies between 10 and 30 cm 1 in the fingerprint range and is often absorbed in the scaling factor, which is then slightly lower than that obtained when considering a linear IR spectrum. Nonetheless, for heavily affected modes this red shift may not fully be compensated for by the scaling factor, as is seen here for the intense aromatic rCN mode (band O), which appears in the IRMPD spectrum 24 cm 1 lower than the value predicted after scaling. Actually, when considering only the m/z = 250 and 223 primary fragments (vide infra), this band occurs at 1626 cm 1, i.e. much closer to the predicted value of 1637 cm 1. A further side effect of anharmonic coupling is that IRMPD intensities for two close-lying transitions are modified such that the lower-frequency mode gains intensity. Two such examples in the IRMPD spectrum of AOH+ are the bands G and N. When considering for the assignment of the IRMPD features only the dominant contributing modes of the spectrum predicted for N10 and neglecting the large shift of band O arising from the IRMPD mechanism, all experimental positions are within ±12 cm 1 of the predicted frequencies, with an average deviation of 4 cm 1. This agreement is compatible with the resolution of the spectroscopic approach, utilizing radiation with 3–9 cm 1 bandwidth, a calibration accuracy of 1–5 cm 1, and step sizes of 3–7 cm 1, which finally leads to peak widths of 10–30 cm 1 for individual transitions. The mean value of all deviations is +1 cm 1, which justifies the scaling factor of 0.975 employed for the B3LYP calculations. The IR spectrum of crystalline AOH+ with its vibrational assignment using semi-empirical PM3 calculations [19] is directly compared to the present IRMPD spectrum of isolated AOH+ in Table 1 and Fig. F4 in Supplementary information. Although there is reasonable correspondence between the positions of the most intense transitions in both experimental IR spectra, there are noticeable frequency shifts and major differences in the IR intensities of the spectra recorded in the gas and condensed phases. More severely, there are a variety of wrong vibrational assignments, which can readily be traced back to the insufficient quality of the structure and vibrational force field of AOH+ at the PM3 level in Ref. [19]. Comparison of the IR spectra calculated for AO and AOH+ in Fig. 4 reveals directly the impact of protonation at N10 on the positions and intensities of the vibrations in the fingerprint range (Table 1). Most significantly, there is a general trend for an increase in rCN and rCC frequencies, as a direct consequence of the geometry changes. As a second general trend, the IR cross sections for most modes in the 1000–1700 cm 1 range are enhanced, in most cases due to contributions of the bNH involving motion of the large

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positive partial charge. Unfortunately, the IR spectrum of AO isolated in the gas phase appears not to be available. A condensed-phase spectrum of AO  HCl taken from the NIST database [74] is compared in Fig. F5 in Supplementary information to the IR spectra calculated for AO and the N10 isomer of AOH+. The AO  HCl spectrum taken in mineral oil mull suffers from the problem that it is unclear, whether the AO  HCl species resembles the spectrum of neutral AO perturbed by hydrogen bonding to HCl or the spectrum of AOH+ perturbed by the presence of Cl counter ions. The comparison in Fig. F5 actually suggests an assignment of the NIST spectrum to a mixture of both AO and AOH+. 3.2. Astrophysical implications Nitrogen-containing heterocyclic PAH molecules have recently been considered as carriers of the UIR bands (Fig. 5) [75]. As both ammonia and methylamine were detected in the interstellar medium (ISM) [76], the postulated presence of acridine(H+) [9] may also lead to consideration of AO(H+). The UIR bands recur in similar intensity ratios in different galactical and extragalactical sources, and are widely believed to arise from IR fluorescence of electronically excited PAHs and their derivatives [7]. The quest for the UIR carriers has recently turned from neutral to ionized PAHs, whose formation in the ISM is likely and whose IR spectra show a better correspondence with the astronomical data [57]. In particular, the high IR activity in the CAH bend and CAC stretch range is only reproduced by charged PAH. As attachment of H atoms to ionized PAHs was measured to be fast, the formation of protonated PAHs in the ISM was hypothesized [77] and recent laboratory IR spectra of H+PAH provide strong support for this scenario [11,12,54, 61,67,78,79]. One major motivation for the suggestion of nitrogen-containing PAH is the 6.2 lm feature observed in the UIR spectra, because the intense high-frequency CAC stretch modes of bare PAH+ and H+PAH appear at longer wavelengths [14], i.e. often below 1600 cm 1 (there are few exceptions such as protonated naphthalene with 1612 cm 1, as estimated from its IRPD spectrum via Ar tagging [12]). For example, this band occurs at 1586 and 1583 cm 1 for ionized and protonated anthracene (Fig. 5a), respectively [11,80]. In contrast, CH ? N substitution of one of the CH units in PAHs shifts this band to higher frequency [7,9]. Indeed,

the high-frequency aromatic CAC stretch band of AOH+ features substantial asymmetric rCN character and occurs with high intensity as band O at 1613 cm 1 in the IRMPD spectrum, i.e. it matches exactly the 6.20 lm band of the UIR spectrum (Fig. 5). As both the IRMPD and the UIR emission mechanism involve the same type of anharmonic vibrational couplings, the direct comparison between UIR and IRMPD spectra is meaningful, although both IR spectra significantly differ from cold single-photon absorption spectra [11,57]. Apart from the 6.20 lm UIR feature, also the intense 8.6 lm band shows a coincidence with the 1168 cm 1 transition in the AOH+ spectrum (band D). Similarly, the weak 6.9 and 11.0 lm UIR bands are close to the weak 1448 (K) and 908 cm 1 (C) bands of AOH+. On the other hand, there are also clear discrepancies between both spectra. In particular, bands G and H occur in the AOH+ spectrum at higher frequency (1340 and 1385 cm 1) than the corresponding 7.6–7.8 lm features of the UIR spectrum (1280–1315 cm 1). As these modes involve contributions of aliphatic CAN stretch motions of the methylamino side chains, the agreement may be better for the ion without these functional groups, and comparison with the IRMPD spectrum of acridineH+ confirms this conclusion [9]. There is in fact a band near 1290 cm 1 in the AOH+ spectrum, however its relative intensity is much weaker than in the UIR spectrum. Overall, the considerable match between the IRMPD spectrum of AOH+ and the UIR spectrum is encouraging and supportive of the hypothesis of the presence of protonated N-containing PAH in the ISM [9]. It is instructive to compare the IR spectra of AOH+ and acridineH+ in order to evaluate the substantial effects of the dimethylamino groups on the appearance of the IR spectrum in the fingerprint range (Fig. F6 in Supplementary information). Significantly, the IR intensities of the intense transitions of AOH+ are much higher than those of acridineH+. For example, 15 transitions of AOH+ have IR oscillator strengths between 50 and 1800 km/mol, whereas only 2 transitions of acridineH+ have intensities in the 50– 300 km/mol range. Moreover, while the out-of-plane CAH bend cCH at 767 cm 1 appears as second most intense band in the acridineH+ spectrum, all intense transitions of AOH+ occur above 1100 cm 1. These conclusions derived from the calculated spectra are fully supported by the corresponding experimental IRMPD spectra (Fig. 2 and Ref. [9]). 3.3. Fragmentation process

a

Fig. 5. Astronomical UIR spectrum compared to laboratory IR spectra of AOH+ and anthraceneH+. IRMPD spectra of protonated anthracene (a, Ref. [11]) and AOH+ (b) compared to an astronomical UIR spectrum (c, Ref. [7]). The dashed lines indicate positions of UIR bands in lm.

IRMPD of AOH+ exhibits a complex fragmentation pattern. As shown in Fig. 2c, seven fragmentation channels are observed under the present experimental conditions. The two predominant channels with m/z = 250 and 234 occur with an approximate ratio of 2:1. The remaining five channels are less intense by factors 10–20 with respect to the m/z = 250 channel. Interestingly, the spectroscopic signatures are different in various fragmentation channels. For example, bands A–C can only be seen in the m/z = 250 and 223 channels. This observation is taken as clear evidence for a sequential photodissociation process [52,56]. In this scenario, the initial photofragment ions resonantly absorb further photons leading to secondary fragments. Thus, signal in a secondary fragment channel can only be observed at resonances of both the parent and the primary fragment. As the IR spectra of parent and primary fragments are usually different, the appearance spectrum in the primary and secondary fragment channels can be rather different. In fact, the action spectrum in the secondary fragment should be a fraction of that of the primary fragment. Fig. 6 details the fragmentation model suggested from the IRMPD signals observed for AOH+ and its fragment ions, and the relevant dissociation energies are collected in Table 4. Initial photodissociation of parent ions (m/z = 266) results in the population of two competing fragment channels with m/z = 250 and 223,

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+

266 N H

N

CH3-N=CH2 (96)

CH4 (54) +

250 H2C

N

N H

(25)

N

CH3-N=CH2

CH4

(59)

(104)

(62)

+

N H

N

(28)

H2C

H (377)

N

N

+

N H

HCN (31)

206 H2C

(110)

207 HCN

CH2

CH3-N=CH2

N H

CH4

N

+

223 HCN

N

234 H2C

N

180

N

HCN (45)

H (391)

+

N H

179 N Fig. 6. Suggested fragmentation route upon IRMPD of AOH+. Structures of fragment ions specified by m/z along with dissociation energies in kJ/mol (Table 4) and neutral fragments. Only dissociation channels with at least one closed-shell daughter ion are considered (whenever possible, i.e. for all reactions except for H loss). Less stable isomers are shown in Figs. F7–F12 in Supplementary information. Thick arrows indicate the main fragmentation path.

which exhibit essentially the same action spectrum (Fig. 2c). The m/z = 250 fragment is attributed to CH4 loss, which is a common fragmentation product of methylated hydrocarbon ions. The m/z = 223 fragment ion corresponds to formal loss of CH3AN@CH2 (N-methyl-methanimine), which appears to be substantially more energy demanding, as its intensity is lower by a factor of 13 compared to the m/z = 250 channel. Indeed, the dissociation energy calculated for loss of CH4 (54 kJ/mol) is substantially lower than for elimination of CH3AN@CH2 (96 kJ/mol). The secondary fragment

with m/z = 234 is generated by loss of a second CH4 molecule from IRMPD of m/z = 250 (59 kJ/mol). Clearly, bands A–C are missing and peaks G/H, K, L, and O are largely reduced, whereas the bands D, M, and N have the same intensity as in the m/z = 250 spectrum. These discrepancies clearly indicate the sequential elimination of two CH4 units involving further IR absorption of the intermediate ion (m/z = 266 ? 250 ? 234). The m/z = 234 and 207 spectra are again nearly identical suggesting that they have the same precursor, namely m/z = 250.

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Table 4 Dissociation energies of the N10 isomer of AOH+ (m/z = 266) into various fragment channels (Fig. 6) obtained at the B3LYP and RI-MP2 levels (in kJ/mol).

a

Species (m/z)

B3LYP

RI-MP2

266 250 + CH4 234 + 2CH4 223 + CH3NCH2 207 + CH3NCH2 + CH4 206 + CH3NCH2 + CH4 + H 180 + 2CH3NCH2 179 + 2CH3NCH2 + H

0 53.8 112.4 96.4 158.1 518.0 206.2 563.2

0 60.8 128.3 109.6 179.7 a

232.4 a

MP2 values unreliable due to spin contamination.

The m/z = 207 ion probably results from the loss of CH3AN@CH2. Similar to AOH+, the CH3AN@CH2 loss from m/z = 250 is more energy demanding than CH4 elimination (104 vs 59 kJ/mol), leading to strongly reduced signal in the m/z = 207 channel. It is also possible that m/z = 207 is generated via the sequential loss of CH4 and HCN (Fig. 6). In contrast, it is unlikely that m/z = 207 is generated from IRMPD of the m/z = 223 primary fragment via CH4 loss, because the action signal observed in m/z = 207 is stronger than that in m/z = 223. The spectra in m/z = 206 and 180 are close to the one of m/z = 207, consistent with the scenario given in Fig. 6 that they have the same precursor, namely m/z = 207. In this case, they arise from loss of H and HCN, respectively. Finally, the spectrum in m/z = 179 exhibits an enhanced and slightly red shifted band around 1590 cm 1, indicating that this ion is again arising from a sequential photodissociation process, probably H atom elimination upon IRMPD of m/z = 180. Thus, fragmentation of the N10 isomer of AOH+ upon IRMPD under the present experimental conditions is observed down to the bare acridine unit, which is a very stable aromatic heterocyclic PAH radical cation, and thus difficult to dissociate by IRMPD [9,57]. Although the fragmentation scenario suggested in Fig. 6 is reasonable and consistent, firm elucidation of the actual structures of the fragment ions is impossible at the present stage from the available data set. Further IRMPD spectra of the mass-selected fragment ions generated, for example, from collision-induced dissociation of AOH+ would certainly provide more definitive information [81]. However, such an investigation is clearly beyond the scope of the present work. Moreover, one has to bear in mind that the dissociation energies reported in Table 4 and Fig. 6 do not take into account any reaction barriers, which may be substantial for fragmentation channels involving considerable molecular rearrangements. Finally, it is noted that the structures shown in Fig. 6 are the most stable ones found for each individual fragment mass. There is a plethora of less stable isomers for each fragment mass (see Figs. F7–F12 In Supplementary information), which also may be populated during the fragmentation cascade of AOH+ upon IRMPD. As no data on the fragmentation of AOH+ in the gas phase appear to be available, no direct comparison of IRMPD with fragmentation arising from collisional activation or electronic photoexcitation can be made. Photodegradation studies of AOH+ deposited on TiO2 films using UV radiation at 254 nm demonstrate the successive loss of all four CH3 groups accompanied by uptake of protons, yielding peaks in the laser desorption mass spectra spaced by 14 u [82]. Thus, the fragmentation processes occurring upon electronic excitation differ largely from that upon IR activation in the ground electronic state.

4. Conclusions The geometric and electronic structure of protonated acridine orange has been characterized by IRMPD spectroscopy and quan-

tum chemical calculations. The calculations suggest that protonation at N10 is by far the most favorable protonation site, leading to a C2v symmetric structure in the 1A1 ground electronic state. Although most of the positive charge is redistributed over the whole molecule, a significant fraction remains on the excess proton. As expected, protonation has a large impact on the bond parameters in the vicinity of the N10 atom and on the corresponding vibrational frequencies and IR intensities. In particular, most aromatic CAC and aromatic/aliphatic CAN stretch frequencies increase, and the IR intensities for many modes in the 1000–1700 cm 1 range are enhanced, because they are coupled to motions of the NAH bend involving large positive partial charge of the excess proton. The measured IRMPD spectrum of AOH+ is fully consistent with the IR spectrum simulated for the N10 isomer, and there is no clear spectroscopic signature for the presence of less stable isomers in the sampled ion cloud, which are calculated to be at least 70 kJ/mol less stable than N10. Protonation at N10 is also expected to be predominant in solution, and thus there is no change in the protonation site upon electrospray ionization. The spectroscopic signatures of AOH+ are also discussed in the context of the astronomical UIR bands. Most significantly, the AOH+ spectrum reproduces a band at 1613 cm 1, which is assigned to an aromatic CAN stretch motion. Hence, the IRMPD spectrum of AOH+ supports the hypothesis that N-containing heterocyclic molecules derived from CH ? N substitution of PAH derivatives may contribute to the intense UIR feature at 6.20 lm (1613 cm 1). IRMPD of AOH+ exhibits a complex fragmentation pattern, which has been investigated in some detail by analyzing the intensities and frequencies observed in action spectra recorded in seven different fragment channels. Apparently, CH4 elimination is thermodynamically favored over loss of CH3AN@CH2 units from the two dimethylamino functional groups present in AOH+. Fragmentation upon IRMPD under the present experimental conditions is observed down to the bare acridine unit, which is a very stable aromatic heterocyclic PAH radical cation. Acknowledgments This work was supported by the Technische Universität Berlin, the Deutsche Forschungsgemeinschaft (DO 729/3 and 729/6), the Fonds der Chemischen Industrie, and the European Community’s Seventh Framework Programme (FP7/2007-2013, Grant 226716). We acknowledge the assistance of J. Langer and the FELIX teams of J. Oomens and G. Berden (FT-ICR) and B. Redlich (FEL laser) during the experimental campaign. Appendix A. Supplementary material Supplementary data for this article are available on ScienceDirect (www.sciencedirect.com) and as part of the Ohio State University Molecular Spectroscopy Archives (http://library.osu.edu/sites/ msa/jmsa_hp.htm). Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jms. 2011.03.024. References [1] M. Shaikh, J. Mohanty, P.K. Singh, W.M. Nau, H. Pal, Photochem. Photobiol. Sci. 7 (2008) 408. [2] S. Agravat, V. Jain, A.T. Oza, Ind. J. Chem. A 47 (2008) 341. [3] S.H. Chou, M.J. Wirth, J. Phys. Chem. 93 (1989) 7694. [4] R.W. Larsen, R. Jasuja, R.K. Hetzler, P.T. Muraoka, V.G. Andrada, D.M. Jameson, Biophys. J. 70 (1996) 443. [5] F. Traganos, Z. Darzynkiewicz, T. Sharpless, M.R. Melamed, J. Histochem. Cytochem. 25 (1977) 46. [6] S. Neidle, Z. Abraham, Crit. Rev. Biochem. 17 (1984) 73.

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