Protomers of Benzocaine: Solvent and Permittivity Dependence Stephan Warnke, Jongcheol Seo, Jasper Boschmans, Frank Sobott, James H. Scrivens, Christian Bleiholder,,? Michael T. Bowers, Sandy Gewinner, Wieland Schöllkopf, Kevin Pagel,,k and Gert von Helden, Fritz-Haber-Institut der Max-Planck-Gesellschaft, Faradayweg 4-6, 14195 Berlin, Germany, Biomolecular and Analytical Mass Spectrometry group, Department of Chemistry, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerpen, Belgium, School of Life Sciences, University of Warwick, Coventry CV4 7AL, U.K, Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106, and Department of Biology, Chemistry, and Pharmacy, Institute of Chemistry and Biochemistry, Freie Universität Berlin, Takustr. 3, 14195 Berlin, Germany E-mail: kevin.pagel@fu-berlin.de; helden@fhi-berlin.mpg.de - Supporting Information - To whom correspondence should be addressed Fritz Haber Institute of the Max Planck Society University of Antwerp University of Warwick University of California, Santa Barbara k Freie Universität Berlin? Current address: Department of Chemistry, Florida State University, Tallahassee, FL 32306-4390 S1
Experimental Collision cross section measurement A detailed description of the ion-mobility method can be found elsewhere. 1,2 Briefly, gasphase ions of different sizes but identical m/z ratios can be separated based on their different velocities when they drift through a buffer gas under the influence of a weak electric field. For comparison with theoretical model structures it is useful to convert drift times from ion mobility experiments into collision cross sections, which are a measure of the molecules overall three dimensional sizes. When homogeneous electric fields are used inside the drift region, this can be achieved using the following equation: 2 = 3ze r 16N 2 µk B T t D E L 1013T 273.16P (1) where z and e correspond to the number of charges on the investigated ion and the elementary charge, µ is the reduced mass of the ion-buffer gas molecule system, k B and T are the Boltzmann constant and the temperature, t D is the drift time, E and L are the electric field and the length of the drift cell, N is the number density of the buffer gas molecules and P is the buffer gas pressure (in mbar) inside the drift cell. For collision cross section determination, the drift peaks were fitted with a sum of three gaussian distributions to match the data. The cross sections were determined to be 135 Å 2 for the O-protonated species I and 155 Å 2 and 164 Å 2 for the two features of the N-protonated species II and II, respectively. Drift-time resolved collision induced dissociation Experimental details. Drift-time resolved collision induced dissociation (CID) experiments were performed on a commercial Waters Synapt G2-S (Manchester, U.K.) MS-IMS- TOF MS traveling-wave instrument. 3 After (nano) electrospray ionization, the species of S2
interest were m/z selected by a quadrupole mass filter following isomer separation in an ion mobility (IM) cell. An argon-gas filled collision cell was used to generate CID fragments of the now drift-time separated ions, which are subsequently mass-analyzed by means of TOF MS. Typical experimental setting were: source temperature, 20 C; capillary voltage, 0.8-1.0 kv; sample cone, 40 V; source offset, 20 V; trap collision energy, 2 V; trap gas flow, 2 ml/min; helium cell gas flow, 180 ml/min; IMS gas flow, 80 ml/min; trap DC bias, 40 V; IMS wave height, 40 V; IMS wave velocity, 1000 m/s. The collision voltage in the transfer cell was raised until fragmentation was observed (12-20 V). Results. Figure S1 (a) shows typical arrival time distributions (ATDs) of benzocaine, electrosprayed from different solvents on a commercial Synapt G2-S MS-IM-TOF MS instrument. 3 Also on this instrument, two well separated drift peaks can be observed, which change in relative intensity when the solvent composition is changed; a feature of high mobility with a drift time of 2.25 ms is predominantly observed for an aqueous solution (CH3OH/H2O, lower panel) and a second, lower mobility feature at 2.90 ms drift time is the strongest signal when the solvent is changed to acetonitrile (CH3CN, upper panel). To further investigate each individual species we performed collision induced dissociation (CID) experiments, which can yield information about molecular structure. In CID, the shape and m/z-selected ions are subjected to collisions with an inert gas (argon) with fragment analysis via TOF MS. The grey bars in Figure S1 (a) represent the drift-time windows from which the fragment information of the two drift peaks I and II were extracted. The resulting fragment spectra of the species I and II for collision voltages of 12 V and 20 V, respectively, are shown in Figure S1 (b). The fragmentation patterns and characteristics observed upon slow collisional heating are virtually identical the the ones observed in the IRMPD experiment. The most abundant fragment for both species is formed by the loss of the ethene molecule (C 2 H 4 ), which results in a strong ion signal at m/z 138. Other fragment signals in the lower m/z range are substantially weaker in intensity and are therefore magnified by a factor of 10 2 and 10 3 in S3
Figure S1: CID of drift-time selected benzocaine species. (a) Arrival time distributions (ATDs) of singly protonated benzocaine from two different solvents, measured on a commercial Synapt G2-S MS-IM-TOF MS instrument, and (b) CID fragment mass spectra of the drift-time separated species I and II. The drift-time windows from which the fragment spectra were extracted are indicated in grey. The lower mass range in the fragment spectra is magnified by a factor of 102 and 103, respectively. The two species exhibit differing dissociation yields, which is accounted for by applying a collision voltage of 12 V for species I and 20 V for species II. Ineachcase,dissociationisobserved,accompaniedbythelossof neutral compounds, as annotated in the upper panel of (b). In the Synapt instrument, m/z selection occurs prior IM analysis, which is why no II species (molecule-water complex) is observed here. S4
Figure S2: Fragmentation of O- and N-protonated benzocaine. Fragmentation pathway of O- and N-protonated benzocaine based on the observed CID and photofragment patterns. the lower and upper panel of Figure S1 (b), respectively. In both cases subsequent loss of CO 2 is observed forming m/z 94 followed by loss of NH 3 to form m/z 77. However, higher collision energies and magnification factors were required for species II than for species I indicating its greater stability. Furthermore, a signal at m/z 120, which indicates the loss of water from the m/z 138 fragment, could only be found in the spectrum of species I. Fragment identities The high mass accuracy of the Synapt G2-S instrument allows an unambiguous identification of the fragments observed in the CID experiment. All observed fragments can be explained by a fragmentation pathway of the O- and N-protonated species depicted in Figure S2. Whenthechargeislocatedattheaminenitrogennowaterlosscan occur from the m/z 138 fragment, which leads to the absence of the m/z 120 fragment in the fragment spectra of the N-protonated species. S5
Computational Assignment of vibrational bands For comparison with the experiment, vibrational frequencies of the theoretical model structures were calculated (shown in Figure 3 of the main manuscript). The most important vibrations are summarized in Tables S1 and S2 for the O- and N-protonated species, respectively. Table S1: Vibrational frequencies of the O-protonated species. The strongest features in the calculated and experimental spectra are listed. IR bands (cm 1 ) observed calculated vibrational mode 1635 1637 1616 NH 2 scissoring NH 2 scissoring + C C stretching (ring) 1550 1559 C C, C OEt asymmetric stretching (ester) + C C stretching (ring) 1530 1510 C C, C OEt asymmetric stretching (ester) + in-plane C H bending (ring) 1450 1459 1448 CH 3 scissoring + CH 2 scissoring CH 3 twisting 1410 1423 C=OH + stretching 1340 1343 CH 2 wagging 1190 1198 1170 in-plane C H bending (ring) in-plane C H bending (ring) + in-plane O=H + bending Table S2: Vibrational frequencies of the N-protonated species. The strongest features in the calculated and experimental spectra are listed. IR bands (cm 1 ) observed calculated vibrational mode 1745 1738 C=O stretching 1450 1470 NH 3 umbrellar 1260 1262 C C, C OEt asymmetric stretching (ester) + CH 2 wagging 1100 1121 1098 C C, C OEt symmetric stretching (ester) CH 3 antisymmetric bending Theoretical IR spectra of ring-protonated species A third possible protomeric species could arise from a protonation of the benzene ring, which plays a role in the gas-phase structures of aniline. 4 To rule out this possibility for benzocaine, theoretical model structures and corresponding spectra were computed. The four different S6
ring-protonated structures considered here, are at least 59.0 kj/mol higher in energy than the lowest energy structure of the O-protonated form and 6.3 kj/mol higher than the most stable N-protonated species (see insets in Figure S3). Additionally, the theoretical spectra do not reproduce the experimental data of species I and II (Figure S3). Energy optimization and calculation of theoretical spectra was carried out in analog to the theoretical spectra shown in the main text. The theoretical spectra were convoluted with a Gaussian profile of 1.5 % width and scaled by a factor of 0.975. Figure S3: Theoretical spectra of ringprotonated species of benzocaine. The energies of the optimized structures with respect to the lowest energy structure of the O- protonated species are annotated to the insets, showing the chemical structures of the ringprotonated species, respectively. None of these calculated spectra give a good match to the experimental data which is shown in the top two panels. S7
References (1) Revercomb, H.; Mason, E. A. Analytical Chemistry 1975, 47, 970 983. (2) Mason, E. A.; McDaniel, E. W. Transport Properties of Ions in Gases;JohnWiley& Sons, 1988. (3) Pringle, S. D.; Giles, K.; Wildgoose, J. L.; Williams, J. P.; Slade, S. E.; Thalassinos, K.; Bateman, R. H.; Bowers, M. T.; Scrivens, J. H. International Journal of Mass Spectrometry 2007, 261, 1 12. (4) Lalli, P. M.; Iglesias, B. A.; Toma, H. E.; de Sa, G. F.; Daroda, R. J.; Silva Filho, J. C.; Szulejko, J. E.; Araki, K.; Eberlin, M. N. Journal of Mass Spectrometry 2012, 47, 712 719. S8