Combining Ion Mobility Spectrometry, Mass Spectrometry, and

Wyttenbach , T.; Batka , J. J.; Gidden , J.; Bowers , M. T. Int. J. Mass Spectrom. ...... Prior , D. C.; Buschbach , M. A.; Li , F.; Tolmachev , A. V...
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Combining Ion Mobility Spectrometry, Mass Spectrometry, and Photoelectron Spectroscopy in a High-Transmission Instrument Matthias Vonderach, Oli T. Ehrler, Patrick Weis,* and Manfred M. Kappes Abteilung f€ur Physikalische Chemie Mikroskopischer Systeme, Institut f€ur Physikalische Chemie, Karlsruher Institut f€ur Technologie (KIT), Fritz-Haber Weg 2, 76128 Karlsruhe, Germany

bS Supporting Information ABSTRACT: We have developed a novel instrument that combines ion mobility spectrometry, mass spectrometry, and photoelectron spectroscopy. The instrument couples an electrospray ion source, a high-transmission ion mobility cell based on ion funnels, a quadrupole mass filter, and a time-of-flight (magnetic bottle) photoelectron spectrometer operated with a pulsed detachment laser. We show that the instrument can resolve highly structured anion arrival time distributions and at the same time provide corresponding photoelectron spectra;using the DNA oligonucleotide ion [dC6 - 5H]5- as a test case. For this multianion we find at least four different, noninterconverting isomers (conformers) simultaneously present in the gas phase at room temperature. For each of these we record well-resolved and remarkably different photoelectron spectra at each of three different detachment laser wavelengths. Two-dimensional ion mobility/electron binding energy plots can be acquired with an automated data collection procedure. We expect that this kind of instrument will significantly improve the capabilities for structure determination of (bio)molecular anions in the gas phase.

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on mobility spectrometry (IMS) in combination with mass spectrometry (MS) is a technique that is widely used to identify structures of ions in the gas phase, as well as a tool for isomer/conformer identification and separation. The systems investigated with IMS-MS comprise a large variety of different species such as peptides,1-7 oligonucleotides,8-11 synthetic polymers,12-15 and metal16-22 and nonmetal23-28 clusters. Compared to mass spectrometry alone, the combination with IMS adds another dimension of information;geometric structure;by way of a determination of the collision cross sections for the ions of interest. With the combination of IMS and fastscanning time-of-flight mass spectrometry (ToF-MS), it has even been possible to rapidly acquire two-dimensional mass vs cross section plots of complex biomolecular mixtures.29 Intensity losses upon ion injection into and extraction out of the IMS cell constitute a major limitation of IMS-MS coupling schemes. To obtain acceptable (cross section) resolution, it is necessary that ions undergo a large number of collisions with the buffer gas present in the cell. Therefore, IMS cells operate in a pressure range of several millibars. High-resolution IMS cells are run even close to atmospheric pressure.30 Mass spectrometers on the other hand need to operate in a pressure regime where collisions with residual gas molecules are negligible, i.e., in the range of 10-4 mbar or lower. As a consequence, the IMS-MS interface has to be differentially pumped and connected via small apertures with typical diameters below 1 mm. Electrostatic lenses that work excellently in the low-pressure regime are ineffective in the 1 mbar range, where ion-buffer gas collisions and electric fields determine the ion trajectories. As a consequence, schemes for focusing transmitted ions onto the exit apertures of IMS cells have not been very efficient until recently. Typically, huge intensity losses (95% or higher) prevailed. This situation changed r 2011 American Chemical Society

dramatically with the invention of the “ion funnel”,31,32 an electrodynamic device that quite effectively focuses ions in the millibar pressure range. By combining such a funnel with an IMS drift cell, the ion transfer through the entrance and exit apertures could be drastically improved. Wyttenbach et al. have adapted an ion funnel to the front aperture of an IMS cell33 and have used it to efficiently store and pulse-inject ions from a continuous electrospray ionization (ESI) source into the drift cell. Smith and coworkers have constructed ion funnel interfaces for both the front and back ends of an IMS cell and have as a result developed the new technique of high-throughput IMS.34 In more recent years, ion funnel-IMS combinations have been implemented in several instruments.35-37 Furthermore, the huge intensity gains associated with such setups have allowed for adding additional IMS dimensions, such as IMS-IMS-MS.35 Even IMSIMS-IMS-MS38 experiments have now been realized. Photoelectron spectroscopy (PES) of ions in the gas phase allows determination of their (occupied) electronic density of states. It is thus a direct measure of electronic stability, which often also correlates with overall thermodynamic stability. While determination of dispersed electron spectra generally allows for extensive analysis of electronic properties, PES can also be used as a complementary tool to more direct geometric structure determination methods such as IMS. As such, measured spectra in combination with predictions from electronic structure theory allow identification, e.g., of different isomers present in the ion beam. Similar to ion mobility, PES has been extensively coupled to mass spectrometry, in particular since the development of Received: November 16, 2010 Accepted: December 15, 2010 Published: January 7, 2011 1108

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Figure 1. Schematic view of the instrument. It comprises four main segments: an electrospray ion source, an IMS drift cell, a quadrupole mass filter, and a photoelectron spectrometer.

time-of-flight PE spectrometers.39,40 With their pulsed operation, they can be easily adapted to pulsed mass spectrometers and used with pulsed high-power lasers. Such setups allow;at least in principle;for the measurement of the PE spectrum of an ensemble of ions within a single light pulse. Practically, however, PES suffers from low detachment cross sections, while at the same time it remains necessary to limit photon fluences to prevent multiphoton processes that complicate interpretation of the spectra. Therefore, data acquisition is usually extended over longer times, and (relatively) stable high-intensity ion sources become a necessity. Despite these restrictions, PES has been applied to basically the same variety of (negative) gas-phase ions as IMS: atomic 41-43 and molecular clusters,44-46 multiply charged anions,47,48 and molecules of biological interest, among others. In particular, PES of biomolecules has developed into a rapidly expanding field, with experiments performed on smaller systems such as single nucleic bases49,50 and extended to biopolymers and their subunits, such as (oligo)nucleotides51,52 and peptides.53-55 A possible complication of MS-PES experiments is the presence of isomer/conformer mixtures in the sample that cannot be separated by MS alone and lead to overlapping and sometimes even featureless PE spectra.56 This is particularly true for polymers in general and biopolymers in particular that owe their intrinsic structural complexity to a multitude of polar functional groups. To still be able to extract meaningful quantities from such spectra, it becomes necessary to somehow separate overlapping spectral signatures. If electron affinities of the various isomers differ strongly, spectral hole burning can be used to deplete the more weakly bound species via near-threshold detachment with an additional laser and subsequent PE measurement of the enriched ensemble.57 Characteristic spectra are then obtained from difference spectra before and after enrichment. In an analogous chemical implementation, “etching” of the molecular beam (e.g., by reactions with molecular oxygen) allows for depletion of isomers with higher reactivity as has recently been demonstrated for metal clusters.43 While both methods have been successfully applied in certain cases, they rely on the presence of specific differences in particular properties of the various isomers, have effectively not been shown to also work on mixtures of more than two isomers, and are too complex to be

implemented in a general fashion to allow for routine determination of PE spectroscopic signatures of particular isomers. The coupling of an IMS cell with a mass spectrometer and a PE spectrometer provides a more general solution for mass- and isomer-resolved ion spectroscopy. Fromherz et al. were the first to realize such a setup in a pioneering study of small carbon cluster anions in the size region of the transition between linear and cyclic structures.58 Since their instrument did not make use of ion funnels for guiding the ions from high pressure (IMS) to low pressure (PES) regions, it was however “limited by its modest mobility resolution and sensitivity”.58 Here we report on a new experimental setup based on ion funnels that allows for mass selection and mobility resolution of gas-phase conformers prior to PE spectroscopy without such severe intensity limitations. We expect that this kind of instrument will significantly improve the future capabilities for structure determination of (bio)molecular anions in the gas phase.

’ EXPERIMENTAL SECTION Our instrument consists of four main components: an electrospray ion source, an ion mobility drift tube with ion funnels, a quadrupole mass spectrometer, and a time-of-flight photoelectron spectrometer (see Figure 1). We describe each in turn. ESI Source. The ESI source consists of an off-axis sprayer, connected via isolating Teflon tubing to a syringe pump (TSE systems), and a heated stainless steel capillary inlet into the vacuum system. All components are electrically isolated from ground and float on top of the IMS cell entrance potential. The capillary inlet consists of a 120 mm long, 0.5 mm i.d. stainless steel tube mounted in a copper housing. This housing can be heated to 150 °C by a Thermocoax heater coil, its temperature being monitored by thermocouples. It is biased to typically 400 V above the IMS cell entrance potential. Ions leaving the capillary first enter the throat of the entrance ion funnel. This primary stage is pumped by a rotary vane pump (Oerlikon Trivac D65B; see Figure 1, pump 1) to a pressure slightly below the IMS cell pressure. Since the inlet flange also floats at the IMS voltage, the rotary pump line is also electrically isolated (by means of 1 m long KF40 plastic tubing). IMS Cell with Ion Funnels. The IMS cell design closely follows that of Tang et al.34 and comprises entrance and exit 1109

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Analytical Chemistry funnels enclosing the drift cell. The entrance ion funnel is of the “hourglass” design.34 It consists of 100 0.5 mm thick electrodes (nickel-coated brass) with an outer diameter of 43 mm. The electrodes are separated by 0.5 mm Teflon sheets. The first 16 electrodes have an inner diameter of 25 mm, followed by 47 electrodes with diameters linearly decreasing in steps of 0.5 mm down to a minimum of 2 mm. The latter represents the limiting aperture between the first pumping stage and the IMS cell. The last 35 electrodes open up again (with the same angle) to a diameter of 20 mm. The electrodes are alternatingly connected to the two phases of a home-built rf power supply (500 kHz, 200-400 Vpp (peak-to-peak)). Simultaneously, dc potentials of typically 350 and 150 V are applied to the first and last electrodes, respectively. The resulting voltage gradient of 200 V guides the ions against the gas flow from the IMS cell. In the front region of the funnel, one (hollow) electrode is replaced by an electrode incorporating an electrically contacted 6 mm disk on the beam axis. This serves as a “jet disrupter” 59 and is held at a dc potential of typically 250 V (without rf voltage). The last electrode of the funnel (held at 150 V) is followed by an “ion gate”, which comprises an electrode covered with a high-transmission mesh (Precision Eforming, MN49, 118 lines per inch). When a voltage of 160 V is applied, the gate is closed and ions are stored prior to injection into the IMS cell. Upon injection, the gate is pulsed to 50 V (by a Behlke HTS21-03GSM switch) for 50 μs, and an ion packet is injected into the cell. The operation frequency of this gate is 15 Hz (half the repetition rate of the detachment laser; see below). The IMS drift cell consists of a series of 200 mm i.d., 200 mm long stainless steel tubes. They are isolated from ground and from each other by 1 cm thick PEEK sandwich flanges with a diameter of 245 cm and a 83.5 mm center bore. The flanges carry a stack of 21 guard rings (1 mm thick, 80 mm o.d., and 55 mm i.d.) mounted on four 209 mm long, 3.2 mm diameter ceramic rods with 9 mm long PEEK spacers. Adjacent rings are electrically connected by 1 MΩ resistors. As a result, a highly homogeneous electrical field is obtained upon application of a voltage across the stack. We typically run the IMS-MS-PES experiment with a stack of three tubes, i.e., a 63 cm long IMS cell and a voltage of 650 V. The cell is operated with 3-4 mbar of He, and the pressure is monitored by an MKS baratron capacitance gauge (MKS627A). At the end of the IMS cell, the ions are focused by the exit ion funnel. It consists of two stacks of electrodes: the first stack has 97 electrodes (nickelcoated brass) and converges from 50 mm i.d. in steps of 0.5 mm to 2 mm i.d. for the last electrode, which also corresponds to the pressure-limiting aperture of the cell. Electrodes of the first stack are separated by Teflon sheets and compressed by springs, producing a gastight arrangement (apart from the 2 mm aperture). The second stack consists of 70 electrodes, each 0.5 mm thick, with a 36 mm o.d. and an 8 mm center bore, separated by 1 mm Teflon washers. This stack is not gastight and allows for pumping of the He gas emerging from the IMS cell (via pump 2, Oerlikon Ruvac WAU 501, 500 m3/h þ Trivac D65B). The electrodes of both stacks are alternatingly connected to the two phases of a 500 kHz rf supply (typically 60 Vpp); simultaneously a dc voltage of 250 V is applied to the first electrode (50 mm i.d., immediately behind the IMS cell) and 50 V to the last electrode of the second stack. Additionally, the bias voltages of the penultimate and ultimate electrodes of the first stack can be adjusted. Typical values are 60 and 55 V, respectively; i.e., the gradient in the first stack is much steeper than that in the second

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stack. The typical IMS resolution (t/Δt) under optimal conditions was 45-50. Quadrupole Ion Guide, Ion Mirror, and Quadrupole Mass Filter. Ions leaving the funnel are focused into an rf-only (140 mm long, 900 kHz, 100 Vpp) quadrupole ion guide by an electrostatic lens with a 7 mm center bore, which represents the limiting aperture to the next stage of differential pumping. This stage is pumped by a diffusion pump (pump 3, Oerlikon DIP3000 þ baffle þ Trivac D45B) to a pressure of 5  10-6 mbar, low enough that ions leaving the quadrupole ion guide do not undergo appreciable collisions with residual gas molecules. As a result they can now be guided by conventional electrostatic lenses. To further reduce the gas load in the PE spectrometer, a direct line of sight between the IMS cell and PES chamber is prevented by deflecting the ions at right angles using a dc quadrupolar electrostatic mirror which consists of electrodes with quarter-circle cross sections held at þ100 and -30 V, respectively. Ions are then focused into the quadrupole mass filter (Extrel, 4000 amu, 880 kHz), and all unwanted masses are removed from the beam. The exit hole of the mass filter (L = 7 mm) represents the limiting aperture to the PES chamber, which is pumped by a turbomolecular pump (pump 4, Oerlikon TD700) to 3  10-7 mbar (during the experiments, i.e., with the IMS cell filled with He). The mass-selected ion beam is focused into the detachment region of the PE spectrometer by a stack of electrostatic lenses, where it is crossed by the detachment laser beam. The intensity of the remaining ions is monitored by a channeltron detector mounted downstream from the PE spectrometer, its output being directly fed into an oscilloscope (TDS 2024, Tektronix). This setup allows for online monitoring of ion intensities and optimization of ion optics while running PE spectra. The rf and dc potentials were adjusted using a LabViewwritten user interface (National Instruments) in combination with a home-built 30-pole high voltage supply. Photoelectron Spectrometer. Our PE spectrometer is a time-of-flight instrument of the magnetic bottle type. It loosely follows the original implementation by Kruit and Read.39,40 Ions leaving the quadrupole mass filter were irradiated in the center of the detachment chamber using a pulsed laser incident perpendicular to the cluster beam. Effective ion and laser beam diameters in the interaction region are discussed in the context of sensitivity in a later section. In the current experiments, the third (355 nm), fourth (266 nm), and fifth (213 nm) harmonics of a pulsed Nd:YAG laser (Spectra Physics, LAB150-30) were used, with pulse durations between 5 and 8 ns. Pulse energies were typically reduced to 20, 3, and 0.5 mJ, respectively, to avoid saturation and nonlinear processes. Selection of the proper arrival time of the desired mass-selected and isomer/conformer-separated ions in the PE spectrometer was achieved by adjusting the temporal delay between the injection of the electrosprayed ion bunch into the drift cell on one hand and the laser pulse on the other hand (SRS DG535 delay generator). Detached electrons were dispersed by time-of-flight kinetic energy analysis in the 1.2 m long drift tube and detected with a dual microchannel plate (MCP; L = 50 mm, Tectra) detector in chevron configuration and using a 50 Ω impedance matched anode. Spectra in the electron binding energy (EBE) domain are then obtained by transformation of kinetic energy (Ekin) distributions according to ð1.1Þ EBE ¼ hν - Ekin with hν the photon energy of the detachment laser. 1110

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Figure 2. (a) Electrospray ionization mass spectrum of a solution of oligonucleotide dC6 in water/methanol. (b) Arrival time distribution of [dC6 5H]5-. Four peaks (isomers or conformers) can be clearly resolved. (c) Photoelectron spectra for the four different conformers at a 266 nm detachment laser wavelength, obtained by adjusting the laser delay to hit the different peaks of the arrival time distribution.

The guiding fields of the magnetic bottle guarantee unity transmission efficiency of the detached electrons onto the detector by bending all trajectories toward the electron drift tube, independent of their initial direction. This is achieved by a strong diverging magnetic field right below the interaction volume of the laser and ion beams, which converges into the weak homogeneous magnetic field lines inside the drift tube. We realize the strong field with four cylindrical NdFeB permanent magnets (L = 10 mm, height 10 mm, IBS Magnet, NE105), placed on a translation feedthrough. The weaker guiding field is realized by winding a coil around the flight tube (2 cm pitch) and applying a current of 3 A. To shield earth and stray magnetic fields, the tube is covered by two layers of μ-metal, each 1 mm thick, and a pair of perpendicular Helmholtz coils that surround the detachment region. Stray electric fields and surface patch potentials are minimized by graphitizing all inner surfaces of flight tube and detachment region. Electrons impinging on the MCP detector create low impedance potential spikes that are amplified with a fast preamplifier (TA 1800, FAST Comtec) and recorded by a high-resolution dual channel digitizer (P7888-2, FAST Comtec), which is triggered synchronously to the emission of the laser. Reduction of background noise due to ionization of residual gas molecules and stray light hitting metal surfaces is an important issue. Apart from keeping the pressure in the PES chamber as low as possible, we run PE spectra alternating with and without ions by opening the ion gate every other laser shot (trigger scheme; see Figure S1, Supporting Information), feeding the background signal into the second channel of the time digitizer, and subtracting the main and reference spectra from each other. Spectra are typically integrated over 20 000 laser shots at a resolution (ΔEkin/Ekin) of ∼3% at 1 eV of kinetic energy.

’ RESULTS AND DISCUSSION One-Dimensional IMS-PES. As a test system, we have chosen the all-cytosine DNA hexanucleotide dC6 (obtained from Jena Bioscience and dissolved in water without further purification). Electrospray conditions were 50-100 μM solution in water/ methanol (1:4) at a flow rate of 0.15 mL/h and a sprayer voltage of 2.5 kV (above the capillary inlet potential). In negative ion mode, we observe a mixture of charge states, i.e., [dC6 - 5H]5-, [dC6 - 4H]4-, and [dC6 - 3H]3- (see Figure 2a). During the mass scan, the ion gate in front of the IMS cell was permanently open; i.e., ions were allowed to continuously enter and drift through the cell. For all further investigations, the quadrupole

mass filter was operated in constant-mass mode, selecting only [dC6 - 5H]5- after passing through the IMS cell. The ion gate in front of the drift cell was then used to accumulate ions prior to injection by applying a 50 μs voltage pulse. Subsequently, ions travel through the IMS cell where their drift time is determined by their collision cross section. For [dC6 - 5H]5- we observe a highly structured ion arrival time distribution (ATD) at the downstream channeltron detector with four clearly distinguishable peaks. Their widths (fwhm) are consistent with the IMS resolution of our instrument of approximately 45. This indicates a mixture of at least four different conformers (see Figure 2b) with collision cross sections of 338, 351, 364, and 377 Å2, respectively. These data can be used in combination with molecular dynamics simulations and density functional theory (DFT) calculations either to assign a unique structure for each peak or at least to resolve structural motifs.60 Photoelectron spectra were obtained by adjusting the delay between ion gate and detachment laser pulse to hit the different conformers as they pass the detachment region. As can be seen from Figure 2c, the various conformers show distinct differences in their PE spectra. The leading edge on the low-energy side of each spectrum can be extrapolated to obtain the adiabatic electron affinity (AEA) of [dC6 - 5H]4-. A common feature in the four curves is a negative AEA. This might at first glance look counterintuitive, but is, in fact, often observed in multiply charged anions (MCAs).47 It reflects the metastability of all [dC6 - 5H]5- conformers against electron loss. The multianions are, however, stabilized against immediate electron emission by a repulsive Coulomb barrier (RCB), which results from the long-range repulsive interaction between the detached electron and the remaining (still 4-fold negatively charged!) ion. This situation is schematically visualized in Figure S2 (Supporting Information). Both the RCB height and the EBE depend strongly on the distribution of the extra charges in the MCA. These observables can thus be used in combination with molecular dynamics and electronic structure theory (e.g., density functional theory) to obtain additional structural information on the conformers.60 Two-Dimensional IMS-PES and Global Fitting Routine. The ATD of [dC6 - 5H]5- is special inasmuch as it consists of four well-separated peaks, and the obvious procedure to obtain conformer-resolved PE spectra is to adjust the detachment laser delay to overlap with each peak in the maximum (see above and Figure 2c). More often, arrival time distributions are more or less 1111

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Figure 3. (a) False color plot of arrival time dependent photoelectron spectra of [dC6 - 5H]5- at a detachment wavelength of 266 nm and (b) 3D view of these data. (c) Residuals and (d) simulated spectrum as obtained from the multivariate fit.

continuous without well-resolvable structure. In such situations it is useful to obtain PE spectra in a (quasi) continuous fashion as well, instead of selecting only a few representative points in the ATD, and to analyze the resulting data to extract the characteristic spectral signature of each conformer/isomer. We have therefore developed a scheme to scan the detachment laser in small steps across the ATD and automate the data acquisition. For this both the adjustment of the settings of the master delay generator (via a GPIB interface) and accumulation and workup of the resulting PE spectra are controlled through home-built software within the LabView (National Instruments) environment. To account for slow fluctuations in the intensity of the ion source, spectra at each arrival time are not recorded at once, but instead the whole ATD is sampled repeatedly to collect the IMS-PES data over many short measurements. For the 2D IMS-PES plot of [dC6 - 5H]5- at a 266 nm detachment wavelength shown in Figure 3a, 15 arrival times were sampled over a total of 1500 measurements. ATD and PES data were collected in bins of 25 μs and 50 meV, respectively, and normalized intensities (i.e., the relative number of collected electrons) are represented by false colors. Each row in the diagram thus corresponds to a PE spectrum and each column to the ATD convoluted with the detachment efficiency at the respective energy. As ions present in the beam are not coupled in any way, intensities in the 2D spectral plot are simply the product of their abundances and individual PE spectra. We therefore use an iterative nonlinear regression procedure to disentangle the multivariate data and obtain the characteristic PE spectrum of each contributing conformer (for details see the Supporting Information). To validate the resulting global fit (Figure 3d), residuals as the difference from the original raw data (Figure 3b)

are obtained. If these residuals are small and distributed statistically over the whole multidimensional plot (as is the case in Figure 3c), a good description of the experimental data set has been found. Collision-Induced Isomerization. Another important aspect of our instrument is the option to investigate collision-induced isomerization. Relative intensities of the different conformers of [dC6 - 5H]5- strongly depend upon the injection conditions (see Figure 4): At high injection electrode potentials, we obtain mostly the first conformer with an early arrival time corresponding to a small collision cross section and thus a compact structure. At low injection potentials, the relative intensity of this conformer decreases drastically, while conformer 4, with the longest drift time and therefore the largest cross section, becomes dominant. The injection electrode potential determines the amount of kinetic energy the ions gain prior to injection into the IMS cell. Lower potentials correspond to higher kinetic energies; i.e., ions are injected faster into the IMS and are consequently heated further due to inelastic collisions with (helium) buffer gas atoms. The same process also results in energy transfer to the inert background gas, and the ions are quickly slowed/cooled back to essentially thermal (room temperature) conditions. This annealing process takes place in close proximity to the injection electrode. Since the pressure in the cell is comparatively high (3-4 mbar), ions undergo on the order of 106 collisions during their overall drift of typically 3 ms. Consequently, we expect that the injected/annealed ions spend by far most of their drift time at room temperature. This is indirectly confirmed by the observation that relative intensities of the different conformers vary while their cross sections (i.e., the peak position in the ATD) remain constant. Further confirmation comes from the fact that 1112

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without ions (see above). Limited by the repetition rate of our current detachment laser (30 Hz), the ion gate therefore operates at 15 Hz. In each cycle, the gate is open for 50 μs and closed for 67 ms; i.e., the duty cycle is less than10-3. Therefore, it is crucial that the entrance ion funnel is capable of accumulating the ions emerging continuously from the ESI source and extracting them as a dense ion packet. The storage efficiency of the funnel can be determined by measuring the ion signal intensity (=output of the channeltron detector) in the pulsed and continuous (=gate always open) mode. We find it close to 44%; i.e., the entrance funnel is a highly efficient storage device. (2) Transfer through the IMS cell and exit funnel. After transfer into the IMS cell, the ion packet spreads both axially and radially. The axial spread translates into the ATD, the fundamental result of IMS. The radial spread; which can be a few centimeters in our 63 cm long IMS cell;can lead to severe intensity losses since only those ions close to the cell axis can emerge through the exit aperture. The exit ion funnel recollimates the radially spread ion cloud onto the cell axis (without disturbing the ATD). To estimate the performance of the funnel, we replaced it by an end plate with a 1 mm aperture (adjusted to achieve the same pressure conditions inside and outside the IMS cell; i.e., the conductivity of the funnel with a 2 mm aperture corresponds to the 1 mm pinhole). With this setup, the ion signal dropped dramatically by at least 3 orders of magnitude, below our detection limit. Likewise, the exit funnel is responsible for an intensity gain of at least 3 orders of magnitude. Its transmission is most likely close to unity.34 (3) Photodetachment efficiency. For a given laser power and wavelength, the number of photoelectrons depends on the geometrical overlap of the ion and detachment laser beams. In our setup, they cross perpendicularly and the laser beam diameter in the detachment region is close to 3 mm. With optimized ion optics (based on SIMION61 calculations), we are confident that we are able to focus the ion beam radially well within this limit. The (spatial) width of the axial distribution is much more crucial. It is determined by the ion velocity and the (temporal) width of the ATD, which is on the order of 50-100 μs for a single peak (see Figure 2b). The ion kinetic energy (per charge) in the detachment region is roughly determined by the potential of the last electrode of the exit funnel (50 V). For an ion of m/z 333 (such as [dC6 - 5H]5-), this translates into a spatial width of a single peak of 25-50 cm; i.e., in our current setup the detachment laser hits only 1% of the ion beam. While this is definitely a severe sensitivity limitation, it is partially compensated for by the (near) unity collection and transmission efficiency of the magnetic bottle PE spectrometer. Various ion bunching schemes provide scope for further improvement, and we hope to address this in the future.

Figure 4. Collision-induced isomerization upon injection into the drift tube.

PE spectra for each peak are independent of the injection electrode potential. Variation of the Detachment Laser Wavelength. Additional insight into the electronic structure of the various isomers/conformers is gained from their PE spectra at different detachment photon energies (Figure S3, Supporting Information). In general, acquisition at higher energies maps out larger fractions of the occupied electronic density of states, as additional continua of excited final states become energetically accessible. Their relative cross sections are determined by the emitted electron partial waves and reflect the symmetry of the involved electronic states. In the case of MCAs, additional information results from the presence of the RCB.47 As described above, the cutoff on the high EBE side of a PE spectrum results from suppression of the emission of slow electrons that cannot overcome this barrier. It is, however, usually not clear whether any electronic state is accessible at the position of this cutoff, and direct determination of the barrier height is not possible from a single spectrum. Comparison of spectra at different photon energies, however, allows extraction of lower and upper limits. Similar to the overall repulsive Coulomb energy of the unshielded negative charge carriers in an MCA, the barrier height reflects the excess charge density and is thus correlated with the geometry of the molecule. Caution has to be taken when PE spectra of biopolymers are measured, as aromatic chromophores in many peptides and oligonucleotides typically lead to strong absorption in the UV spectral range. In addition to single-photon detachment, various multiphoton processes (e.g., two-photon detachment or delayed autoemission) can become prominent and lead to additional contributions in the spectra that complicate their interpretation.53 At wavelengths shorter than 355 nm, we therefore work at rather low photon fluences to prevent such processes while maintaining sufficiently high electron yields to reduce statistical noise. Comparison of spectra at different detachment wavelengths and power-dependent measurements and the analysis of the resulting detachment yields exclude unwanted multiphoton contributions to the spectra. Sensitivity Considerations. An important aspect of our instrument is its sensitivity, i.e., the ratio of the number of photoelectrons detected to the number of ions (of the mass of interest) produced in the ESI source. It is the product of several factors. (1) Storage efficiency of the ion gate. Background correction is an important aspect in our experiment, and we use a shot to shot reference by recording PE spectra with and

’ CONCLUSIONS We have developed a novel instrument that allows for conformer-selective photoelectron spectroscopy of biomolecules in the gas phase. It combines an electrospray ion source, a hightransmission ion mobility cell34 for isomer selection based on the 1113

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Analytical Chemistry collision cross section, a quadrupole mass filter, and a (magnetic bottle) time-of-flight photoelectron spectrometer. Key features of the ion mobility cell are (pulsed) entrance and exit ion funnels that drastically minimize ion losses between different pressure regions. With this high-transmission setup, the ion intensity is high enough that we can record arrival time distributions in real time with an oscilloscope and, more importantly, add a photoelectron spectrometer directly into the transmitted pulsed ion beam, without the need for any (further) ion storage device that would perturb the arrival time distribution. With the oligonucleotide ion [dC6 - 5H]5- as a test case, we record a highly structured arrival time distribution with four well-resolved peaks. For each of these we determine photoelectron spectra at different detachment laser wavelengths. By automatically scanning the detachment laser delay, we are able to record two-dimensional ion mobility/electron binding energy plots that are analyzed by a multivariate fit procedure to directly yield the spectrum of each isomer/conformer present in the ion beam. We expect that this new type of instrument will significantly improve the capabilities for structure determination of ions in the gas phase.

’ ASSOCIATED CONTENT

bS

Supporting Information. Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

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