Differentiating between Capillary and Counter Electrode Processes

and Counter Electrode Processes during Electrospray Ionization by Opening the Short Circuit at the Collector ... Publication Date (Web): March 16,...
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Anal. Chem. 1999, 71, 1585-1591

Differentiating between Capillary and Counter Electrode Processes during Electrospray Ionization by Opening the Short Circuit at the Collector Florence Charbonnier,*,†,‡ Ludovic Berthelot,† and Christian Rolando*,§

De´ partement de Chimie, Ecole Normale Supe´ rieure, URA 1679 du CNRS, Processus d’Activation Mole´ culaire, 24 rue Lhomond, 75231 Paris Cedex 05, France, and UFR de Chimie, Universite´ des Sciences et Technologies de Lille (Lille 1), Baˆ timent C4, URA 351 du CNRS, Chimie Organique et Macromole´ culaire, 59655 Villeneuve d’Ascq Cedex, France

The chemical reactivity of an electroactive neutral compound in the conditions of electrospray mass spectrometry was studied by carrying out preparative experiments. The residue obtained by spraying a micromolar methanolic solution of 9-nitroanthracene onto a metallic plate in negative mode (cathodic capillary) was collected and then analyzed by GC/MS, showing that both reduction and oxidation products were formed. To distinguish between the two processes, the two functions of the metallic plate, definition of the electric potential and storage of products, were separated by using a copperbronze grid as counter electrode and a silica TLC plate, located a few millimeters behind, as collector. The collected products were thus stored away from the counter electrode, avoiding their possible reoxidation at this site. The amount of anthracene, a reduction product of 9-nitroanthracene, which was detected only in trace amounts with the metallic plate collector, increased significantly with the new device. Thus, anthracene can be viewed as the result of a capillary process whereas 9,10-anthraquinone can be estimated as mainly due to further oxidation at the collector. Furthermore, in deuterated solvents no incorporation was detected, suggesting that some reaction steps take place at the collector. Despite an explosive development of electrospray mass spectrometry (ESI-MS) as an analytical tool, the basic principles that govern the droplet charging process remain mostly unknown.1 As pointed out by Kebarle,2 the existence of a spray current implies electron transfers involving electroactive species both at the capillary and at the collector. Negative ionization for instance * Corresponding author: (phone) 33-4-76-51-41-86 or -79; (e-mail) Florence. [email protected]. † Ecole Normale Supe ´ rieure. ‡Current address: Universite ´ Joseph-Fourier (Grenoble I), EA 582, BP 53, 38041 Grenoble Cedex 9, France. § Universite ´ des Sciences et Technologies de Lille (Lille 1). (1) Cole, R. B., Ed. Electrospray Ionization Mass Spectrometry, Fundamentals, Instrumentation & Applications; John Wiley & Sons: New York, 1997. (2) Blades, A. T.; Ikonomou, M. G.; Kebarle, P. Anal. Chem. 1991, 63, 21092114. 10.1021/ac980799l CCC: $18.00 Published on Web 03/16/1999

© 1999 American Chemical Society

involves reduction at the capillary and oxidation at the counter electrode (Figure 1). For neutral compounds, which are not ionized in the starting solution, electron transfers either heterogeneous (at the capillary tip) or homogeneous (inside the capillary tip or within droplets) must be responsible for the ions observed. On this basis, a description of ESI-MS as a controlled-current electrolytic cell has been comprehensively developed by Van Berkel3-5 and Cole.6 The ions formerly produced are partly consumed in reactions leading to neutrals and partly lost during the transfer from atmospheric pressure to collector. Finally, the ratio of the ions detected by the mass spectrometer per electrons consumed at the capillary is approximately only 1 per 10 000.7 Thus, ESI-MS analysis requires that neutral compounds be sufficiently electroactive to get ionized in a significant amount by electron transfer at the capillary tip and that the created ions be stable enough in order to reach the collector before subsequent reaction. Very few experiments have been devoted to studying the hidden electrochemistry occurring within the spray. Van Berkel and co-workers have been able to detect redox processes by fluorescence spectroscopy analysis of the electrospray mist.8 With the aim of unraveling hidden parts of the electrochemistry occurring during ESI-MS analysis, we chose to study the fate of neutral electroactive compounds during electrospray ionization. In a typical experiment, a solution of the electroactive neutral compound is sprayed onto a metallic plate in ESI conditions and the deposit is extracted with a solvent and analyzed by GC/MS. In a preliminary report, we demonstrated that such preparative electrospray experiments can be performed on halogenonitroaromatic compounds.9 The global behavior of the analyte can then (3) Van Berkel, G. J.; McLuckey, S. A.; Glish, G. L. Anal. Chem. 1992, 64, 1586-1593. (4) Van Berkel, G. J.; Zhou, F. Anal. Chem. 1995, 67, 2916-2923. (5) Van Berkel, G. J.; Zhou, F. Anal. Chem. 1995, 67, 3958-3964. (6) Xu, X.; Nolan, S. P.; Cole R. B. Anal. Chem. 1994, 66, 119-125. (7) Smith, R. D.; Loo, J. A.; Loo, R. R. O.; Busman, M.; Udseth, H. R. Mass Spectrom. Rev. 1991, 10, 359-451. (8) Chillier, X. Fr. D.; Monnier, A.; Bill, H.; Ulac¸ ar, F. O.; Bush, A.; McLuckey, F. A.; Van Berkel, G. J. Rapid Commun. Mass Spectrom. 1996, 10, 299304. (9) Charbonnier, F.; Nicolas, J.-P.; Eveleigh, L.; Hapiot, P.; Pinson, J.; Rolando, C. C. R. Acad. Sci. Paris, Ser. IIc 1998, 449-456.

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Figure 1. Schematic description of the ESI-MS process: negative ion mode.

be inferred on the basis of the components identified in the collected residue. We report here on 9-nitroanthracene (HArNO2) submitted to negative ionization in methanol. First, we used a metallic plate as the collecting surface. In such conditions, an oxidized derivative of 9-nitroanthracene, 9,10-anthraquinone, was isolated as the major product. This result led us to consider the anodic activity of the collector. Since the collecting plate also worked as the counter electrode in these experiments, it was likely to involve the compounds formerly produced within the spray in further oxidation reactions. To study selectively the sequence of electrolytic processes initiated at the capillary, we designed a twopiece collector set, made of a metallic grid connected to the highvoltage supply and a silica trapping surface independent from the electrospray circuit. With this device, we were able to isolate anthracene, a typical reduction product of the analyte. We propose a mechanism for its formation within the electrospray mist. EXPERIMENTAL SECTION Reagents and Materials. Reagent grade dichloromethane, methanol, 9-cyanoanthracene, 9-nitroanthracene, 9-H-anthrone, 9,10-anthraquinone, trifluoroacetic acid, and 25% aqueous trimethylamine (TMA) were purchased from the Aldrich Chemical Co. (Milwaukee, WI) and used without further purification. Due to the low concentrations used (in the micromolar range), great care was taken to avoid contamination (by phthalates, fingerprints, etc.) throughout the process. Electrospray Device. The high voltage was provided by a (10-kV, 1-mA power supply (SDS, Paris, France). It was connected to the collecting plate through the secondary of a transformer (obtained from the high-voltage unit of a Dupont mass spectrometer). The primary of the transformer was connected to a rf generator with a maximum amplitude of 10 V peak to peak. With this device, an alternative voltage (voltage, 0-300 V peak to peak; frequency, 100-3000 Hz) was added for securing the synchronization of the spray. The instantaneous electrospray current was measured at the capillary with an AD843JN current transducer (Analog Device, Norwood, MA), with a rising time less than 1 µs and a 106 amplification ratio. The image of the synchronization voltage was detected by a capacitor plate located behind the collector and subtracted from the electrospray current. Data were recorded using a TDS 320 digital oscilloscope (Tektronix, Beaverton, OR) with 1000 data points and an 8-bit dynamic, in the peak detect mode.10,11 (10) Charbonnier, F.; Rolando, C.; Saru, F.; Hapiot, P.; Pinson, J. Rapid Commun. Mass Spectrom. 1993, 7, 707-710.

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The capillary was a stainless steel 22s gauge (o.d., 0.72 mm; i.d., 0.15 mm) needle (Hamilton, Reno, NV) adapted by a Teflon tube to a 5-mL glass syringe driven by a syringe pump (model A-99 Razel Scientific Instrument, Stanford, CT). The collector was a 5 × 5 cm copper plate (for printed board). The distance between capillary tip and collector was fixed to 6 mm by using an optical bench (Microcontrole, Evry, France). The whole device was placed into a glass enclosure where the temperature (∼35 °C) was controlled by means of a warming belt and measured with a thermocouple. The capillary was always connected to the grounded side of the high-voltage supply (positive high voltage (HV) in reduction mode, i.e., cathodic capillary). The solution was sprayed under ambient atmosphere at a flow rate of 1.25 mL‚h-1. The time required for infusing 5 nmol of analyte (5 mL of a 1 µM solution) was ∼4 h. After spraying, the plate was washed with dichloromethane (50 µL) and 9-cyanoanthracene (2 µL of a 1 mM methanolic solution) was added as an internal standard to the collected solution before GC/MS analysis. In the new device, the metallic plate was replaced by a 5 × 5 cm copper-bronze grid (2-mm mesh) positioned at the same place. A 5 × 5 cm TLC silica plate (Silicagel 60, Merck, Darmstadt, Germany) separated by a Teflon spacer was placed 1.6 mm away. After spraying, the silica plate was observed under UV light and the absorbing zone was collected, extracted with dichloromethane (1 mL), and filtered on glass wool. The resulting solution was concentrated to 50 µL under argon and GC/MS analysis was performed as before. GC/MS Analysis. GC/MS analyses were performed on a JMS700 MStation mass spectrometer (JEOL, Tokyo, Japan), equipped with a HP 6890 (Hewlett-Packard, Palo Alto, CA) gas chromatograph. The chromatograph was fitted with a CPSIL-5 CB (ChromPack, Middelburg, The Netherlands) low-bleed column (30 m × 0.25 mm, 0.25-µm film thickness). Injection was performed in splitless mode. The temperature varied from 80 (15 min) to 280 °C, at a 5 °C‚min-1 rate. Ionization mode was electron impact (70 eV) and the mass/charge range from m/z 50 to 550 was scanned in 2 s. ESI/MS Analysis. ESI/MS was performed on a API 100 instrument (Perkin-Elmer, Norwalk, CT), with the analyte solution infused by means of a Hamilton syringe pump (Hamilton). RESULTS AND DISCUSSION Preparative Electrospray with a Copper Plate Collector. We selected HArNO2 as a probe for electron transfers involved at different stages of the ESI process in negative ionization mode as its electrochemical behavior as well as its photochemical reactivity has been described comprehensively in reducing conditions.12-16 On the other hand, we had observed during previous preparative electrospray experiments with 9-bromo-10(11) Beaugrand, C.; Charbonnier, F.; Rolando, C.; Sablier, M.; Saru, F. Proceedings of the 43th ASMS Conference on Mass Spectroscopy and Allied Topics, Atlanta, May 21-26, 1995; p 668. Details concerning the synchronization of the spray will be described in a forthcoming publication. (12) M’Halla, F.; Pinson, J.; Save´ant, J. M. J. Am. Chem. Soc. 1980, 102, 2, 41204127. (13) Chapman, O. L.; Heckert, D. C.; Reasoner, J. W.; Thackaberry, S. P. J. Am. Chem. Soc. 1966, 88, 5550-5554. (14) Hamanoue, K.; Nakayama, T.; Kajiwara, K.; Yamanaka, S.; Ushida, K. J. Chem. Soc., Faraday Trans. 1992, 88, 3145-3151. (15) Cheng, E.; Sun, T. C.; Su, Y. O. J. Chin. Chem. Soc. 1993, 40, 551-555. (16) Hammerich, O.; Parker, V. D. Acta Chem. Scand. 1981, B35, 341-347.

Figure 2. Schematic overview of the preparative ESI device.

nitroanthracene (BNA) a progressive increase of the conversion yield with increasing dilution in the bulk, reaching a 50% conversion yield for micromolar solutions and a near complete transformation for submicromolar solutions.9 Thus, we used here micromolar solutions for all experiments, expecting in this concentration range conversion yields will be both fair and sensitive to experimental conditions. During a typical experiment, a micromolar methanolic solution of HArNO2 containing trifluoroacetic acid (TFA) or 25% aqueous trimethylamine (TMA) as the pH modifier (10-3 (v/v) in CH3OH), was sprayed onto a copper plate at 35 °C under atmospheric pressure at a flow rate of 1.25 mL‚h-1. Figure 2 shows the three parts of the device, designed for spraying the analyte solution, measuring the instantaneous current, and stabilizing the spray. In its free mode, the spray instantaneous current observed on the oscilloscope appears as a pseudoperiodic phenomenon.10 A typical steady-state pattern shows sharp peaks, of which the periodicity can be measured, allowing one to define a given spraying mode by a mean pulsation frequency of the instantaneous spray current. Since we scanned a very narrow range of concentrations, as close as possible to routine ESI-MS conditions, to correlate analytical results with functioning modes, we had to ensure the stability of spraying modes during several hours. By superimposing to the main HV an ac voltage of which the frequency is chosen close to that of the free stable spraying mode, the spray is assisted and forced to trigger on the ac voltage maximums.11 The resulting current proves perfectly periodic. A typical pattern characterizing the time dependence of the electrospray current in such conditions is shown in Figure 3. In comparison to neutral solutions, the spray was rather difficult to stabilize for both acidic and basic conditions. For acidic solutions, spraying conditions were optimized with a high voltage of ∼5 kV (interelectrode distance, 6 mm) at a pulsation frequency of ∼1 kHz. A pulsation period proceeds in two stages, a sharp peak, followed by a plateau at a much lower intensity (Figure 4), and for the usual high-voltage range (3-5 kV), no current is observed without feeding the capillary. For higher values of the high voltage,

Figure 3. Time dependence of the instantaneous electrospray current for a solution of 9-nitroanthracene (1 µmol‚L-1; TFA 2.5 × 10-4 (v/v) in CH3OH; 3 kV). The synchronization voltage is drawn in thin lines.

the plateau is no longer detected, as peak succeeds peak at high frequency, and this simpler pattern is also observed in the absence of spray (without feeding the capillary). Since, at the highest HV values, corona discharges can be considered as mainly responsible for the electrospray current, we assigned the peak preceding the plateau to a corona discharge, also at lower HV values. The discharges probably involve the solvent molecules (gaseous methanol) at the lowest HV values and, at the highest HV values (“all-corona” mode), the atmospheric components of the interelectrode space (oxygen and nitrogen). On the contrary, the halfperiod at steady intensity is likely to originate in electron transfers occurring between electroactive components of the solution and the metallic surface at the capillary tip. These processes allow recovery of electroneutrality in the liquid phase. The height of a plateau is rather independent of the nature of analyte and additives and we observed almost no change in current, either peak intensities or mean current, while changing the methanolic solution of analyte for “pure” solvent. This general result leads to the conclusion that most redox processes mainly involve the solvent or impurities. On the basis of the easy reduction of methanol into methylate anion and hydrogen, the main part of Analytical Chemistry, Vol. 71, No. 8, April 15, 1999

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Figure 4. Time dependence of the synchronized spray current: enlargement showing the detailed sequence of events during a pulsation period, for the spraying conditions of Figure 3.

the current can be assigned to the solvent electroactivity. Moreover, the resulting methylate anion, as an oxygenated nucleophile, is possibly responsible for initiating the transformation of the anion radical derived from the analyte into varied oxygenated products. Throughout the experiment, the regularity of the spraying mode is controlled by observing and measuring the instantaneous current frequency on the oscilloscope. Immediately after the end of spraying, the residue deposited on the collector is extracted with dichloromethane, filtered, and concentrated under argon. Then the internal standard (9-cyanoanthracene, HArCN) is added before GC/MS analysis is performed. With this collecting device, the major components containing the three condensed rings of anthracene were residual 9-nitroanthracene (molecular peak, m/z 223; retention time, 48.2 min) and 9,10-anthraquinone (molecular peak, m/z 208; retention time, 45.1 min) in approximately equal amounts. 9-H-Anthrone (molecular peak, m/z 194; retention time, 49.4 min) and anthracene (molecular peak, m/z 178; retention time, 41.1 min) were also detected, both in trace amounts. The products were identified by their mass spectra and by comparing the retention times with those of authentic samples. A typical chromatogram consisting of reconstructed ionic currents at m/z 178 (anthracene), 180 (9,10-anthraquinone; fragment ion, M CO•+), 203 (HArCN), and 223 (HArNO2) is shown in Figure 5, for acidic conditions. Comparative results of GC/MS analyses in acidic and basic conditions are reported in Table 1. The overall recovery yield, calculated by comparison with the internal standard (HArCN: molecular peak, m/z 203; retention time, 48.2 min) was ∼3%. Based on the recovered amount of the four components, the global conversion yield of 9-nitroanthracene in acidic (respectively basic) conditions was 35% (respectively 70%), with a selectivity in favor of oxygenated derivatives (9,10-anthraquinone and 9-H-anthrone) of 91% (respectively 94%). The increased conversion yield (oxidation) in basic conditions can be accounted for by proposing for the production of 9,10anthraquinone and 9-H-anthrone SRN-type reactions of the anion radical with oxygenated nucleophilic species, the concentration of which is largely increased in basic conditions. Furthermore, it must be noticed that such processes are catalytic in electrons. On the other hand, the very high selectivity in favor of 9,10anthraquinone even in acidic conditions led us to consider the 1588 Analytical Chemistry, Vol. 71, No. 8, April 15, 1999

Figure 5. GC/MS analysis of the residue from an acidic methanolic solution of 9-nitroanthracene (1 µmol‚L-1; TFA 10-3 (v/v) in CH3OH) collected onto a copper plate.

possible anodic origin of this compound (thus produced at the counter electrode as a result of oxidation processes) as an alternative pathway. Opening the Short Circuit at the Collector. Assuming that some steps could occur at the collector, we designed a new experimental device allowing prevention of further oxidations at this site. The metallic plate was replaced by a two-piece set: a metallic grid with large mesh acting as the counter electrode and a silica plate acting as the collector (Figure 6). The components of the electrospray mist were supposed to be sorted out, ionic components being mainly stopped at the metallic grid and neutral products being mainly carried out through the grid onto the silica surface. On the other hand, silica gel trapping was expected to improve the recovery yield by reducing sublimation of the stored products. Such processes could previously be favored during the 4-h stay of the residue on the metallic collector in the warmed enclosure. In the context of a study of the pH dependence of chargestate distributions of amino acids, strongly acidic conditions have been shown to produce intense [M - H]- ions in negative ESI-MS, in the so-called “wrong-way-round” processes.17 Similarly, on the basis of our previous results (Table 1), we chose TFA as the pH modifier but a lower concentration (2.5 × 10-4 v/v), to favor reduction processes and at the same time to avoid excess corona discharges. In such conditions, the spray was stabilized at a pulsation frequency of 200 Hz for a high voltage of 3 kV. Coupled Copper-Bronze Grid Counter Electrode and Silica Plate Collector. No difference was observed between the two collecting systems concerning the nature of the collected products. Though, with the new device, the overall recovery yield almost doubled (∼6%) and the proportion of reduction product, anthracene, increased by a factor 3 (from ∼10% to ∼30%). The global conversion yield remained around 30-35%. These effects are illustrated in Figure 7, where anthracene is made visible with a magnifying ratio of only 10 whereas a ratio of 100 was necessary in the case of the copper plate collector (Figure 5). A significant percentage of analyte is still transformed into 9,10-anthraquinone, supporting the hypothesis of a dual origin for oxygenated products. Such products would be issued both from electrochemi(17) Mansoori, B. A.; Volmer, D. A.; Boyd, R. K. Rapid Commun. Mass Spectrom. 1997, 11, 1120-1130.

Table 1. Preparative ESI of 9-Nitroanthracene in Methanol

a TFA 10-3 (v/v). b TMA 10-3 (v/v). c Global recovery yield calculated by using 9-cyanoanthracene as the internal standard. d Global conversion yield of 9-nitroanthracene into 9,10-anthraquinone, 9-H-anthrone, and anthracene. e Product ratio calculated from reconstructed ionic currents.

Figure 6. Schematic description of the open short circuit device.

Figure 7. GC/MS analysis of the residue from a methanolic solution of 9-nitroanthracene (CF3COOH 2.5 × 10-4 M (v/v) in CH3OD; 1 µmol‚L-1), collected onto a silica plate through a copper-bronze grid.

cal processes at the counter electrode and from chemical reactions with reactive species formed and transported within the mist. The results of GC/MS analyses, including experiments in deuterated solvents, are reported in Table 2. No incorporation could be detected in CH3OD or in CD3OD. In particular, monodeuterated anthracene (molecular peak, m/z 179; retention time, ∼41.1 min) could not be observed. DISCUSSION As a nitroanthracenic derivative, 9-nitroanthracene exhibits on one side the typical reactivity of nitroaromatic compounds and

on the other side the specific behavior of anthracenic compounds, where the internal cycle has a rather weak aromatic character, favoring oxidation processes at this site.18 Thus, for this substrate, in reducing conditions, in addition to reactions of the anion radical with hydrogen and proton donors or nucleophilic species, leading in particular to SRN reaction products, monomeric or dimeric oxime derivatives have also been identified, giving rise to oxygenated products.15,16 Under classical electrochemical conditions, 9-nitroanthracene reacts at a cathode by a reversible one-electron transfer giving 9-nitroanthracen-10-yl-9-ide anion radical HArNO2•- at a half-wave potential of ∼-1 V (vs SCE), followed by a second irreversible one-electron transfer at a half-wave potential of ∼-1.5 V (vs SCE) leading to the dianion.19,20 In negative ESI ionization only neutral compounds with half-wave potentials more positive than -0.8 V (vs SCE) are observed.21,22 Thus, only the first one-electron transfer at lower potential can be expected to occur in the ESI of 9-nitroanthracene. We analyzed the behavior of 9-nitroanthracene in negative ESI-MS in order to compare detected ions with neutrals from preparative ESI. The detection of the anion radical in ESI-MS would require its transfer with limited decomposition from capillary tip to droplets and within the electrospray mist to the collector. Neither the anion radical HArNO2•- nor any other anionic organic fragment could be detected. Changing methanol for a less reactive solvent such as acetonitrile did not allow us to observe a signal. In the same conditions, the ESI mass spectrum of 9-bromo-10nitroanthracene showed no peak corresponding to an anthracenyl fragment but exhibited an intense bromide ion peak, in agreement with the known reactivity of 9-bromo-10-nitroanthracene anion radical.9 With the separated counter electrode and collector set, the yield in isolated anthracene was largely improved, confirming that 9,10-anthraquinone results at least in part from oxidation taking (18) Sainsbury, M. In Rodd’s Chemistry of Carbon Compounds; A ModernComprehensive Treatise, 2nd ed.; Coffey, S., Ed.; Elsevier: Amsterdam, 1979; Vol. III, Part H, Chapter 28, pp 5-93. (19) Kitagawa, T.; Ichimura, A Bull. Chem. Soc. Jpn. 1973, 46, 3792-3795 (20) Verniette, M.; Pouillen, P.; Martinet, P. Bull. Soc. Chim. Fr. Ser. I 1984, 141-144. (21) Dupont, A.; Gisselbrecht, J.-P.; Leize E.; Wagner, L.; Van Dorsselaer, A. Tetrahedron Lett. 1994, 35, 6083-6086. (22) Van Berkel, G. J. In Electrospray Ionization Mass Spectrometry, Fundamentals, Instrumentation & Applications: The Electrolytic Nature of Electrospray; Cole, R. B., Ed.; John Wiley & Sons: New York, 1997; pp 65-105.

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Table 2. Preparative ESI of 9-Nitroanthracene in Methanol with TFA (2.5 × 10-4 v/v) as the pH Modifier (Pulsation Frequency, 200 Hz)

a Global recovery yield calculated by using 9-cyanoanthracene as the internal standard. b Global conversion yield of 9-nitroanthracene into 9,10anthraquinone, 9-H-anthrone, and anthracene. c Product ratio calculated from reconstructed ionic currents.

Scheme 1. Plausible Mechanism for Reduction of 9-Nitroanthracene into Anthracene in Negative ESI

Figure 8. Rough estimate of ESI parameters.

place at the counter electrode and that this competitive process is efficiently avoided by securing the redox inertness of the collecting surface. On the other hand, the absence of incorporation when deuterated solvents were used (neither in anthracene nor in remaining 9-nitroanthracene) corroborates that 9-nitroanthracene dianion is not formed and that reduction stops after the first one-electron transfer at lower potential, the anion radical being less prone to protonation. Assuming that the electroactive volume inside the capillary is roughly equal to the disturbing volume formed at the capillary tip at each pulsation, we used the spray cone length as an estimate of the electrolysis electrode length. The calculated residence time of the solution during an electroactive period is then ∼50 ms. Assuming a typical value for the speed of droplets, the time of flight of droplets from capillary tip to collector can be estimated at 0.6 ms (Figure 8).23 On the other hand, the average current density can be calculated by assuming that the spray current remains roughly constant during an electroactive period and equal to the steady-state value of the plateau (Figure 4). The estimated value is roughly 0.43 mA‚cm-2, which is in the common range for electrochemical synthesis processes. In condensed phase, the lifetime of the anion radical HArNO2•is greater by at least 1 order of magnitude than the residence time in the electroactive capillary tip.19,20 As the time of flight of droplets to the collector is even shorter, the reversible character of the formation of the anion radical will lead back to 9-nitroan(23) Kozhenkov, V. I.; Fuks, N. A. Russ. Chem. Rev. 1976, 45, 1179-1184. (Translated from Usp. Khim. 1976, 45, 2274-2284).

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thracene by anodic oxidation in the case of solution chemistry. The formation of anthracene implies breakdown of the anion radical HArNO2•- into nitrite anion and anthracen-9-yl radical, which must occur mainly within droplets and not inside the capillary, where the anthracenyl radical would be further reduced into anthracenyl anion and then protonated or deuterated. On the other hand, the formation of antracenyl radical in the gas phase may be driven by the better solvation of the nitrite anion in the methanolic droplets compared to that of the hydrophobic anion radical.24-27 By comparison with other aromatic radicals, the anthracenyl radical is known to have a moderate reactivity in hydrogen abstraction processes.28 Thus, it is supposed to be transported as a gaseous component of the electrospray mist to the silica surface where it abstracts an hydrogen. Finally the results are globally consistent with the following sequence: (i) formation of the anion radical HArNO2•- at the capillary; (ii) breakdown of this intermediate within droplets into nitrite ion in condensed phase and anthracen-9-yl radical HAr•, which desorbs in the gas phase; (iii) hydrogen abstraction by the anthracenyl radical from silica gel at the collector. (Scheme 1). (24) Kebarle, P.; Ho, Y. In Electrospray Ionization Mass Spectrometry, Fundamentals, Instrumentation & Applications: On the Mechanism of Electrospray Mass Spectrometry; Cole, R. B., Ed.; John Wiley & Sons: New York, 1997; pp 3-63. (25) Kebarle, P.; Tang, L. Anal. Chem. 1993, 65, 972A-986A. (26) Iribarne, J. V.; Thomson, B. A. J. Chem. Phys. 1976, 64, 2287-2294. (27) Thomson, B. A.; Iribarne, J. V. J. Chem. Phys. 1979, 71, 4451-4463. (28) Chen, R. H.; Kafafai, S. A.; Stein, S. E. J. Am. Chem. Soc. 1989, 111, 14181423.

Instead of the expected nitro or anthracenyl reduction products of 9-nitroanthracene (hydroxyamino and amino or dihydro derivatives), we observed mainly the elimination of the nitro group. Such nonclassical chemical transformations within the methanolic electrospray mist can be related to novel compound syntheses designed by extension of gas-phase electrical discharge reactions to the liquid phase, which has been described elsewhere. For instance, the formation of perchlorinated fullerene fragments at copper electrodes immersed into liquid chloroform or carbon tetrachloride (10 kV-20 kHz ac voltage, 1 mm interelectrode distance, 20-50 mA arc current) was found surprisingly highly selective.29 CONCLUSION We had previously shown that synchronization allows large durations and secures the reproducibility of results of preparative electrospray experiments, with yields becoming quantitative in the micromolar range, which is 10-100 times lower than the (29) Huang, R.; Huang, W.; Wang, Y.; Tang, Z.; Zheng, L. J. Am. Chem. Soc. 1997, 119, 5954-5955.

routine concentrations of ESI-MS.11 We describe here a new device where the spraying residue is trapped on a silica plate placed behind a metallic grid acting as the counter electrode instead of being collected directly onto a metallic plate. Thus, electron transfers between the collected products and the counter electrode are avoided. With 9-nitroanthracene as the analyte, whereas using the simple metallic plate collector most of the detected products were oxidation derivatives, this new device led to a dramatic increase of the proportion of isolated reduction product. Thus, the chemical inertness of the new trapping surface and the possibility of getting by this means a more straightforward access to the specific redox processes initiated at the capillary during negative electrospray ionization were confirmed. The existence of a redox chemistry even when no ion from the analyte is detected in ESI-MS was also demonstrated. Received for review July 21, 1998. Accepted December 16, 1998. AC980799L

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