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Anal. Chem. 2003, 75, 1620-1627

Electrospray-Atmospheric Sampling Glow Discharge Ionization Source for the Direct Analysis of Liquid Samples Christine N. Dalton and Gary L. Glish*

Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599

Coupling electrospray with atmospheric sampling glow discharge ionization for the direct analysis of liquid-phase samples is demonstrated. Electrospray is utilized for nebulizing and transporting intact sample molecules into the glow discharge where ionization occurs through various pathways, including electron ionization and ionmolecule reactions with reagent ions. Reagent ions are formed through ionization of air molecules in an area of reduced pressure. The effects of discharge current, electrospray voltage, and solution flow rate on the absolute and relative ion intensities observed in the mass spectra are discussed. This technique is applicable to compounds containing various functional groups and encompassing a range of volatility. Analysis of organic compounds with varying volatility and polarity is discussed to illustrate the utility of this ionization technique. Over the past several decades, many approaches have been developed for coupling liquid chromatography to mass spectrometry. For a liquid sample to be analyzed by mass spectrometry, the sample molecules must be transferred into the gas phase, and a large portion of the solvent must be diverted from the mass spectrometer inlet. Interfaces available for liquid chromatography mass spectrometry (LC/MS) can be divided into two groups: interfaces that remove solvent before ionization and those that introduce all of the solvent into the ionization source. Interfaces removing solvent before ionization include the moving wire,1 the moving belt,2 and the particle beam.3-5 Direct liquid introduction (DLI),6-9 atmospheric pressure ionization (API),10-13 atmospheric * Corresponding author. E-mail: [email protected]. (1) Scott, R. P. W.; Scott, C. G.; Munroe, M.; J. Hess, J. J. Chromatogr. 1974, 99, 395-405. (2) McFadden, W. H.; Schwartz, H. L. J. Chromatogr. 1976, 122, 389-396. (3) Willoughby, R. C.; Browner, R. F. Anal. Chem. 1984, 56, 2626-2631. (4) Creaser, C. S.; Stygall, J. W. Analyst 1993, 118, 1467-1480. (5) Gibeau, T. E.; Marcus, R. K. Anal. Chem. 2000, 72, 3833-3840. (6) Talroze, V. L.; Karpov, G. V.; Gorodetskii, I. G.; Skurat, V. E. Russ. J. Phys. Chem. 1968, 42, 1658-1664. (7) Baldwin, M. A.; McLafferty, F. W. Org. Mass Spectrom. 1973, 7, 11111112. (8) Tsuge, S.; Hirata, Y.; Takeuchi, T. Anal. Chem. 1979, 51, 166-169. (9) Apffel, J. A.; Brinkman, U. A. T.; Frei, R. W.; Evers, E. A. I. M. Anal. Chem. 1983, 55, 2280-2284. (10) Horning, E. C.; Horning, M. G.; Carroll, D. I.; Dzidic, I.; N. Stillwell, R. Anal. Chem. 1973, 45, 936-943. (11) Kambara, H. Anal. Chem. 1982, 54, 143-146. (12) Mitchum, R. K.; Korfmacher, W. A. Anal. Chem. 1983, 55, 1485A-1499A. (13) Proctor, C. J.; Todd, J. F. J. Org. Mass Spectrom. 1983, 18, 509-516.

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pressure chemical ionization (APCI),10,13,14 thermospray,15,16 and electrospray ionization (ESI)17-20 are interfaces that introduce the LC effluent into the source without prior enrichment. The moving wire interface was introduced in 1974 to allow LC effluents to be introduced into low-pressure sources, such as electron ionization (EI) and chemical ionization (CI), while chromatographic information is maintained. After the LC effluent is deposited onto a moving wire, the wire moves through a heated transport region toward the ionization source, allowing the solvent to evaporate. After solvent removal, the wire passes through the heated ionization chamber, where the residual analyte is vaporized and ionized via electron ionization or chemical ionization.1 However, only 1% of the sample is carried into the ionization source. Consequently, the moving belt interface was introduced two years later to improve transport efficiency into the mass spectrometer.2 While the operation is similar in the two transport techniques, the moving belt allows almost 100% of the analyte to be carried into the source due to the higher surface area available for effluent deposition. Solvents with high boiling points are not amenable to analysis because the elevated temperature necessary to remove the solvent could evaporate the analyte prior to transport into the ionization source. The elevated temperature could also be detrimental to the analysis of thermally labile analytes. Similarly, the vaporization chamber is heated, which could lead to thermal degradation of the analytes. However, neutral/nonpolar analytes with low volatility have been successfully analyzed via the moving belt interface.21 In the early 1980s, particle beam mass spectrometry (PBMS), originally known as MAGIC-LC/MS (monodisperse aerosol generation interface for combining liquid chromatography with mass spectrometry), was developed. Similar to the moving wire interface, PBMS allows LC effluents to be introduced into low-pressure sources, while chromatographic integrity is maintained.3,5 How(14) vanderHoeven, R. A. M.; Tjaden, U. R.; Greef, J. v. d. Rapid Commun. Mass Spectrom. 1996, 10, 1539-1542. (15) Blakley, C. R.; McAdams, M. J.; Vestal, M. L. J. Chromatogr. 1978, 158, 261-276. (16) Blakley, C. R.; Vestal, M. L. Anal. Chem. 1983, 55, 750-754. (17) Whitehouse, C. M.; Dreyer, R. N.; Yamashita, M.; Fenn, J. B. Anal. Chem. 1985, 57, 675-679. (18) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990, 9, 37-70. (19) VanBerkel, G. J.; McLuckey, S. A.; Glish, G. L. Anal. Chem. 1992, 64, 15861593. (20) Gaskell, S. J. J. Mass Spectrom. 1997, 32, 677-688. (21) Abian, J. J. Mass Spectrom. 1999, 34, 157-168. 10.1021/ac026087j CCC: $25.00

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ever, PBMS is mechanically less complex than moving wire interfaces because the mechanical device (moving wire or belt) is not necessary and the analyte does not have to be desorbed from a wire. The LC mobile phase is dispersed into a fine mist by a nebulizer. As the aerosol passes through a heated two-stage desolvation chamber, the volatile solvent evaporates and gets pumped away, while heavier analyte particles continue traveling in a beam to the exit aperture and enter a heated electron ionization or chemical ionization source.3,4 Neutral/nonpolar compounds can be ionized and detected after LC separation. While the particle beam interface allows complete solvent removal for LC flow rates up to 3 mL/min, analytes must be neutral in solution and have boiling points higher than the temperature used in the desolvation chamber. A particle beam interface also has been used to interface LC to a glow discharge ionization source for atomic emission spectroscopy.22, 23 DLI, was first demonstrated, but flow rates less than 1 µL/ min were employed, limiting chromatographic application.6,21 Subsequently, several DLI techniques were introduced.7-9 Regardless of the solution introduction method (i.e., restricted capillaries, vacuum nebulization, and gas nebulization), ionization occurs in a CI source with the LC effluent serving as the reagent gas. The nebulization interface is heated to 300 °C to assist in solvent evaporation, and the ionization source is heated to 350 °C for analyte vaporization. Similar to the moving belt and PBMS interfaces, increasing the temperature limits the analysis of thermally labile compounds. Maximum mobile-phase flow rates are from 10 to 100 µL/min, restricting DLI use to microbore HPLC. Also, mobile-phase composition is constrained to solvents that can serve as reagent gases for CI. While water can be used as a reagent gas, nebulization of pure water is difficult, and mobile phases with greater than 50% water composition can lead to vacuum system problems. These difficulties with DLI, along with the introduction of other LC/MS interfaces, have diminished the use of DLI.21 API was first introduced in 1973 as an interface for gas chromatography/mass spectrometry (GC/MS)10 to analyze neutral, nonpolar compounds and then was introduced a year later as an interface for LC/MS.24 Operating at atmospheric pressure, the API source consists of a pneumatic nebulizer and a radioactive foil or a corona discharge needle. The heated pneumatic nebulizer is used to vaporize the liquid sample and help transport the sample to the corona discharge needle. API is initiated by either βparticles emitted from the radioactive foil or by an electrical discharge, called a corona discharge, at the tip of a metal needle. Heated carrier gas molecules form reagent ions, which undergo ion-molecule reactions with the analyte to form analyte ions.10,13 Reagent ions can also be formed by doping a reagent gas, such as ammonia, into the carrier gas or by introducing the reagent gas into the system separately. Because the formation of reagent ions is similar to reagent ion formation in chemical ionization, the technique is called atmospheric pressure chemical ionization.13,14 API and APCI have been shown to be sensitive techniques for the detection of various compounds.10-13 Since the publication (22) You, J.; Fanning, J. C.; Marcus, R. K. Anal. Chem. 1994, 66, 3916-3924. (23) You, J.; Patrick, A. D. J.; Marcus, R. K. J. Anal. At. Spectrom. 1996, 11, 483-490. (24) Horning, E. C.; Carroll, D. I.; Dzidic, I.; Haegele, K. D.; Horning, M. G.; Stillwell, R. N. J. Chromatogr. Sci. 1974, 12, 725-729.

of these applications, API has been developed into an LC/MS interface that can handle liquid flow rates up to several milliliters per minute. Unlike thermospray and ESI, API and APCI can ionize neutral, liquid-phase compounds, which vary in volatility and polarity. However, the temperatures used in the nebulizer and source can be detrimental to the analysis of some compounds. Thermospray was developed in the late 1970s to eliminate problems with introduction and vaporization of high liquid flow rates by converting the liquid into a gaseous sample prior to introduction into the mass spectrometer. The liquid is completely vaporized as it passes through a heated tube, resulting in a superheated mist of charged droplets. Clusters of ionized molecules and solvent molecules evaporate from the superheated droplets and diffuse to the mass spectrometer inlet. Although thermospray allows direct sampling from solution with HPLC flow rates up to 1-2 mL/min, nonvolatile compounds are retained more than volatile compounds in the droplets of the mist.15,16 Also, sensitivity for large compounds, such as peptides and saccharides, is worse for thermospray than other techniques.21 ESI was introduced in the mid-1980s as a practical interface between liquid chromatography and mass spectrometry for ionizing highly polar, thermally labile, and nonvolatile compounds. Generally, ESI transfers liquid-phase ionic species into the gas phase. The analyte solution passes through the electrospray needle into a high electric field generated by a potential difference between the needle and the mass spectrometer inlet. A fine spray of charged droplets forms and enters the mass spectrometer.17,20 Compounds with basic sites can readily accept protons or cations in solution for analysis in positive-ion mode, while acidic compounds are often detected as deprotonated molecules in negativeion mode. Neutral, nonpolar compounds are not usually ionized via ESI.18,19 Atmospheric sampling glow discharge ionization (ASGDI),25-28 introduced in the late 1980s, is an ionization technique similar to APCI and has potential to be an interface for solution introduction into the mass spectrometer. Analyte molecules in the atmosphere are sampled through the inlet aperture and ionized in the glow discharge region. Before ionization of analyte molecules can occur, reagent ions must be formed from the components of air, the discharge support gas. Initially, ionization of air molecules occurs through electron ionization, generating secondary electrons that can ionize other molecules present in the glow discharge region. Subsequently, formation of reagent ions (O2•+, NO+, NO2+, H3O+) occurs through charge-exchange and proton-transfer reactions. Ionization of analyte molecules occurs through various pathways, including election ionization, electron capture, and ion-molecule reactions with the reagent ions.25,28 Although ion formation results from ion-molecule reactions in both ASGDI and APCI, the reagent ions in ASGDI are directly formed from untreated air, while APCI reagent ions are usually formed from an added carrier gas or solvent.10 There are several differences between APCI and ASGDI that have an effect on the observed spectra. Both techniques use a (25) McLuckey, S. A.; Glish, G. L.; Asano, K. G.; Grant, B. C. Anal. Chem. 1988, 60, 2220-2227. (26) Asano, K. G.; McLuckey, S. A.; Glish, G. L. Spectrosc. Int. J. 1990, 6, 191210. (27) VanBerkel, G. J.; Glish, G. L.; McLuckey, S. A. Anal. Chem. 1990, 62, 12841295. (28) Bogaerts, A.; Gijbels, R. Anal. Chem. 1997, 69, 719A-727A.

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discharge for ionization but in different current regimes. A corona discharge is used in APCI, while a glow discharge is used in ASGDI. In APCI, the discharge current is in the microampere range, whereas the discharge current is in the milliampere range with ASGDI. In addition, ASGDI operates at reduced pressure (0.5-1 Torr) and results in a lower analyte number density than is available with APCI, which is operated at atmospheric pressure. The lower analyte number density of ASGDI is offset by more efficient extraction of ions into the mass spectrometer, reducing the difference in sensitivity between the two techniques.26 Another operational difference between the two techniques is that the APCI source is normally heated to 250-500 °C,10,21,29 while the ASGDI source is at ambient temperature.25 The elevated temperature in the APCI source can have a deleterious effect on the analysis of thermally unstable compounds. Also, compounds with low boiling points could be volatilized and pumped away before ionization and extraction into the analyzer. APCI is also prone to chemical interferences, such as adduct formation between the matrix component and the analyte. The matrix component can also compete for charge, resulting in decreased ionization efficiency for the analyte of interest. These matrix effects are observed in APCI because the most thermodynamically stable ions form as a result of the large number of ion-molecule collisions occurring at atmospheric pressure. Chemical interferences can also manifest in the form of cluster ions (i.e., analyte molecules clustered with water, solvent molecules, or another analyte molecule), which can decrease instrument sensitivity due to the analyte signal being divided among several ions.25,26 Cluster ion formation occurs when ions are cooled during expansion from an area of high pressure to an area of low pressure. To reduce cluster formation in APCI, instruments have been equipped with an intermediate-pressure region, where collisions between the cluster ions and the background gas can occur to destabilize the cluster ions.14,30,31 With ASGDI, ions are formed at lower pressure, where reduced collision frequency suppresses ion-molecule reactions that could form cluster ions. Expansion from the intermediate pressure area of ASGDI into the low-pressure region of the analyzer does not cause cluster formation due to lower neutral number densities in the ASGDI source.26,32 Reduced pressure in the ASGDI source also results in the ionization being under kinetic control. As a result, chemical (matrix) interferences are substantially diminished. Although ASGDI mass spectra show few peaks related to chemical interferences, ions of interest can fragment in the ASGDI source prior to extraction into the mass spectrometer, causing mass spectra to possibly be complex. Upon ionization, the molecule’s internal energy can be increased above the critical energy necessary to observe fragmentation. Because ionization occurs at reduced pressure, collisions between the background gas and the analyte ions remove some, but not all, of the excess internal energy. Consequently, the ion’s internal energy remains above the critical energy threshold, and fragmentation is observed. By increasing the source pressure, ion internal energies can be reduced, decreasing the amount of fragmentation observed.25 (29) Niessen, W. M. A. J. Chromatogr., A 1998, 794, 407-435. (30) Kambara, H.; Kanomata, I. Anal. Chem. 1977, 49, 270-275. (31) Kambara, H.; Mitsui, Y.; Kanomata, I. Anal. Chem. 1979, 51, 1447-1452. (32) Chambers, D. M.; McLuckey, S. A.; Glish, G. L. Anal. Chem. 1993, 65, 778-783.

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ASGDI spectra for chlorinated hydrocarbons show fragmentation patterns similar to those observed in electron ionization spectra.14,26 Thus, ASGDI can form structurally informative ions for some compounds to allow compound identification, while exhibiting less susceptibility to chemical interferences. Utilizing these advantages, ASGDI has been shown to be a sensitive method for detecting trace organics in air.25 Although there are many advantages to using ASGDI, application of this ionization technique has mainly been for the analysis of contaminants in ambient air. This paper describes coupling ES to ASGDI for the analysis of liquid-phase samples. In ES-ASGDI, the electrospray process serves as the interface between liquid-phase introduction and the mass spectrometer by nebulizing and transporting sample molecules into the ASGDI source. Other interfaces can be coupled to the glow discharge source such as the particle beam interface for liquid-phase sample introduction.22,23 As explained previously, the particle beam system uses a pneumatic nebulizer to generate a fine aerosol of the analyte and solvent. The aerosol passes through a heated (30-200 °C) desolvation chamber and then a two-stage momentum separator. This process is used to remove the solvent prior to the analyte entering the ion source. While the particle beam efficiently desolvates the analyte prior to ionization, the particle beam interface is more complex than ES as an interface between liquid-phase introduction and ASGDI. ES is easily interfaced with the ASGDI source and can be interfaced quite easily to liquid chromatography, capillary electrophoresis, or flow injection analysis for solution-phase introduction. Additionally, the particle beam interface is operated at elevated temperatures, which can lead to thermal degradation or volatilization of low boiling point compounds prior to entering the ion source. This is in contrast to the ES-ASGDI interface, which is operated at ambient temperature. Several characteristics of ES-ASGDI include fast response, generation of positive or negative ions, low maintenance requirements, and high sensitivity. The purpose of this paper is to describe the operation and characteristics of the ES-ASGDI system and its application to the analysis of compounds containing various functional groups and encompassing a range of volatility. Operational conditions are being investigated to develop a sensitive method of detection for analysis of trace organic compounds present in drinking water. The effects of several parameters, including the discharge current, electrospray voltage, and flow rate on the relative and absolute ion intensities observed in the mass spectra, are discussed. For illustration, the analytical responses for the analysis of several compounds with different functional groups are presented. EXPERIMENTAL SECTION Instrumentation. The ES-ASGDI source is composed of a custom-built ES source coupled to a custom-built ASGDI source. Figure 1 shows a side view of the ES-ASGDI source interfaced with a quadrupole ion trap mass spectrometer. Two methods are utilized for conventional ES. The first method uses the nanoES setup built in our laboratory. A glass capillary (0.169-cm o.d., 0.135cm i.d.; Drummond Scientific, Broomall, PA) pulled to a fine tip (5-12-µm i.d.) serves as the ES needle. Voltages of +600 to +1500 V are applied to a wire in contact with the sample solution in the glass needle to initiate the electrospray process. The voltage difference between the ES needle and the ASGDI source inlet (A1 in Figure 1) sustains the electrospray. Although no pump is

Figure 1. Side view of the custom nanoelectrospray source coupled to a custom atmospheric sampling glow discharge ionization source. Aperture plates A1 and A2 are attached to the flanges with PEEK screws and metal screws, respectively, and each plate is sealed with O-rings. The high voltage is applied to A1 to initiate the glow discharge, which occurs in the region between A1 and A2.

connected to the ES needle, the flow rate is in the conventional ES range (1-20 µL/min) when the apparatus is used for ES-ASGDI, because the potential difference between the ES needle and the mass spectrometer inlet (A1) is 1100-1800 V. This is in contrast to when the same setup is used for nanoESI, and the potential difference between the ESI needle and A1 is only 600-800 V, resulting in nanoliter per minute flow rates. By changing the inner diameter of the ES needle tip, the flow rate can be varied for ES-ASGDI. The second ES method is the conventional ES technique, which uses a constant flow of solution supplied via a syringe pump to the ES needle. In this mode, a Harvard Apparatus model 22 syringe pump (Cambridge, MA) connected through PEEK tubing to a metal needle (26 gauge, 0.47-mm o.d., 0.11-mm i.d., 70 mm long; Scientific Instrument Services, Ringoes, NJ) is used for sample delivery into the glow discharge. Voltages of +1500 to +3500 V are applied directly to the metal needle for conventional ES. The ASGDI source is similar to that previously reported.25 As shown in Figure 1, ASGDI occurs in the source at a pressure of ∼0.3 Torr. The discharge is sustained between two stainless steel plates that are separated by 3.3 cm. A potential of -350 to -450 V is applied to the first plate, which contains a 100-µm-diameter aperture (A1). The second plate, with a 500-µm-diameter aperture (A2), is maintained at ground potential for ion extraction into the analyzer region. The range of voltages applied to the first plate results in a current in the range of 3-12.5 mA, the maximum current output of the power supply (Department of Chemistry Electronics Facility, The University of North Carolina). The discharge is self-sustained between these two plates (A1 and A2) with a continuous flow of air at 1.4 mL/s entering the source to serve as the discharge gas. All ES-ASGDI experiments were performed on a customized Finnigan (San Jose, CA) quadrupole ion trap mass spectrometer (ITMS) controlled by ICMS Ion Trap Software Version 2.20.33 The base pressure in the ion trap region is 3.0 × 10-5 Torr. Helium bath gas is subsequently added to obtain an operating pressure of ∼1 mTorr. In routine analysis, ions are allowed to accumulate in the ion trap volume for 300 µs-300 ms. Shorter accumulation (33) Yates, N.; Yost, R., 2.20 ed.; unpublished; University of Florida, Gainesville, FL, 1992.

times (50 ms) are used for semivolatile compounds. Then, the ions of interest are ejected from the quadrupole ion trap for detection. During the ion accumulation period, a broadband frequency waveform generated via stored waveform inverse Fourier transform (SWIFT)34-36 is applied to remove the reagent ions from the ion trap to reduce space charge effects.37,38 SWIFT waveforms are generated with a program written in Labview (National Instruments, Austin, TX) and transferred to a 40-MHz arbitrary waveform generator card (NI 5411 for PCI, National Instruments, Austin, TX) through a second program written in Labview. The trigger to initiate the waveform is provided by the ITMS electronics. The 1-V waveforms pass through an amplifier with a gain of 4.5 and are applied 180° out of phase to the end cap electrodes. Chemicals and Sample Preparation. Solutions of n-butylbenzene (Aldrich, Milwaukee, WI), 2,3-butanedione (Acros Organics, Fair Lawn, NJ), 2-chloroacetaldehyde (Aldrich), and 2,2dichloroacetamide (Aldrich) were prepared at various concentrations in HPLC-grade methanol (Fisher Scientific, Pittsburgh, PA), 50/ 50 (v/v) HPLC-grade methanol/water mixture (Fisher), or 100% HPLC-grade water. Solutions with a concentration of 0.1 mg/mL were used to obtain the data for characterization of the operating parameters. To obtain calibration curves, 5-µL injections of solutions ranging in concentration (0.04-280 µg/mL for semivolatiles; 0.07-100 µg/mL for volatiles) were sequentially injected into the glass needle and analyzed via ES-ASGDI. Mass spectra and chronogram data (analyte ion intensity plotted over a period of time) were acquired. Calibration curves were calculated using two methods. In method A, the areas for the peaks in the chronograms were calculated in Origin 5.0 software (Microcal Software, Northampton, MA) and plotted versus the quantity injected. With method B, the analyte ion intensity in the mass spectrum was plotted versus the quantity injected. Safety. The voltages used on the front plate of the source region result in a current in the milliampere range, which can result in injury if the front plate is touched. Likewise, the voltages used for the electrospray are also a shock hazard and can cause injury if the ES needle is touched during operation. The analytes chosen for quantification are potential carcinogens, and solutions of these analytes should be prepared in a hood. RESULTS AND DISCUSSION Characteristics of Mass Spectra. ES-ASGDI can ionize many compounds that have varying functionality and volatility and are not ionized by ES. Most compounds that are ions in solution enter the ASGDI source and are neutralized through charge transfer with another molecule or ion, or at the first aperture (cathode). However, some compounds, such as caffeine, that are ionized via ESI also give the same ion in the ES-ASGDI source. It is not possible to determine whether the ions generated by ESI remain (34) Marshall, A. G.; Wang, T.-C. L.; Ricca, T. L. J. Am. Chem. Soc. 1979, 51; 1985, 107, 7893-7897. (35) Guan, S.; Marshall, A. G. Int. J. Mass Spectrom, Ion Phys. 1996, 157/158, 5-37. (36) Asam, M. R.; Ray, K. L.; Glish, G. L. Anal. Chem. 1998, 70, 1831-1837. (37) McLuckey, S. A.; Goeringer, D. E.; Glish, G. L. Anal. Chem. 1992, 64, 14551460. (38) Williams, J. D.; Cooks, R. G. Rapid Commun. Mass Spectrom. 1993, 7, 380382.

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Figure 2. ES-ASGDI mass spectra for (a) n-butylbenzene, an example volatile compound, in methanol; (b) 2,3-butanedione, an example semivolatile compound, in water; and (c) air, showing the reagent ions. Ion accumulation time: (a) 300 µs; (b) 500 µs; (c) 1.5 ms.

ions or are neutralized and subsequently ionized in the ASGDI source. Compounds with high volatility, such as n-butylbenzene, acetone, and methanol, are easily ionized using ES-ASGDI, and compounds with aldehyde, ketone, ether, amide, halogen, and aromatic hydrocarbon functionality have been analyzed via ESASGDI. While some of these compounds contain moieties that should be amenable to protonation, ionization does not occur with ES. The mass spectra for n-butylbenzene (example of a volatile compound) and 2,3-butanedione (semivolatile compound) are shown in Figure 2a and b, respectively, demonstrating the ability to generate ES-ASGDI mass spectra for both volatile and semivolatile compounds. While n-butylbenzene can be analyzed via GC/MS, the remaining compounds under study are not amenable to GC/MS analysis without derivatization prior to analysis. 1624 Analytical Chemistry, Vol. 75, No. 7, April 1, 2003

Additionally, the compounds under study are pollutants found in drinking water, and direct analysis of these compounds in water is not possible using GC/MS. Direct analysis of water samples is possible using LC/MS, and ES-ASGDI is a potential interface for analysis of liquid-phase samples. A typical mass spectrum of the reagent ions is shown in Figure 2c. To trap all ions of interest, the ion trap is operated such that ions less than m/z 50 are ejected during ion accumulation to remove the low-mass reagent ions. This is shown in Figure 2a and b, where ions with m/z less than 50 are absent from the spectra. There is a maximum number of ions that can be stored within the ion trapping volume before the charges repel each other, causing ions to be prematurely ejected from the ion trap. This phenomenon, called space charging, can be detrimental to ion trap performance.39-42 To prevent space charging and allow ions of interest to be preferentially trapped, the reagent ions must be ejected from the ion trap during ion accumulation.43 While space charging is a problem in the ion trap, it usually does not occur in beam-type instruments, like the quadrupole mass analyzer. While the presence of reagent ions can be a problem with the ion trap, the use of a broadband frequency waveform generated via SWIFT34-36 to remove reagent ions during ion accumulation allows preferential trapping of the analyte ion of interest. If the reagent ions are not removed during ion accumulation, the absolute ion intensities for the n-butylbenzene fragment ions (m/z 91 and 92) are ∼2% of the reagent ion (O2•+) intensity (data not shown). Because fragmentation can be a prominent process in the glow discharge, structural information can be obtained in addition to the molecular weight information for certain compounds. In Figure 2a, the n-butylbenzene molecular ion at m/z 134 is observed, while the two largest peaks at m/z 91 and 92 correspond to the two predominant fragment ions observed with electron ionization. As shown in this spectrum, ES-ASGDI is capable of generating spectra, which are similar to electron ionization spectra and could be library searchable.26 While ES-ASGDI generates EI-like spectra for many small organic compounds, several of the compounds analyzed do not give EI-like spectra, as shown in the spectrum for 2,3-butanedione in Figure 2b. Compounds containing aldehyde, ketone, and amide moieties analyzed with ES-ASGDI form predominantly [M + H]+ ions instead of molecular ions (M•+) and fragment ions. Although structural information is not obtained, molecular weight information is retained. If structural information is desired, tandem mass spectrometry (MS/MS and MSn) can be performed to elucidate compound structure. Another characteristic of ES-ASGDI shown in Figure 2 is the ability to use different solvent systems, enabling coupling of ES-ASGDI to LC. For Figure 2a, the n-butylbenzene solution was prepared in methanol, while the 2,3-butanedione solution was prepared in water for the spectrum in Figure 2b. Other solvents that have been used in ES-ASGDI analysis include ethanol, butanol, acetonitrile, tetrahydrofuran, N,N′-dimethylformamide, chloroform, dichloromethane, (39) Vedel, F.; Andre´, J. Phys. Rev. A 1984, 29, 2098-2101. (40) Vedel, F. Int. J. Mass Spectrom. Ion Processes 1991, 106, 33-61. (41) Guan, S.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1994, 5, 64-71. (42) Cox, K. A.; Cleven, C. D.; Cooks, R. G. Int. J. Mass Spectrom, Ion Phys. 1995, 144, 47-65. (43) McLuckey, S. A.; Goeringer, D. E.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1991, 2, 11-21.

and acetone. The main criteria for solvent choice are the ability to undergo ES and analyte solubility. As a result, there is a lot of flexibility in solvent choice. So, a solvent can be chosen that will not interfere with analysis of the analyte. Operating Parameters. The discharge current, source pressure, and discharge voltage are the operating parameters that control the performance of the glow discharge.44 For ES-ASGDI, the ES voltage and the solution flow rate are additional parameters that can affect the performance of the glow discharge. These parameters are interdependent and must be optimized to achieve the best results. Throughout the experiments discussed here, the source pressure was held constant at 0.3 Torr, and the discharge voltage to initiate the glow discharge was the dependent variable. The current, ES voltage, and flow rate were varied in the initial experiments to determine the effect of each parameter on the signal. The discharge current determines the number of electrons available in the source region to ionize air molecules, which affects the number of reagent ions available to ionize sample molecules. The ES voltage affects the stability of the electrospray plume, and the flow rate affects the amount of material (solvent and analyte) introduced and thus the stability of the plasma. Each parameter is discussed below in detail. Because the discharge current controls the electron density in the source, an increase in the discharge current should give a corresponding increase in ion intensity. An incremental increase of the discharge current from 2.0 to 12.5 mA (maximum current output of the discharge power supply) produced a linear increase in the reagent ion intensities as shown in Figure 3a. The effect of the discharge current on the 2,3-butanedione signal is shown in Figure 3b and c (squares) and is representative for the compounds studied. When conventional ES is used, an increase in the ion intensity of 2,3-butanedione is observed as expected with a corresponding increase in the discharge current (Figure 3b). Likewise, if the nanoES apparatus is used and the measured flow rate is 5 µL/min or greater, an increase in ionization is observed similar to that shown in Figure 3b (data not shown). If the inner diameter of the needle tip used for the nanoES setup is decreased to give a flow rate less than 5 µL/min, an initial increase in ion intensity is observed with increasing discharge current as shown in Figure 3c (squares). However, the ion intensity decreases above 9 mA of current. The total ion intensity decreases along with the protonated molecule intensity. The inconsistency between reagent ion behavior and analyte ion behavior is due to a change in the flow rate from the electrospray process. The flow rate when the nanoES setup is used appears to be related to the discharge current as well as the needle aperture size. To increase the discharge current, the voltage on the source inlet (A1 in Figure 1) becomes more negative, causing the potential difference between the needle and the source inlet to increase. The effect of increasing discharge current on the flow rate with ES needles of varying diameter is shown in Table 1. The flow rate decreases with increasing discharge current when ES needles with a diameter of less than 8 µm are used, corresponding to flow rates of 5 µL/min or less. The flow rate decreases when the discharge current is above 6 mA for ES needle A (5-µm i.d.) and above 8 mA for ES needle B (6-µm i.d.). Because the flow rate decreases when the discharge current is above 8 mA (for 6-µm(44) Marcus, R. K. Glow Discharge Spectroscopies; Plenum Press: New York, 1993.

Figure 3. Effect of discharge current on the absolute intensity of (a) air; (b) combined effect of discharge current and flow rate on 2,3butanedione; (c) effect of discharge current on the absolute intensity of 2,3-butanedione. In (a), the closed square (9) represents H3O+, the open circle (O) N2•+, the closed triangle (2) NO+, the open triangle (4) O2•+, and the star (*) NO2+. In (c), the closed square (9) represents varying the voltage difference between the ES needle and A1, and the closed triangle (2) represents a constant voltage difference between the ES needle and A1.

i.d. ES needles), the ion intensity in turn decreases, as shown in Figure 3c (squares). The data for ES needles C and D (8- and 12-µm i.d., respectively) in Table 1 indicate that the flow rate does not decrease with increasing discharge current when needle apertures larger than 8 µm are used. If only the electrospray voltage is changed (Figure 4b; discharge current, 4 mA), the flow rate and ion intensity do not decrease with increasing discharge current, because only the potential difference changes, not both the potential difference and discharge current. Likewise, if the potential difference between the electrospray needle and A1 is held constant while the discharge current increases, the flow rate and thus ion intensity continually increases (Figure 3c, triangles). When conventional ES with flow rates of 5 µL/min or greater is used, a decrease in ion intensity is not observed, because the flow Analytical Chemistry, Vol. 75, No. 7, April 1, 2003

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Table 1. Change in Flow Rate with Increasing Discharge Current current (mA)

A

2 4 6 8 10 12

1.7 2.4 3.0 2.5 2.0 1.7

flow rate (µL/min)a B C 1.3 2.9 3.6 5.0 4.4 4.2

3.5 7.6 8.6 8.6 8.6 10.0

D 20.0 20.0 20.0 20.0 23.0 23.0

a Inner diameters of needles A-D are 5, 6, 8, and 12 µm, respectively.

Figure 4. Absolute intensity of 2,3-butanedione with (a) varying flow rate and (b) varying electrospray voltage but keeping a constant discharge current (4 mA). The closed square (9) represents [M + H]+.

is held constant by the syringe pump. Because the flow rate decreases at higher discharge currents when ES needles with an inner diameter less than 8 µm were used, the discharge current was maintained at 4-5 mA and ES needles with an inner diameter of 8 µm or greater were used for evaluating the analytical characteristics of the system. Increasing the flow rate increases the number of molecules transported into the glow discharge. The effect of the solution flow rate on the intensity of 2,3-butanedione when conventional ES was used is shown in Figure 4a and can also be observed in Figure 3b. In Figure 4a, an increase in the flow rate from 5 to 100 µL/min produces a corresponding increase in the ion intensity. Solution flow rates of 80 µL/min or higher resulted in discharge instability and a decrease in the precision of measuring the analyte ion intensity. Therefore, to obtain maximum intensity while 1626

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precision and discharge stability are maintained, the flow rate should be less than 80 µL/min. Flow rates greater than 20 µL/ min are difficult to achieve with the nanoES apparatus. As a result, a flow rate of 10 µL/min with the nanoES apparatus was used for further experimentation as a compromise between ease of use and maximum signal. The effect of ES voltage on the ion intensity for 2,3-butanedione while a constant discharge current was maintained is shown in Figure 4b. The ion intensity was evaluated over the range of +500 to +1500 V applied to the ES needle. The voltage on A1 was decreased as the ES voltage increased to maintain the discharge current at 4 mA, resulting in a voltage difference between the ES needle and the sampling aperture (A1 in Figure 1) of 870-1845 V. Due to the nature of the electrospray process, 870 V was the minimum voltage difference needed to initiate the electrospray when the discharge current was maintained at 4 mA. Voltage differences greater than 2800 V caused a corona discharge between the ES needle and the ASGDI source inlet (A1 in Figure 1). As a result, voltage differences greater than 2000 V were not used for any of these experiments to ensure no interference from a corona discharge. The ion intensity for the protonated molecule of 2,3-butanedione became constant with a voltage difference greater than 1550 V. At higher voltage differences, the ES continues to provide more analyte molecules to the glow discharge, but the maximum ionization has been reached. Without increasing the discharge current, more ionizing electrons are not available to ionize the excess analyte, resulting in constant analyte ion intensity above 1550 V. These trends were representative for all compounds used in this study. To optimize analytical response, the electrospray voltage for the remaining studies was chosen to maximize the intensity of the molecular ion or protonated molecule. Application to Liquid-Phase Sample Analysis. When operational parameters for ES-ASGDI are chosen, a compromise must be made between maximum signal intensity and the discharge stability. A discharge current of 4 mA was utilized with the optimal electrospray conditions (sample dependent) to investigate the analytical response of the ES-ASGDI system for several compounds, as shown below. Two methods were employed to quantitate the compounds under study to determine the figures of merit for ES-ASGDI. With quantitation method A, 5-µL aliquots of sample solutions ranging from 40 ppb to 200 ppm were infused using the nanoES setup, and the analyte chronograms were plotted. The chronogram for a 5-µL injection of n-butylbenzene is presented in Figure 5 and is representative of injections for all compounds studied. The signal for the n-butylbenzene rearrangement fragment ion C7H8•+ (m/z 92) is plotted with respect to time, while the protonated molecule signal is plotted versus time for the remaining compounds. To obtain quantitative information from the chronograms, the area under the main peak in the chronogram was calculated. The peak areas were then plotted versus concentration to obtain an analytical response curve. In quantitation method B, a mass spectrum was acquired for each sample used in quantitation method A. The analyte intensity was obtained from the mass spectrum for the same m/z values as used in quantitation method A. The ion intensities or peak areas for triplicate analyses of each sample

Table 2. Analytical Response Data for Various Compounds Analyzed via ES-ASGDI peak height

peak area

compound

equation

R

equation

R

LOD (ppb)

n-butylbenzene 2,3-butanedione 2-chloroacetaldehyde 2,2-dichloroacetamide

y ) 850x - 19.7 y ) 5.49x + 351 y ) 290x + 4990 y ) 146x+279

0.9998 0.9918 0.9859 0.9991

y ) 776x + 1381 y ) 40.6x + 8110 y ) 261x + 7335 y ) 2582x-4554

0.9996 0.9977 0.9822 0.9934

32 119 26 130

drinking water, and detection limits of ∼1 ppb are needed for obtaining health risk assessment data for these compounds. Some possible approaches to improve the detection limit are to increase the temperature of the source and analyzer regions and to preconcentrate the drinking water sample prior to ES-ASGDI analysis. Increasing the temperature of the source and analyzer regions will allow more desolvation of the analyte molecules, increasing analyte ionization. Solid-phase extraction can be used to preconcentrate the water sample prior to instrumental analysis, and a 100×-1000× concentration of the analyte will allow the required detection limits to be possible. Figure 5. Representative chronogram for 5-µL injection of 0.252 µg/mL n-butylbenzene.

solution were averaged and plotted versus concentration to obtain analytical response curves. Table 2 summarizes the analytical response curves obtained from linear least-squares fit of chronogram peak areas and mass spectral peak intensities. While there is little difference in the R values for the two quantitation methods, method B (mass spectral peak intensity) is the preferred method due to better precision, shorter instrumental analysis time, and less complex data analysis. All of the compounds have limits of quantitation in the low-ppb range (72-175 ppb), except 2,2-dichloroacetamide, which has a limit of quantitation of 2 ppm. Compounds with higher volatility (n-butylbenzene and 2-chloroacetaldehyde in this study) have lower limits of quantitation. 2,3-Butanedione and 2,2-dichloroacetamide have linear ranges of 4 and 2 orders of magnitude, respectively, with upper limits of 200 ppm. 2-Chloroacetaldehyde and n-butylbenzene have linear ranges of 3 orders of magnitude and exhibit upper limits of 100 ppm. As observed in the analytical response curves, the ionization sensitivity is higher for 2-chloroacetaldehyde and n-butylbenzene, causing the discharge to become saturated at a lower concentration than with the other two compounds. Limits of detection (LOD ) 3sblank/m) for the compounds studied range from 26 to 130 ppb (Table 2), with the volatile compounds having lower detection limits than the semivolatile compounds. The data for n-butylbenzene and 2-chloroacetadlehyde demonstrate that analyte loss during transfer of the analyte from ES to the ASGDI source is not significant for volatile compounds. The compounds under study are pollutants found in

CONCLUSION Analysis of liquid-phase samples using ES coupled to ASGDI has been demonstrated. ES is utilized for nebulizing and transporting intact sample molecules into the glow discharge where ionization occurs. While other forms of sample nebulization may be coupled with ASGDI, ES was used due to ease of operation and coupling to HPLC. ES-ASGDI can be advantageous in providing structural information in addition to molecular weight information. Several operational parameters, specifically the discharge current, ES voltage, and flow rate, are shown to be an integral part of optimization of ES-ASGDI. An investigation is currently underway to determine the optimal conditions for several groups of compounds. Although not demonstrated here, ESASGDI can be coupled to an HPLC to extend ES-ASGDI to the analysis of complex mixtures. ES-ASGDI is also a viable technique for the quantitation of various types of compounds. The detection limits ranged from the nanogram to picogram level for the compounds studied here. More studies are underway to improve the detection limits and extend analysis to more classes of compounds. ACKNOWLEDGMENT C.N.D. was funded through the U.S. Environmental Protection Agency’s Science to Achieve Results (STAR) program (Fellowship U-91579501-1).

Received for review August 29, 2002. Accepted January 16, 2003. AC026087J

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