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Dielectric Barrier Discharge Ionization for Liquid Chromatography/Mass Spectrometry Heiko Hayen,* Antje Michels, and Joachim Franzke ISAS-Institute for Analytical Sciences, Bunsen-Kirchhoff-Strasse 11, 44139 Dortmund, Germany An atmospheric pressure microplasma ionization source based on a dielectric barrier discharge with a helium plasma cone outside the electrode region has been developed for liquid chromatography/mass spectrometry (LC/MS). For this purpose, the plasma was realized in a commercial atmospheric pressure ionization source. Dielectric barrier discharge ionization (DBDI) was compared to conventional electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI) in the positive ionization mode. Therefore, a heterogeneous compound library was investigated that covered polar compounds such as amino acids, water-soluble vitamins, and nonpolar compounds like polycyclic aromatic hydrocarbons and functionalized hydrocarbons. It turned out that DBDI can be regarded as a soft ionization technique characterized by only minor fragmentation similar to APCI. Mainly protonated molecules were detected. Additionally, molecular ions were observed for polycyclic aromatic hydrocarbons and derivatives thereof. During DBDI, adduct formation with acetonitrile occurred. For aromatic compounds, addition of one to four oxygen atoms and to a smaller extend one nitrogen and oxygen was observed which delivered insight into the complexity of the ionization prossesses. In general, compounds covering a wider range of polarities can be ionized by DBDI than by ESI. Furthermore, limits of detection compared to APCI are in most cases equal or even better. The coupling of liquid chromatography and mass spectrometry (LC/MS) has been established as one of the most powerful tools in analytical chemistry and has resulted in important advances, especially in biomedical and biochemical research. This is the result of extensive basic research on atmospheric pressure ionization (API) techniques, which today offer a robust way to couple LC to MS. The main API techniques are electrospray ionization (ESI),1,2 atmospheric pressure chemical ionization (APCI),3,4 and less frequently applied atmospheric pressure * To whom correspondence should be addressed. E-mail: hayen@ isas.de. Fax: +49-231-1392120. (1) Dole, M.; Hines, R. L.; Mack, L. L.; Mobley, R. C.; Ferguson, L. D.; Alice, M. B. J. Chem. Phys. 1968, 49, 2240–2249. (2) Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 4451–4459. (3) Horning, E. C.; Horning, M. G.; Carroll, D. I.; Dzidic, I.; Stillwell, R. N. Anal. Chem. 1973, 45, 936–943. (4) Horning, E. C.; Carroll, D. I.; Dzidic, I.; Haegele, M. G.; Horning, M. G.; Stillwell, R. N. J. Chromatogr. Sci. 1974, 12, 725–729. 10.1021/ac902176k 2009 American Chemical Society Published on Web 11/13/2009
photoionization (APPI).5,6 ESI, APCI, and APPI differ in their ionization process and their applicability.7 ESI revolutionalized biochemical research by offering a highly sensitive method for the analysis of large biomolecules.8 ESI has been widely used also for smaller polar organic molecules, and it is the most widely used API technique today. The ionization in an ESI interface is considered primarily a liquid-phase ionization technique: preformed ions in solution are desorbed or evaporated to the gas phase and can subsequently be mass analyzed. The ionization efficiency tends to be poor for more nonpolar compounds. For these, APCI and APPI are more suitable. The ionization conditions in APCI are considered to be somewhat “harder” than those in ESI.9 The ionization in APCI is understood to be primarily based on gas-phase ion-molecule reactions between analyte molecules and a solvent-based reagent gas, generated by a series of ion-molecule reactions initiated by electrons from the corona discharge needle. Owing to the short free pathway of ions at atmospheric pressure, ion-molecule reactions in the gas phase play an important role during the ionization process. The ions undergo several collisions before reaching the mass analyzer. The HPLC solvent is used as reactant gas and serves for the chemical ionization of the analyte molecule. As an alternative ionization technique to APCI-MS for nonpolar compounds, APPI has been introduced.5,6 The APPI interface can be considered as a modified APCI source, with the corona discharge being replaced by a gas discharge lamp except that other ionization mechanisms will take place. In case of APPI, the analytes will be ionized by resonant light excitation. Most often, a krypton discharge lamp, which emits 10.03 eV (1s4, 80 912.561 cm-1, 123.590 nm) and 10.64 eV (1s2, 85 847.501 cm-1, 116.486 nm) photons, is used. The compounds that can be directly ionized by the photons must possess ionization energies below 10.03 eV (or 10.64 eV). As the common LC solvents are characterized by high first ionization potentials, selective ionization of the analytes may occur.5 In APCI, the initial ionization in corona discharge takes place in a very small volume near the needle tip. Therefore, it can be expected that an increased dielectric barrier discharge (DBD) plasma volume results in a larger fraction of ionized analytes, leading to improved sensitivity. Compared to APPI, where in most cases a krypton discharge lamp is applied as emission source, (5) Robb, D. B.; Covey, T. R.; Bruins, A. P. Anal. Chem. 2000, 72, 3653–3659. (6) Syage, J. A.; Evans, M. D.; Hanold, K. A. Am. Lab. 2000, 32, 24–29. (7) Niessen, W. M. A. Liquid Chromatography-Mass Spectrometry, 3rd ed.; CRC Press: Boca Raton, FL, 2006. (8) Mann, M.; Meng, C. K.; Fenn, J. B. Anal. Chem. 1989, 61, 1702–1708. (9) Niessen, W. M. A. J. Chromatogr. A 1999, 856, 179–197.
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ionization by DBD plasma takes place by collisions of higher excited states. Due to the fact that the two wavelengths 116.486 and 123.590 nm are in the vacuum UV range, the radiation will be absorbed by air and therefore a low ionization yield can be expected. Analytes with energy levels higher than 10.64 eV cannot be excited efficiently. Therefore, often a large concentration of a substance that is easily ionized (i.e., dopant) may be added to increase the number of ions via initial photoionization of the dopant and subsequent charge exchange with the analyte (dopant-assisted APPI).5 However, there is no universal dopant working for all applications making individual optimization (e.g., kind of dopant, flow rate) necessary. It is therefore desirable to find an alternative to the most common ionization techniques in LC/MS for efficient ionization of compounds characterized by a wide range of polarities. The latter is in particular important for metabolomics applications, where a broad range of metabolites (amino acids vs. lipids) have to be analyzed. As alternative to corona discharge generated plasmas, a DBD can be used to generate low-temperature plasmas at atmospheric pressure.10,11 The DBD is typically formed between two electrodes, with at least one dielectric layer which separates the electrode from the plasma. The DBD plasmas are suitable for the atomization of volatile species.12-14 Furthermore, different geometrical arrangements have also served as an ionization source for ambient MS15-18 and ion mobility spectrometry.19-21 In this respect, DBD offers a new approach for efficient ionization for LC/MS applications which has not been described in the literature before and which is explored in this work. Consequently, we implemented a microplasma ionization based on dielectric barrier discharge (DBDI) into commercial API interface for LC/MS applications. Therefore, a heterogeneous compound library was investigated by DBDI to illustrate the potential use of the miniaturized plasma as an alternative ionization technique to ESI, APCI, and APPI. EXPERIMENTAL SECTION Chemicals. The following compounds were purchased from Sigma Aldrich (Steinheim, Germany): acenaphthene (CAS-No. 8332-9), 2-aminoanthracene (2-anthracenamine, CAS-No. 613-13-8), (10) Miclea, M.; Kunze, K.; Musa, G.; Franzke, J.; Niemax, K. Spectrochim. Acta Part B 2001, 56, 37–43. (11) Snyder, H. R.; Anderson, G. K. IEEE Trans. Plasma Sci. 1998, 26, 1695– 1699. (12) Zhu, Z.-L.; Zhang, S.-C.; Lv, Y.; Zhang, X.-R. Anal. Chem. 2006, 78, 865– 872. (13) Yu, Y.-L.; Du, Z.; Chen, M.-L.; Wang, J.-H. J. Anal. At. Spectrom. 2008, 23, 493–499. (14) Zhu, Z.-L.; Liu, J.-X.; Zhang, S.-C.; Na, X.; Zhang, X.-R. Spectrochim. Acta Part B 2008, 63, 431–436. (15) Na, N.; Zhao, M.-X.; Zhang, S.-C.; Yang, C.-D.; Zhang, X.-R. J. Am. Soc. Mass Spectrom. 2007, 18, 1859–1862. (16) Na, N.; Zhang, C.; Zhao, M.-X.; Zhang, S.-C.; Yang, C.-D.; Fang, X.; Zhang, X.-R. J. Mass Spectrom. 2007, 42, 1079–1085. (17) Zhang, Y.; Ma, X.; Zhang, S.; Yang, C.; Ouyang, Z.; Zhang, X. Analyst 2009, 134, 176–181. (18) Ma, X.; Zhang, S.; Lin, Z.; Liu, Y.; Xing, Z.; Yang, C.; Zhang, X. Analyst 2009, 134, 1863–1867. (19) Michels, A.; Tombrink, S.; Vautz, W.; Miclea, M.; Franzke, J. Spectrochim. Acta Part B 2007, 62, 1208–1215. (20) Vautz, W.; Michels, A.; Franzke, J. Anal. Bioanal. Chem. 2008, 391, 2609– 2615. (21) Olenici-Craciunescu, S.-B.; Michels, A.; Meyer, C.; Heming, R.; Tombrink, S.; Franzke, J. Spectrochim. Acta Part B, in press (http://dx.doi.org/10.1016/ j.sab.2009.10.001).
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anthracene (CAS-No. 120-12-7), 9,10-anthraquinone (CAS-No. 8465-1), azulene (CAS-No. 275-51-4), benzo[a]pyrene (CAS-No. 5032-8), biotin (vitamin B7; CAS-No. 58-85-5), biphenylene (CASNo. 259-79-0), 9-carboxyanthracene (9-anthroic acid, CAS-No. 723-62-6), fluoranthene (CAS-No. 206-44-0), fluorene (CAS-No. 86-73-7), glutamic acid (CAS-No. 142-47-2), lysine (CAS-No. 5687-1), 9-methylanthracene (CAS-No. 779-02-2), phenanthrene (CAS-No. 85-01-8), pyrene (benzo[d,e,f]phenanthrene, CAS-No. 129-00-0), tryptophan (CAS-No. 73-22-3), threonine (CAS-No. 72-19-5), and riboflavin (vitamin B2; CAS-No. 83-88-5). LC/MSgrade acetonitrile and water were obtained from Carl Roth (Karlsruhe, Germany). Instrumentation. Flow-injection analysis and HPLC separations were performed using a Surveyor MS pump and Surveyor autosampler (Thermo Fisher Scientific, San Jose, CA). For flowinjection analysis, 2 µL of diluted standard solution were injected into a 200 µL/min flow of acetonitrile/water (70:30, v/v). Three different concentrations were measured (1 × 10-6, 1 × 10-5, and 1 × 10-4 mol/L) of the following compounds: 2-aminoanthracene, anthracene, 9,10-anthraquinone, biotin, 9-carboxyanthracene, glutamic acid, lysine, 9-methylanthracene, tryptophan, threonine, and riboflavin. HPLC separation was carried out on a reversed-phase C-18 column (Hypurity Aquastar, 150 × 2.1 mm, 3 µm, 190 Å) of the following polycyclic aromatic hydrocarbons: acenaphthene, 2-aminoanthracene, anthracene, 9,10-anthraquinone, azulene, benzo[a]pyrene, biphenylene, 9-carboxyanthracene, fluoranthene, fluorene, 9-methylanthracene, phenanthrene, and pyrene. A dilution series of these compounds was prepared from a 1 × 10-3 mol/L stock solution ranging from 5 × 10-5 to 1 × 10-8 mol/L. A binary gradient consisting of acetonitrile and water containing acetonitrile (5.0%, v/v) was used for elution: 15% acetonitrile isocratic for 2 min, from 15 to 95% in 36 min, 95% isocratic for 7 min, from 95 to 15% in 3 min, and then 15% acetonitrile isocratic for 12 min. The flow rate was 200 µL/min and the injection volume was set to 2 µL. The column oven was set to 30 °C. Ionization Sources. The DBD microplasma ionization was realized by modification of a commercial API source (Ion Max source, Thermo Fisher Scientific) because it was anticipated that, for efficient DBD microplasma ionization, the HPLC eluent would require nebulization and vaporization in the same manner as for APCI and APPI. Furthermore, this approach has the advantage that the new source could be directly connected to the mass analyzer, without having to modify the vacuum interface. Comparisons between the new source and the standard APCI and APPI source were also facilitated, because their housings were identical. Additionally, the ESI probe head fits into the same housing, which guarantees a similar geometry and hence comparability to DBDI, APCI, and APPI, respectively. Ionization was carried out by a DBD with a plasma cone outside the electrode region. Helium applied to the plasma at atmospheric pressure had a purity of 99.999%. The plasma and its spectroscopic characterization are described in detail elsewhere.19,21 The plasma was operated with a helium flow of 150 mL/min, by applying high-voltage pulses of 5 kV and 35 kHz. Details of the DBDI source are depicted in Figure 1.
Figure 1. Photographs of the DBD microplasma that is implemented into the Ion Max source.
Figure 1A shows a photograph of the DBD with a plasma cone outside the electrode region. In order to show the inner arrangement of this discharge, a photomontage of the discharge and the housing is presented in Figure 1B. The discharge consists of a 3 cm long glass capillary with an inner diameter of 500 µm and an outer diameter of 1.2 mm. Rings with an inner diameter of 500 µm are located around the capillary, forming electrodes with a separation distance of 12 mm. The distance of the electrode to the end of the capillary is 2 mm. A periodic positive voltage pulse (5 kV with a frequency of 35 kHz and a pulse width of 2 µs) is applied. The plasma electrodes are enclosed in a Teflon tube (see Figure 1B) not only for safety precautions but also to prevent a discharge between the electrodes outside the capillary. For this Teflon tube, a probe head of PEEK was manufactured at the machine shop of ISAS for direct installation into the original API source housing. Therefore, only the window on the front side of the Ion Max source is replaced by the PEEK probe head. An installed O-ring within the PEEK body ensured proper fitting of the Teflon tube and easy adjustment of the distance to the mass spectrometer inlet. The position of the tube was axial to the mass spectrometer inlet. This ion source, like the unmodified APCI and APPI source, is working with a heated nebulizer (see Figure 1C), which was maintained at 450 °C for all experiments (except ESI). Nitrogen (5.0, 99.999%) was used to nebulize the liquid eluent (nebulizer gas) and also to transport the finely dispersed sample droplets through the heated ceramic tube in which they were vaporized (auxiliary gas). The ion current for the APCI and APPI experiments was set to 5 µA. For ESI the capillary voltage was set to 4 kV. Mass Spectrometer. Mass spectrometric detection was carried out using a LTQ FT (Thermo Fisher Scientific, Bremen, Germany) Fourier transform ion cyclotron resonance hybrid-mass spectrometer (FTICR-MS). For flow-injection measurements of model compounds and HPLC separation of PAH standard mixture, survey MS spectra in the mass range m/z 100-500 were acquired in the FTICR-MS with a resolution r ) 50 000 (full width at halfmaximum, fwhm). During the experiments comparing the DBDI to APCI, APPI, and ESI, the operating parameters of the mass spectrometer were unchanged. RESULTS AND DISCUSSION A characterization of the DBD plasma utilized for ionization of organic molecules is already presented when the DBD plasma was used in an ion mobility spectrometer.19-21 The source is based on a He plasma jet established at the end of a capillary dielectric barrier discharge at atmospheric pressure. Spectroscopic emission measurements were carried out along the plasma jet in and outside the capillary. The intensity variation of N2+ lines, for example 391.4 and 427.8 nm, were associated with the protonation
process, which is the basis for the soft ionization.21 In corona discharge ionization, N2+ is also the primary ion, which reacts with additional nitrogen to give N4+. The latter ionizes water by charge transfer to give H2O+, which in a subsequent step reacts with another molecule of water to give H3O+. By association of H3O+ with additional water molecules ion clusters H+(H2O)n are formed. H3O+ and N4+ are responsible for the chemical ionization of the analytes by either proton transfer reactions or by charge transfer reactions.22-24 Nitrogen plays an important role for soft ionization mechanisms using DBD plasma. Nitrogen can be excited to the upper level of the N2+ first negative system (B 2Σu+) by Penning ionization due to the He metastables (HeM). Therefore, the population of the excited upper level of N2+ first negative system (B 2Σu+) cannot exceed the population density of the metastables. The reaction of Penning ionization should be in the vicinity of the protonation process. This means that the Penning ionization between He metastables and nitrogen should happen in the ionization chamber outside the plasma capillary in the vicinity of the plasma jet. On the basis of these findings, the DBDI was anticipated to serve as an efficient ionization means for LC/MS. Therefore, a heterogeneous set of model compounds was investigated to evaluate the performance of DBDI also in comparison to APCI, APPI, and ESI. DBDI Mass Spectra. In Figure 2, DBDI high-resolution mass spectra of selected polar model compounds are depicted. Ionization was carried out in the positive mode with acetonitrile/water as solvent. The observed signals will be discussed in the following sections. The hydroxyl-containing amino acid threonine is shown in Figure 2A. The signal at m/z 120.0654 is the protonated molecule [M + H]+, and no fragmentation was observed (e.g., neutral loss of H2O or NH3). Therefore, DBDI can be considered a soft ionization technique. Interestingly, a signal at m/z 161.0923 is the base peak in the full scan mass spectrum. The difference of 41.0269 Da reveals addition of acetonitrile (CH3CN) to the protonated molecule (theoretical value, 41.0266 Da). The adduct formation with acetonitrile was also observed during APCI and APPI (
