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Characterization of an atmospheric pressure ion lens for electrospray ionization sources in mass spectrometry Manisali, Irina 2005

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CHARACTERIZATION OF AN ATMOSPHERIC PRESSURE ION LENS FOR ELECTROSPRAY IONIZATION SOURCES IN MASS SPECTROMETRY by IRLNA MANISALI B .Sc , The University of British Columbia, 2002 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Chemistry) THE UNIVERSITY OF BRITISH C O L U M B I A June 2005 © Irina Manisali, 2005 Abstract The purpose of this project is to characterize the properties of an atmospheric pressure ion lens used to facilitate and improve the operation of electrospray ionization (ESI) sources for mass spectrometry (MS). This work is in part motivated by the inefficient transmission of ions from atmospheric pressure into vacuum, characteristic of current electrospray ion source designs. The first chapter of this thesis will be an introduction to the basic theory and the historical developments of ESI sources for MS from early designs to today's commercial geometries. The second chapter of this thesis summarizes previous research that has been done with respect to atmospheric pressure electrostatic devices for ESI sources, including work done in our laboratory. The objective of the present project is also described in more detail in this section. Descriptions of the instruments and their operation, as well as the experimental procedures used in this project are presented in the third chapter. The fourth chapter presents some of the results and it attempts to explain the observed trends with the help of simulations. The results show that the mechanism of the ion lens is not trivial. The effect 6f the ion lens is different for analytes of various charge states and is highly dependent on a number of instrumental and environmental parameters. The size and shape of the lens is also important. The ion lens was shown to stabilize the electrospray, as well as improve the signal-to-noise ratio (S/N) in some instances, while at other times it was difficult to observe any effect at all. Future work that may help optimize the operation of the ion lens is discussed in the final chapter. ii Table of Contents Abstract ii Table of Contents . iii List of Tables . .....v List of Figures... vi List of Symbols and Abbreviations , viii Acknowledgment . ix Chapter 1. Electrospray Ionization Source Geometry for Mass Spectrometry...*...... 1 1.1. Brief introduction to electrospray ; / 1.2. Milestones in the evolution of electrospray 1 1.3. Theory 2 1.4. Historical Highlights 6 1.4.1. The Development of the Traditional ESI Source 6 1.4.2. The CE/MS Interface 10 1.4.3: The Miniaturization of Electrospray: Initial. Developments of Micro and Nandelectrospray 12 1.5. Currently Available Commercial Sources —14 1.5.1. Source Configuration and Its Relationship to Flow Rate 14 1.5.1.1. . Low flow rate. 14 1.5.1.2. High Flow Rate 16 1.5.2. Interface 20 1.6. Multiple Sprayers '.' 20 1.7. Recent Developments to the ESI Source 22 1.8. Remarks 23 1.9. References .-. 24 Chapter 2. Atmospheric Pressure Ion Lens for ESI Sources in Mass Spectrometry , 29 2.1. Early work involving atmospheric pressure focusing devices 29 2.2. Previous work from our laboratory 29 2.3. Other recent developments 32 2.4. Objectives of the present project 32 iii 2.4. References 33 Chapter 3: Instrumentation, Experimental Procedures and Sample Preparation... 35 3.1. Instrumentation , : 35 3.1.1. Operation principles of quadrupole mass filters 35 3.1.2. The Toby single quadrupole mass spectrometer 38 3.1.2.1. The ion source 38 3.1.2.2. Features of the Toby prototype mass spectrometer 39 3.1.2.3. Ion detection 39 3.1.3. The PE SCIEX API III triple quadrupole mass spectrometer 39 3.1.3.1. The ion source 40 3.1.3.2. Features of the API III mass spectrometer 42 3.1.3.3. Ion detection 42 3.2. Sample Preparation 42 3.3. Experimental set-up and procedures 43 3.3.1. Ion spray source equipped with ion lens 43 3.3.2. Ion lens for nanoelectrospray source 46 3.3.3. Instrument Parameters 48 3.4. References .i... • 49 Chapter 4. Effects of an Atmospheric Pressure Ion Lens for ESI Sources 50 4.1. Impact of the ion lens on the charge states of gas-phase molecules 50 4.1.1. Ion lens vs. original source configuration, and the effect on charge state.... 50 4.1.2. Variation of ion spray and ion lens voltages, and the effects on cytochrome c charge states 54 4.1.2.1. Results for cytochrome c .... 54 4.1.2.2. Discussion of results for cytochrome c 58 4.1.3. Variation in ion spray and ion lens voltages, and the effects on insulin charge states ;......... 62 4.1.3.1. Results for insulin 62 4.1.3.2. Discussion of results for insulin 65 4.1.4. Variation in ion spray and ion lens voltages, and the effects on bradykinin charge states : r • 65 4.2. The Effect of Ion Lens Size 67 4.3. Spray Stability and Signal-to-Noise Measurements 73 4.4. Nanoelectrospray ion source equipped with an atmospheric pressure ion lens.... 73 4.4.1. Results and Discussion 76 4.4.1.1. Signal intensity and nanoelectrospray stability , 76 4.4.1.2. Relationship between distance of the emitter to orifice and ion lens effect '. 78 4.5. References .'. 80 Chapter 5. Conclusions and Future Work 81 iv List of Tables Table 3. 1 Summary of the solvent compositions and sample concentrations used for this project 43 Table 3. 2 Solvents used for sample preparation 43 Table 3. 3 Diameter of lenses tested on the ion spray source 45 Table 3. 4 Summary of parameters optimized for each analyte on the Toby mass spectrometer 48 Table 3. 5 Summary of parameters optimized for bradykinin on the API III mass spectrometer 49 Table 4. 1 Improvements in cytochrome c peaks intensity using the ion lens 51 Table 4. 2 Comparison of spray stability for the conventional nanoelectrospray source vs. using the ion lens at various voltage combinations 77 v List of Figures Figure 1.1 Schematic diagram of the electrospray process 3 Figure 1.2 Configuration of Dole's first electrospray-based mass analysis system. Reprinted with permission from [3] , Copyright 1968, American Institute of Physics ; 6 Figure 1.3 Configuration of the electrospray ion source coupled with a quadrupole mass analyzer designed by Fenn and cp-workers in 1984. Reprinted with permission from [22], Copyright 1984, American Chemical Society 7 Figure 1.4 Schematic of the Ion Max source from Thermo Electron using a heated metal capillary for ion desolvation and transfer 8 Figure 1.5 Schematic of the ion spray configuration developed by Bruins in 1987 9 Figure 1.6 Schematic of the three most common ways to interface CE/MS: a) coaxial sheath-flow interface; b) sheathless interface; c) liquid-junction interface 11 Figure 1.7 Schematic of an ESI source configuration developed by Agilent 1.7 Figure 1.8 Schematic of the Zspray™ source configuration used by Waters/Micromass 18 Figure 1.9 Schematic of the TurboV™ source used by MDS SCIEX 19 Figure 1.10 Schematic of the particle discriminator interface developed by MDS Sciex 23 Figure 2. 1 a) Schematic of a typical reduced flow rate ESI source demonstrating the defocusing nature of the equipotential lines near the tip of the sprayer; b) reduced flow rate ESI source with an ion lens around the tip of the sprayer, reducing the defocusing of the equipotential lines; c) schematic of equipotential lines generated for a standard ion spray source; d) equipotential lines generated for an ion spray source with an atmospheric pressure ion lens. The ion trajectories are qualitative. .31 Figure 3. 1 Schematic of a quadrupole mass filter 35 Figure 3. 2 The first stability region of a quadrupole field, m,, m 2, and m 3 represent ions with different masses on an operating line. m 2 is within the stability region, while m, and m 3 are not 37 Figure 3. 3 Schematic of the Toby prototype mass spectrometer 38 Figure 3. 4 Schematic of the API III triple quadrupole mass spectrometer 40 Figure 3. 5 a) schematic of the off-line nanoESI interface; b) image of the emitter positioned in front of the interface orifice as seen through the microscope 41 Figure 3. 6 Schematic of the ion spray source with an ion lens. The ion spray mount (1) is attached to the mass spectrometer with a stud inserted through the mounting hole (2). The electrospray potential is applied to a stainless steel tee (3) through the conductive mount (1). Two concentric stainless steel capillaries (4) hold the inner glass capillary and allow the nebulizer gas flow from the nebulizer gas line (5). The sample is introduced through the fused silica capillary (6) to the inner stainless steel tube (7). The ion lens (8) was located near the tip of the inner stainless steel tube (7). The ion lens had a mounting bracket (9) and an adjustable arm (10). Pivots (11 and 12) allowed the lens to be positioned in different locations. The length of protrusion of the inner stainless steel tube from the outer tube is labeled " X " 44 vi Figure 3. 7 Front view of a) an oblong shaped lens and b) a circular lens (8); the figure also demonstrates the location of the stainless steel sprayer tube (7) within the lens. 45 Figure 3. 8 The nanoelectrospray source fitted with an adjustable ion lens 46 Figure 3. 9 Two-dimensional and three-dimensional views of the nanoelectrospray source and lens positioned in front of the mass spectrometer orifice 47 Figure 4. 1 Mass spectra of the analytes tested for this project: a) horse heart cytochrome c; b) horse heart cytochrome c (at a different time period); continued on pg. 53 52 Figure 4. 2 Trends in the intensity of various charge states of cytochrome c obtained by varying the ion spray voltage while holding the ion lens at fixed voltages; continued. pg.56 55 Figure 4. 3 Simulation of the ion spray source equipped with the atmospheric pressure ion lens; the electrospray voltage was set to 4000 V, the interface plate was set at 1000 V and the orifice plate at 100 V; a) ion transmission without a lens; b) ion transmission with lens at 0 V; continued on pg. 61 60 Figure 4. 4 Trends in the intensity of various charge states of bovine insulin obtained by varying the ion spray voltage while holding the ion lens at fixed voltages; continued on pg. 64 63 Figure 4. 5 Trends in the intensity of two charge states of bradykinin obtained by varying the ion spray voltage while holding the ion lens at fixed voltages 66 Figure 4. 6 Changes in cytochrome c peaks intensity with different ion lens diameters; the ion lens voltage was optimized for each ion spray voltage; continued on pg. 70 69 Figure 4. 7 Trends in cytochrome c peak intensity for various ion lens diameters at fixed ion spray voltage and varying ion lens voltage; continued on pg. 72 71 Figure 4. 8 Potential energy view of the a) conventional nanoelectrospray set-up with nanospray at 600 V, interface plate at 100 V and orifice plate at 20 V; b) nanoelectrospray equipped with the ion lens at 800 V 75 Figure 4. 9 Changes in bradykinin +2 charge state peak intensity while varying the ion lens voltage at fixed nanospray voltages. Note that the "0 V " point indicates "no ion lens" being used in this case 76 Figure 4. 10 Changes in bradykinin peak intensity with distancing of the nanoelectrospray emitter from the orifice combined with the use of the ion lens at various voltages. Distance increments of 0.25 mm were used 79 vii List of Symbols and Abbreviations Symbol or Abbreviation Definition ESI Electrospray ionization MS Mass spectrometry M A L D I Matrix assisted laser desorption ionization HPLC High pressure liquid chromatography LC/MS Liquid chromatography coupled with mass spectrometry CE Capillary electrophoresis CZE Capillary zone electrophoresis CE/MS Capillary electrophoresis coupled with mass spectrometry JEM Ion evaporation model C R M Charge residue model RF Radio frequency DC Direct current SIMS Secondary ion mass spectrometry S/N Signal-to-noise ratio QO Quadrupole ion guide OJ First quadrupole (mass filter) Q2 Second quadrupole (collision cell) Q3 Third quadrupole (mass filter) C E M Channel electron multiplier viii Acknowledgment I would like to thank my supervisor Dr. David Chen for his guidance and support during my courses and research at UBC. I have joined a wonderful group of people that have inspired me and helped me constantly. I would particularly like to thank my lab mates David McLaren, Wuyi Zha, Ning Fang, Rong Yi, Jingyan Zeng and Stefan Buster for making this experience a memorable one. Many thanks to Dr. Damon Robb for his advice and help with the instrument, as well as to Dr. Jiirgen Kast for allowing me to use the triple quadrupole mass spectrometer in his laboratory. Thank you Dave Tonkin for all the hours you have dedicated to repairing our mass spectrometer. I also would like to express my gratitude to my parents and my sister for being so supportive in all of my decisions, as well as to my grandparents for their love. ix Chapter 1. Electrospray Ionization Source Geometry for Mass Spectrometry 1.1. Brief introduction to electrospray Electrospray ionization (ESI) is a soft ionization method, allowing the formation of gas-phase ions through a gentle process that makes possible the sensitive analysis of non-volatile and thermolabile compounds. Consequently, the use of the ESI source in the field of mass spectrometry (MS) has greatly facilitated the study of large biomolecules, as well as pharmaceutical drugs and their metabolites. Thus, the ESI source has evolved with the growth of proteomics and drug discovery research [IJ. The analysis of carbohydrates, nucleotides, and small polar molecules are a few of the many other applications in which ESI/MS is presently used. Together with matrix-assisted laser desorption ionization ( M A L D I ) , another soft ionization technique, ESI has revolutionized biomedical analysis, and culminated in 2002 with the award of the Nobel Prize to its developer, Dr. John Fenn. This prize was shared with Koichi Tanaka for his outstanding contribution to the development of M A L D I . The concept of the ESI source is deceivably simple, as some aspects of its functioning are still not well understood. As the name suggests, the basis of this technique lays in using a strong electric field to create an excess of charge at the tip of a capillary containing the analyte solution. Charged droplets exit the capillary as a spray and travel at atmospheric pressure down an electrical gradient, to the gas conductance limiting orifice or tube. Gas-phase ions are then transported through different vacuum stages to the mass analyzer and ultimately the detector. ESI has successfully been coupled with a variety of mass analyzers. Each analyzer has different advantages over other types and its use is based on the required application. Comprehensive information about the coupling of ESI with various mass analyzers can be found in Cole's 1997 compilation of review articles on the topic [2|. 1.2. Milestones in the evolution of electrospray Since 1968, when Dole used ESI and a nozzle-skimmer system to produce charged gas-phase polystyrene [3], the ESI source has continued to undergo various changes in its size, material, and geometry. These transformations are made to optimize 1 both the ionization efficiency and the transfer efficiency of gas-phase ions into the mass analyzer. The evolution of the electrospray source design has also reflected the coupling of the ion source to separation systems such as high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). This has brought about new challenges with generating ions at different flow rates, minimizing contamination of the interface, and optimizing overall efficiency. Leading instrument manufacturers are directly involved in providing innovative solutions to the industry's demand for more sensitive and reliable mass spectrometers. 1.3. Theory When a large potential difference is applied between an electrode shaped as a wire and a counter electrode, a strong electric field is created at the tip. In the case of ESI, a high voltage is applied to a capillary containing the analyte solution. For simplicity, we only consider the application of positive potential. Due to the electric field gradient at the tip, charge separation occurs in the solution as anions migrate towards the capillary walls, and cations travel towards the meniscus of the droplet formed at the tip (Fig.l . 1) [4]. 2 ions and neutrals offspring droplets counter electrode Figure 1.1 Schematic diagram of the electrospray process In order to direct charged species into the mass spectrometer, a series of counter electrodes in order of decreasing potential are used. Typically, the principal counter electrode can be either the curtain plate of the mass spectrometer or a transport capillary, which will be discussed later. The optimum potential difference between the sprayer and the principal counter electrode is dependant on experimental parameters such as the charge state of the analyte, solution flow rate, solvent composition, and distance between the tip and the counter electrode. While different mass spectrometers may require different applied voltages on the sprayer and counter electrode, the potential difference between them is similar (typically 2000 V - 5000 V) [5]. In the presence of an electric field, liquid emerges from the tip of the capillary in the shape of a cone also known as the "Taylor cone" (Fig. 1). When the electrostatic repulsion between charged molecules at the surface of the Taylor cone approaches the surface tension of the solution -phenomenon known as reaching the Rayleigh limit - charged droplets are expelled from the tip. The droplets containing excess positive charge generally follow the electric field 3 lines at atmospheric pressure toward the counter electrode. However, trajectories will also be affected by space charge and gas flow effects. Charging of analyte molecules can occur through more than one process [6]. Charge separation (in the ESI source), adduct formation, gas-phase molecular reactions, and electrochemical reactions may contribute to ionization during the electrospray process. The mechanism of formation of the Taylor cone is not entirely understood but it is known that under certain conditions the morphology of the spray emitted from the capillary tip can change [7]. The various spray modes are strongly dependant on the capillary voltage and are related to pulsation phenomena observed in the capillary current [7]. Juraschek and Rollgen showed that liquid flow rate, capillary diameter, and electrolyte concentration can all impact the spray mode. Controlling the spray mode is thus crucial in achieving a stable spray and optimum signal. This, however, becomes particularly difficult when the mobile-phase composition changes during gradient elution conditions necessary in many LC/MS applications. To address this problem, Valaskovic and Murphy have developed an orthogonal optoelectronic system capable of identifying many spray modes under different conditions [8]. The system automatically optimizes the ESI potential in response to changes in flow rate and solvent composition. The size of the spray droplets released from the Taylor cone, highly dependent on flow rate and capillary diameter, is critical to the efficient ionization of the analyte. Since a small droplet contains less solvent, desolvation and ionization can be more efficient. Due to less fission required to produce ions, the salt concentration in the final offspring droplets may be lower compared to a droplet that has undergone more evaporation - fission cycles. As a result, the background noise in the mass spectrum may be reduced [9j. In addition, with smaller droplets analytes that are not surface active will have a greater chance of being transferred to gas phase rather than being lost in the bulk of the parent droplet residue [9]. The solvent is typically a combination of acidified water and an organic modifier. The role of the organic solvent is to lower the surface tension of the liquid, facilitating the formation of gas-phase ions. As the solvent evaporates, the droplets shrink and the electrostatic repulsion between charges within the droplet increases. When the Rayleigh 4 limit is approached, offspring droplets begin to break away unevenly in a process also known as "coulombic fission" [4|. Evaporation of the offspring droplets leads to a new fission series and the process repeats itself producing smaller and smaller droplets. The eventual creation of gas-phase ions from these droplets is thought to be a combination of two mechanisms known as the "ion evaporation mechanism" (IEM) initially proposed by Iribarne and Thomson [10] and the "charged residue model" (CRM) put forward by Dole et al. [3, 11] and supported by Rollgen et al. [12]. The fundamental difference between the two lays in the mechanism of formation of gas-phase ions. IEM suggests that when the electric field on a charged droplet is high enough, single solvated analyte molecules carrying some of the droplet charge become ejected into the gas phase. This happens because the potential energy of the ions near the surface becomes high enough to allow evaporation to occur [13]. In contrast, C R M maintains that gas-phase ions are formed when successive fissions lead to a charged droplet containing a single analyte. Although difficult to determine which of the two mechanisms is more accurate, extensive studies [14-16] seem to suggest that C R M is the preferred mechanism in the case of formation of gas-phase charged globular proteins. Kebarle also concluded that charging of a single protein in the evaporating droplet is due to small ions found at the surface of the droplet 115]. The mechanism of formation of small analyte ions is still not clear. Efficient transport of ions and charged droplets from the sprayer into the mass spectrometer is challenging and depends on parameters such as interface arrangement and gas throughput into the instrument [5|. As gas and ions are transported from atmospheric pressure into vacuum, strong cooling of the mixture occurs during expansion. Under these conditions, polar neutral molecules may cluster with analyte ions and therefore it is very important to achieve efficient desolvation within the atmospheric pressure region. One instrument manufacturer (MDS SCIEX) introduced a method of desolvation whereby heated auxiliary nitrogen gas is directed at an angle from the direction of the spray (TurboIonSpray™) [17, 18]. In addition, counter-current nitrogen gas flow (Curtain Gas 1 M) emanating from in front of the gas conductance-limiting orifice provided additional desolvation [5]. The advantage of the Curtain Gas is that neutral molecules, such as solvent, are carried away from the sampling orifice by the nitrogen flow, while charged molecules are directed through the gas flow under the influence of the electric 5 field. Since nitrogen is inert, it cannot form covalent bonds with the ions and clustering during transfer to vacuum is prevented. While its name varies depending on the manufacturer, the counter-current gas flow is an important feature of the ESI ion source. In addition to using inert gas flow, heating the ion source or gas conductance limiting orifice/capillary is also a common way to aid declustering and desolvation [19]. 1.4. Historical Highlights .1.4.1. The Development of the Traditional ESI Source Although the work of Fenn and colleagues demonstrated the applicability of ESI for generation of biomolecular ions, it was Dole and co-workers who led the way in the late 1960s by building the first electrospray-based mass analysis system [3] as shown in Fig. 1.2. N 2 gas out Pump Pump Figure 1.2 Configuration of Dole's first electrospray-based mass analysis system. Reprinted with permission from [3] , Copyright 1968, American Institute of Physics A sharp hypodermic needle was used to spray polystyrene solution into an evaporation chamber. The chamber was made from a plastic film and measured 12 cm in diameter and 70 cm in length. A Teflon plate supported the needle as well as a gas inlet introducing nitrogen as a bath gas. The evaporation chamber also contained two brass-collimating plates with adjustable positions. A nozzle-skimmer system and two stage differential pumping were used to reduce pressure in the analyzer region and permit sampling of the supersonic free jet without disturbing the molecular flow [20|. The voltage applied on the needle was maintained at approximately -1.0 kV (negative polarity 6 used), while the voltages on the first and second collimating plates were -3kV, and -1.4kV, respectively. Dole measured mass to charge ratios (m/z) by determining the retarding potential of ions through a grid preceding a Faraday cup detector. Intrigued by Dole's ESI work, and having himself been a pioneer of supersonic free jets as molecular beam sources, Fenn reproduced Dole's experiments with the same observations but discontinued research in this area due to technical limitations [21]. Years later, Fenn reprised his experiments on molecular beams and used ESI coupled to a quadrupole mass analyzer for ion detection. Recognizing flaws in Dole's ion source design as well as results interpretation, Fenn modified the electrospray ion source by reducing the distance between the hypodermic needle and end plate containing the nozzle [22] (Fig. 1.3). As a result, the applied sprayer voltage could be significantly lower. | Niirogan Figure 1.3 Configuration of the electrospray ion source coupled with a quadrupole mass analyzer designed by Fenn and co-workers in 1984. Reprinted with permission from [22], Copyright 1984, American Chemical Society. A cylindrical electrode surrounding the needle to end plate region was maintained at 500-600 V relative to ground. The purpose of the cylindrical electrode was not described, but intuitively, its purpose was to limit radial expansion of the electrospray plume. In contrast with Dole's ion source, Fenn's ESI chamber was built with metal walls to prevent charge build-up, and had significantly smaller dimensions. It also contained a nozzle-skimmer configuration similar to Dole's experiments. Fenn's ESI source later evolved to what is known as the Fenn-Whitehouse design [23]. The additional characteristic is a glass capillary that allows ions from atmospheric 7 pressure to enter the first vacuum stage of the instrument. The capillary dimensions can be chosen to allow the same flux of drying gas and ions as a regular thin plate orifice [20]. Increasing the capillary diameter may be desirable in order to increase the acceptance and decrease the chance of blockage. However, for a fixed gas throughput this must be associated with a corresponding increase in capillary length, potentially lowering performance. An alternative to using the counter-current gas for ion desolvation is the heated metal capillary, introduced by Chowdhury et al. [19]. With this configuration, gas throughput depends on both capillary dimensions and temperature. The heated metal capillary inlet (ion pipe) as shown in Fig. 1.4 is currently used on Thermo Electron instruments. However, a number of other manufacturers have developed modified versions of this device, including heated resistive tubes [24] and heated metal capillaries with different inlet/outlet dimensions [25]. The combination of a heated metal capillary and the counter-current gas makes possible even more efficient ion desolvation. Figure 1.4 Schematic of the Ion Max source from Thermo Electron using a heated metal capillary for ion desolvation and transfer 8 ESI/MS interfaces consisting of multiple capillaries have been developed for various applications such as ion-ion reaction monitoring [26, 27], separate introduction of analyte and calibrant using a multiple sprayer source [28], and for improved ion transmission while used in combination with an electrodynamic ion funnel [29]. Other modifications to the heated capillary inlet include offsetting the exit of the capillary from downstream ion optics devices (such as a skimmer) |30, 311. The advantage of this design is that some large solvent clusters exiting the transfer capillary are prevented from entering the next vacuum stage, while the lighter analyte ions follow the gas flow through the ion optics. Recently, Varian also adopted this modification by positioning a heated capillary outlet off-axis from a subsequent RF multipole ion guide [32]. In 1987, Bruins et al. introduced the pneumatically assisted nebulizer for the electrospray interface, also known as ion spray. Fig. 1.5 shows the details of the ion spray interface [33]. s i l i ca capi l lary con ta in ing s a m p l e solut ion s a m p l e so lut ion • T s ta in less s tee l cap i l la ry ho lder s ta in less s tee l tub ing | ^ / N 2 c h a r g e d drop le ts 3 A i r / N 2 neut ra ls N2 IIL UT atmospher ic pressure N2 Figure 1.5 Schematic of the ion spray configuration developed by Bruins in 1987 9 The nebulizer gas was beneficial for the coupling of L C with ESI/MS because it helped to stabilize electrospray for flow rates up to approximately 0.2 ml/min. It also tolerated larger distances between the sprayer and the counter electrode, reducing the occurrence of corona discharge [33]. Bruins observed that the spraying process was less dependent on the sprayer position relative to the orifice than without nebulizer assistance, and that better sensitivity was obtained if the sprayer was pointed off-axis instead of directly at the orifice. The reasoning behind the off-axis geometry was that by sampling the periphery of the spray, finer droplets entered the mass spectrometer while the larger droplets struck the curtain plate. This lead to improved performance because finer droplets are easier to desolvate. Currently, the off-axis sprayer geometry has evolved to an orthogonal sampling position. More detail will be presented in the Current Commercial ESI Sources section. 1.4.2. The CE/MS Interface While ion spray became standard for LC/MS at higher flows, CE/MS coupling was more complicated due to the large variety of solvent composition required for separation and problems associated with electrochemistry. In addition, differences in flow rate requirements also presented problems. Three common ways of interfacing CE/MS are shown in Fig. 1.6. 10 a) nebulizer gas nebulizer gas tube sheath liquid I analyte/buffer solution to M S stainless steel separation capillary capillary/electrode for CZE / ESI voltage application b) r lanalvte/buffer solution _> to M S nebulizer gas separation capillary with conductive coating for ESI voltage application c) H.V. nebulizer gas IS analyte/buffer solution liquid junction to M S separation capillary Figure 1.6 Schematic of the three most common ways to interface CE/MS: a) coaxial sheath-flow interface; b) sheathless interface; c) liquid-junction interface The most common method of CE/MS interfacing is the coaxial sheath-flow interface, first described by Smith et al. in 1988 134]. In 2003, approximately 77% of CE/MS users employed this type of interface due to its reproducibility and robustness [35]. By allowing a conducting liquid to provide electrical contact between the stainless steel capillary maintained at high voltage and the fused silica capillary carrying the analyte solution, the interfacing of capillary zone electrophoresis (CZE) with ESI/MS could be achieved with many buffer systems that were previously not practical. This is 11 significant because aqueous buffers of high ionic strength are normally not tolerated by ESI/MS. Buffer ions can lead to charge neutralization and corona discharge as well as formation of undesirable adducts with the analyte resulting in loss of signal sensitivity and high background. By using organic solvents such as acetonitrile or methanol as the sheath liquid, buffers of ionic strength up to 0.2 M can be used. The sheath liquid prevents direct contact between the high voltage electrode and the analyte solution avoiding electrochemical modification of analytes. An additional advantage of using the sheath-flow interface is minimization of corona discharge as a result of reduction of the ionic strength of the sprayed solvent [34]. Olivares et al. developed the sheathless interface in 1987 by using a stainless steel capillary to create electrical contact with the analyte solution [36]. Accounting for about 11% of CE/MS interfaces in 2003 [35], this method evolved with a focus on low flow rates for improved sensitivity. Other sheathless methods were subsequently developed, such as sharpening the CE capillary tip in order to produce a stable spray, while the ESI electrode was made by installing a piece of gold wire [37] or coating the capillary end with a metal or alloy [38]. The advantages of the sheathless interface are that analyte dilution and ion suppression due to the sheath-flow can be eliminated. Sensitivity can also be improved by up to an order of magnitude over sheath-flow designs [39]. A third method of interfacing CE/MS was the liquid-junction interface developed by Henion and colleagues [40, 41]. A liquid junction between the CE capillary and the ESI emitter was built using a stainless-steel tee equipped with a buffer reservoir. This interface reduced the complication brought about by the different flow rate requirements of CE and ESI [42], but peak broadening and mechanical difficulties limited the general applicability of this technique [38]. 1.4.3. The Miniaturization of Electrospray: Initial Developments of Micro and Nanoelectrospray In an attempt to reduce flow rates and thus produce smaller droplets to improve the ionization efficiency, various miniaturized ESI sources were developed [43]. Caprioli et al. [44, 45] developed an on-line ESI source equipped with a sprayer tip of 10-20 [im internal diameter and capable of operating at flow rates of 300 - 800 nl/min. The emitter was created by burning off the polyimide coating of a fused silica capillary tip and 12 tapering it by etching with hydrofluoric acid to give droplets in the micron diameter range. Caprioli named his technique microelectrospray, a term which currently describes a flow rate range of approximately 200 nl/min - 4|ll/min [46]. Operation in this flow rate regime may involve various means for pulling silica capillaries and applying the electrospray voltage [47]. In 1995, Caprioli introduced the idea of using the L C column tip combined with the microelectrospray technology [48] to spray directly towards the mass spectrometer inlet. This was significant because it triggered the evolution of analytical L C columns to miniaturized versions of micro and nanobore capillary columns necessary for low flow rate electrospray [46]. Nanoelectrospray, introduced by Wilm and Mann more than a decade ago [49], was a particularly successful technique due to its ability to form droplets 100-1000 times smaller in diameter than droplets formed by conventional ESI. The technique was named to reflect the nanometer sized droplets as well as the flow rate of nanoliters per minute [50]. Essentially, the solution infusion system was eliminated as a few microliters of solution were transferred to the tip of a gold-coated glass capillary. By applying a low potential onto the conductive capillary surface, a fine spray was generated and the charged droplets were directed towards the counter electrode under the effect of the electric field gradient. Despite an estimated efficiency of 500 times greater than traditional ESI [50], nanoelectrospray has some limitations including plugging of the emitter tip and lower reproducibility associated with differences in tip morphology. While the term nanoelectrospray initially referred to Wilm and Mann's off-line technique, evolving technology has made possible the use of on-line flow rates down to tens of nanoliters/minute. The term nanoelectrospray has thus expanded to encompass both on-line and off-line techniques. The effects of the tip geometry, flow rate, as well as solvent composition for nanoelectrospray have been investigated by a number of groups [51-53]. Motivated by the fact that at certain solvent compositions the sensitivity decreased dramatically, Vanhoutte et al. compared the effects of various mobile phase compositions using several types of electrospray capillaries. These included combinations of uncoated or gold-coated, tapered or non-tapered fused silica capillary tips, as well as stainless steel emitters. He found that the gold-coated, tapered tips were the most effective because they 13 covered the most extensive range of solvent composition for the tested conditions without loss of sensitivity [51]. Schmidt and Karas used combinations of model compounds to examine the effect of flow rate on ion signal [52]. They determined that at flow rates of a few nl/min, signal suppression caused by differences in surface activity of the analytes was insignificant compared to higher flow rates (> 50 nl/min). L i and Cole studied the often-observed shift in charge state as a result of changing experimental parameters for nanoelectrospray [53]. Parameters such as tip diameter, flow rate, analyte concentration and solvent composition can all affect the observed ions and charge states. 1.5. Currently Available Commercial Sources Since its recognition as an invaluable bioanalytical tool in the late 1980's, research groups have attempted to exploit the capabilities of ESI by modifying the source geometry in order to allow a wider range of flow rates and in-source fragmentation, as well as improved sensitivity, efficiency and practicality. Niessen's 1998 and 2003 review articles [54, 55] are a good starting point for those interested in the trends that have stimulated the ESI source evolution. Despite numerous improvements, today's commercial ESI sources still retain many features of the original configuration. 7.5.7. Source Configuration and Its Relationship to Flow Rate A parameter of critical importance in ESEMS is the solution flow rate. Depending on the application for which the mass spectrometer is used, the ESI source needs to be adapted to handle the in-coming flow of solution, whether it is direct infusion, LC, or CE eluent. 1.5.1.1. Low flow rate While flow rates from approximately 1 | iL/min up to several mL/min are typically used with the assistance of a nebulizer, traditional nanospray relied simply on the electrostatic attraction between the mobile phase and the counter-electrode to generate and disperse the charged droplets. The nanoflow regime can be generally defined as using flow rates of less than 1 nX/min and can extend to levels of less than 1 nL/min [51]. As mentioned previously, nanoflow ESI can be used independently (off-line) or coupled 14 to a separation system (on-line) such as CE or HPLC. Depending on experimental requirements, the commercially available nanospray emitters vary in material, tip shape, diameter, and the configuration used for electrical contact. On-line analyses typically use higher flow rates (100 nL/min - 1000 nL/min), and the tips are usually fabricated from fused silica or metal. Off-line analyses typically use flow rates in the low nL/min range, with coated or uncoated glass or quartz emitters. There are many different configurations for nanoflow LC/MS coupling [56], although they all include an L C column and a narrow diameter emitter tip. The simplest combination involves coupling an upstream L C column to the emitter tip using some type of low dead volume union [57]. The union can also serve as the electrical contact for the electrospray process, although it is also possible to use conductive tips or other electrode configurations as well [56, 58]. The advantage of decoupling the L C column from the sprayer is simple and inexpensive replacement of sprayer tips should they become damaged or plugged. However, the coupling must be done carefully to maintain chromatographic performance. An alternate combination involves packing the L C column material directly into the sprayer tip to eliminate any dead volume after the column [59-62]. Tapered sprayers containing L C column packing are available commercially from companies such as New Objective Inc. The column preparation typically involves passing a slurry containing column packing material through a tapered sprayer [63, 64]. Sometimes a small frit is included inside the tip of the sprayer to help confine the packing material. The elimination of post-column dead-volume helps to ensure optimal chromatographic performance. In addition, it has been suggested that the presence of packing material in the tapered sprayer acts similar to a filter, extending tip lifetimes due to prevention of plugging. The drawback with this configuration is that tip replacement due to blockage or damage requires replacement of the entire column. Electrical contact can be established at the sprayer tip or distal to the tip end. Amirkhani and co-workers recently compared the efficiency of four different sheathless electrospray emitter configurations for a nanoflow liquid chromatography system [65]. Two of the configurations used on-column emitters with the applied voltage either at the outlet or the inlet end of the column, and the other two configurations had emitters coupled to columns, with the electrical connection at either the sprayer tip or the low dead volume 15 union. It was demonstrated that all of the configurations worked equally well provided that the connections were made with minimal dead volume [65]. Presently, most major mass spectrometry manufacturers make the nanospray source available. The number of applications using electrospray at low flow rates is very large, and outside the scope of this paper. Wood et al.'s 2003 review article is a good starting point and a source of references [43]. 1.5.1.2.High Flow Rate If the flow rate is in the range of 0.05 - 3 mL/min, sensitivity can be an issue due to decreased ionization efficiency resulting from large droplet size. ESI/MS is widely interfaced with liquid chromatography, so high flow rates are often necessary. One solution to this problem is to employ a splitter that only allows a limited volumetric flow rate to reach the MS interface. Under these conditions, part of the eluent is wasted and connection dead volume is a common problem. A more practical approach adopted by manufacturers is to sample the spray from a region peripheral to the main droplet trajectory where the mist is much finer. This is done by simply re-orienting the sprayer relative to the interface such that the fine droplets from the exterior of the spray plume can enter the sampling inlet, while the majority of large droplets are directed away from the entrance [66]. To minimize contamination, many major MS manufacturers now sample orthogonal from the spray plume. An example of this is shown in Fig. 1.7 for an Agilent Technologies source incorporating an additional asymmetrical lens. 16 HPLC eluent orthogonal nebulizer asymmetrical lens nebulized analyte solution • atmospheric pressure 1.5 torr ion transfer capillary to MS Figure 1.7 Schematic of an ESI source configuration developed by Agilent The asymmetrical lens is essentially a 50% full, partial cylinder electrode operated at high voltage during the electrospray process [67]. The purpose of the electrode is to help initiate and sustain electrospray. Conversely, other groups have attempted to use various types of auxiliary electrodes [68-70] to focus ions at atmospheric pressure. Also exploiting the advantages of orthogonal sampling is the Waters/Micromass Zspray™ [71, 72] interface shown schematically in Fig. 1.8. 17 vent cleanable baffle * ESI probe I ! sampling cone isolation valve -— + t H 3 <• * - • Si ion block qjllr^l P extraction cone hexapole ion guide —1 cone gas Figure 1.8 Schematic of the Zspray™ source configuration used by Waters/Micromass The spray is first sampled orthogonally through the sampling cone into a low-pressure chamber. An extraction cone (skimmer) is oriented at a right angle relative to the axis of the spray to sample for a second time into the next differentially pumped vacuum stage. The double orthogonal sampling system prevents solvent and neutral molecules from entering the analyzer, resulting in reduced chemical background [73]. Larger cone apertures are used to compensate for transmission losses. The ability to disassemble and clean the sampling cone without breaking vacuum is an additional advantage contributing to the ruggedness of the Zspray™ source. Although some MS manufacturers such as MDS SCIEX, Waters/Micromass, and Agilent have opted for the orthogonal sampling, Thermo Electron uses a 60° sampling angle from the ion optics axis. In their Ion Max™ source, the function of the angled position of the sprayer is similar to the orthogonal one. The sprayer orientation is not the only important parameter when it comes to optimizing sensitivity while using high flow rates. For conventional high flow rate ESI 18 sources, the nebulizer gas is an essential component. Air or nitrogen is typically used to disperse the emerging solution into small droplets, and to direct the droplets on the trajectory chosen for optimal sampling. To vaporize the large amounts of solvent emerging from the sprayer as efficiently as possible, manufacturers have introduced additional features to assist the nebulizer gas. MDS SCIEX developed the TurboIonSpray™ source [17, 18], in which heated nitrogen gas that is released from a unit external to the sprayer, is used to assist evaporation of the spray droplets at atmospheric pressure. More recently, MDS SCIEX took this design a step further, creating the TurboV™ source shown in Fig. 1.9. In this case, two heated auxiliary nitrogen sources are oriented to achieve very efficient desolvation. The improved desolvation permits stable and sensitive operation for flow rates of greater than 1 ml/min [74]. Figure 1.9 Schematic of the TurboV™ source used by MDS SCIEX Thermo Electron also integrated auxiliary nitrogen gas for desolvation purposes, except it flows directly from the nozzle of the Ion Max™ source [75]. Similar to the 19 TurboV™, the Ion Max™ uses a high temperature source heater incorporated into the housing of the ion source, allowing the use of higher flow rates than previously possible. 1.5.2. Interface Various ion sampling interface configurations have been developed [5]. The sampling orifice can be built in a disc, in the top/bottom of a cone, or in glass/metal tubes. Some mass spectrometer manufacturers now equip their instruments with conical interfaces. Intuitively, the conical shape is meant to improve the electric field density at the sampling orifice in an attempt to improve ion transmission. To avoid potential contamination, the sampling cone can be heated to evaporate residuals from its surface. Since high temperatures are required for this purpose, ceramic materials have often been used for the interface. Ceramic produces no outgassing, and is thought to have low sample adsorptivity, thus reducing memory effects on the interface surface [76]. 1.6. Multiple Sprayers Commercial multiple sprayer systems for ESI have evolved mainly as a result of industry's increasing requirements for high throughput sample analysis, particularly for pharmaceutical samples. With a multiple sprayer source and suitable equipment, LC/MS analyses can be done quickly and efficiently. The first attempts at building multiple ESI sprayers, however, were encouraged by other reasons. Smith et al. [26, 77] were the first to describe the coupling of two ESI sprayers in order to generate ions of opposite polarity and study ion-ion chemistry. McLuckey et al. also interfaced up to three ion sources (two ESI sources and one atmospheric sampling glow discharge ionization (ASGDI) source) onto a quadrupole ion trap mass spectrometer in order to control charge states of reactant and product ions [78, 79]. Using a linear array of capillary electrodes, Rulison et al. demonstrated the feasibility of increasing sample throughput by using parallel capillaries [80] He also observed that the Taylor cones at the ends of the array were being deflected due to end effects caused by the electric field. In 1994, Kostiainen and Bruins proposed that multiple sprayers could be used to improve the ion current stability under high flow rate (above 200 _l/min) conditions [81]. Four years later, Kassel and Zeng developed a dual ESI source for the purification and 20 the simultaneous analysis of combinatorial libraries using two separate LC/MS eluent streams [82]. Although this development paved the way for further efforts in high-throughput parallel analysis, Kassel's system required the knowledge of the particular analyte each sprayer was generating. Dual ESI sources have also been used to introduce an internal calibration solution separately from the analyte solution [83-86]. This configuration provides benefits over a single solution containing both analyte and calibrant because it avoids preferential ionization. In 1999, Kassel et al. [87] and De Biassi et al. [88] described indexed multiple sprayer systems that allow sequential sampling of each spray by a mass spectrometer, thus making possible the identification of ions from their respective eluent stream. Presently, publications in which fast LC/MS analyses of samples are discussed indicate that the Waters/Micromass multiplexed electrospray ion source (MUX) is the most commonly used for the analysis of multiple LC eluent streams [89-91]. Tiller's 2003 review paper provides useful references regarding biological applications using the M U X system as well as other types of parallel analysis [92]. The M U X system attaches to the Zspray™ source of the mass spectrometer and is fitted with either four or eight channels. As a rotating aperture allows only one spray at a time to reach the sampling cone, spectra of compounds eluting from individual separation columns can be quickly acquired. However, a significant problem with MUX-type systems is a decrease in sensitivity of approximately three fold at high flows [93]. Yang et al. attributed the drop in sensitivity to several reasons such as difficulty in optimizing sprayer position and lower desolvation efficiency due to large amounts of solvent introduced into the source in combination with a desolvation gas that was counter-current relative to the spray instead of the traditional coaxial flow. The sensitivity decrease is expected to be larger for nanoflow operation, where sprayer position can be more critical. In 2002, Schneider et al. [94] developed an alternative multiple sprayer system that appeared to solve the sensitivity problem. By installing an atmospheric pressure ion lens [68, 95] on each sprayer of a dual and a four-sprayer ESI source, they were able to maintain high sensitivity by optimizing the applied lens potential as well as sprayer position. An additional advantage of the system was that sprayers could be enabled or 21 disabled by changing the lens potential, potentially a faster method than using mechanical devices. 1.7. Recent Developments to the ESI Source The quest for a superior source design capable of effectively focusing more of the electrospray-generated ion cloud through the sampling aperture of the instrument is still on going. Due to gas-phase collisions occurring under atmospheric pressure conditions and space-charge effects, only a small percentage of the spray plume is actually sampled. There is still significant room for improvement in ion sampling efficiency. One approach developed by Zhou et al. [96] involved installation of an industrial air amplifier between the electrospray probe and the sampling orifice. The concentric, high-velocity gas flow was meant to control the expansion of the spray plume as well as assist in the desolvation of ions. In addition, focusing of ions could be achieved by applying an optimized voltage of up to 3 kV onto the amplifier/Thus, when both the air amplifier and the high voltage were used, Zhou's ion source configuration seemed to provide some improvement in ion sampling efficiency. Suppression of background chemical noise was also observed. Schneider et al. developed a novel interface for nanoflow ESI (Fig. 1.10) that provides two separate atmospheric pressure regions for droplet desolvation as well as two regions for unwanted particle removal [97]. 22 Curtain Plate Heated Laminar Flow Chamber Curtain Gas / o Orifice Tee and Nanospray Tip with Nebulizer Particle Discriminator Space Teflon Spacer Figure 1.10 Schematic of the particle discriminator interface developed by MDS Sciex The first desolvation stage makes use of the traditional counter-current gas to eliminate neutral particles and solvent. Next, charged particles, ions and gas travel through the heated laminar flow chamber where additional desolvation takes place at optimum temperature. Once they reach the particle discriminator area, ions are carried by the gas streamlines through the discriminator space and into the sampling orifice, while large charged particles with high momentum escape from the gas streamline trajectory under the influence of the strong electric field. This source design provided improved signal and ion current stability for flow rates from a few nL/min up to 1 LtL/min as well as improved performance for gradient elution nanoLC/MS in both positive and negative ion mode 1.8. Remarks The introduction to this thesis has depicted some of the most significant developments in the geometry of the ESI source from the time when it was first coupled with a mass analyzer, to current commercial designs. What it has also attempted to show is the great potential that still lies behind this essential analytical tool. Modifying the ESI [98]. 23 source for improving the transmission of ions from atmospheric pressure into vacuum is still an active research area. Research done in our group has also focused on developing a solution to this well-known problem. An atmospheric pressure ion lens installed behind the ESI emitter and operated at high voltage has previously shown promise at stabilizing the spray and improving the ion signal. More details on earlier work from our group in this area will be presented in the next chapter. 1.9. References [I] N . Mano, J. Goto, Anal. Sci. 19 (2003) 3. [2] R. B. Cole, I. Electrospray Ionization Mass Spectrometry Fundamentals, and Applications ; John Wiley &Sons, Inc.: New York, 1997. [3] M . Dole, L. L. Mack, R. L. Hines, J. Chem. Phys. 49 (1968) 2240. [4] R. B. Cole, J. Mass. Spectrom. 35 (2000) 763. [5] A . P. Bruins, in Cole, R. B.; Electrospray Ionization Mass Spectrometry Fundamentals Instrumentation & Applications; John Wiley & Sons, Inc.: New York, 1997; pp 107. [6] N. B. Cech, C. G. Enke, Mass Spectrom. Rev. 20 (2001) 362. [7] R. Juraschek, F. W. Rollgen, Int. J. Mass Spectrom. 177 (1998) 1. [8] G. A . Valaskovic, J. P. I. Murphy, J. Am. Soc. Mass Spectrom. 15 (2004) 1201. [9] M . Karas, U . Bahr, T. Dulcks, Fresenius J. Anal. Chem. 366 (2000) 669. [10] J. V . Iribarne, B. A . Thomson, J. Chem. Phys. 64 (1976) 2287. [II] L. L. Mack, P. Kralik, A . Rheude, M . Dole, J. Chem. Phys. 52 (1969) 4977. 112] G. Schmelzeisen-Redeker, L. Buffering, F. W. Rollgen, Int. J. Mass Spectrom. Ion Process. 90 (1989) 139. [13] B. A . Thomson, J. V . Iribarne, J. Chem. Phys. 71 (1979) 4451. [14] P. Kebarle, M . Peschke, Anal. Chim. Acta 406 (1999) 11. [15] N . Felitsyn, M . Peschke, P. Kebarle, Int. J. Mass Spectrom. 219 (2002) 39. [16] M . Gamero-Castano, F. J. de la Mora, J. Mass. Spectrom. 35 (2000) 790. [17] L. Y . T. L i , D. A . Campbell, P. K. Bennett, J. Henion, Anal. Chem. 68 (1996) 3397. 24 [18J J. P. Allanson, R. A . Biddlecombe, A . E. Jones, S. Pleasance, Rapid Commun. Mass Spectrom. 10 (1996) 811. [19] S. K. Chowdhury, V. Katta, B. T. Chait, Rapid Commun. Mass Spectrom. 4 (1990)81. [20] C. N . McEwen, B. S. Larsen,- in Cole, R. B.; Electrospray Ionization Mass Spectrometry; John Wiley and Sons, Inc., 1997; pp 177. [21] J. B. Fenn, M . Mann, C. K. Meng, S. F. Wong, C. M . Whitehouse, ; Mass Spectrometry Reviews; John Wiley & Sons, Inc., 1990; pp 37. [22] M . Yamashita, J. B. Fenn, J. Phys. Chem. 88 (1984) 4671. [23] C. M . Whitehouse, R. N . Dreyer, M . Yamashita, J. B. Fenn, Anal. Chem. 57 (1985)675. [24] J. Franzen ; Bruker Franzen Analytik G M B H : US5736740, 1998. [25] M . J. Tomany, J. A. Jarrell ; Millipore Corp.: US5304798, 1994. [26] L. R. R. Ogorzalek, H. R. Udseth, R. D. Smith, J. Phys. Chem. 95 (1991) 6412. [27] L. Ogorzalek, R. D. Smith, J. Am. Chem. Soc. 5 (1994) 207. [28] L. Jiang, M . Moini, Anal. Chem. 72 (2000) 20. [29] K. Taeman, H. R. Udseth, R. D. Smith, Anal. Chem. 72 (2000) 5014 . [30] I. C. Mylchreest, M . E. Hail ; Finnigan Corp.: US5171990, 1992. [31] I. C. Mylchreest, M . E. Hail ; Finnigan Corp.: USRE35413E, 1996. [32] A . Mordehai, S. J. Buttrill ; Varian Associates: US5672868, 1997. [33] A . P. Bruins, T. R. Covey, J. D. Henion, Anal. Chem. 59 (1987) 2642. [34] R. D. Smith, C. J. Barinaga, H. R. Udseth, Anal. Chem. 60 (1988) 1948. [35] P. Schmitt-Kopplin, M . Frommberger, Electrophoresis 24 (2003) 3837. [36] J. A . Olivares, N . T. Nguyen, C. R. Yonker, R. D. Smith, Anal. Chem. 59 (1987) 1230. [37] M . Moini, Anal. Bioanal. Chem. 373 (2002) 466. [38] A . von Brocke, G. Nicholson, E. Bayer, Electrophoresis 22 (2001) 1251. [39] M . Mazereeuw, A . J. Hofte, U . R. Tjaden, J. van der Greef, Rapid Commun. Mass Spectrom. 11 (1997)981. [40] E. D. Lee, W. Muck, J. D. Henion, T. R. Covey, J. Chromatogr. 458 (1988) 313. 2 5 [41] E. D. Lee, W. Muck, J. D. Henion, T. R. Covey, Biomed. Environ. Mass Spectrom. 18 (1989) 844. [42] J .Cai . J . Henion, J. Chromatogr. A 703 (1995) 667. [43] T. D. Wood, M . A . Moy, A . R. Dolan, P. M . J. Bigwarfe, T. P. White, D. R. Smith, D. J. Higbee, Applied Spectroscopy Reviews 38 (2003) 187. [44] M . R. Emmett, R. M . Caprioli, J. Am. Soc. Mass Spectrom. 5 (1994) 605. [45] E. Andren, M . R. Emmett, R. M . Caprioli, J. Am. Soc. Mass Spectrom. 5 (1994) 867. [46] E. Gelpi, J. Mass Spectrom. 37 (2002) 241. [47] J. Abian, A . J. Oosterkamp, E. Gelpi, J. Mass Spectrom. 34 (1999) 244. [48] P. E. Andren, R. M . Caprioli, J. Mass Spectrom. 30 (1995) 817. [49] M . S. Wilm, M . Mann, Int. J. Mass Spectrom. Ion Process. 136 (1994) 167. [50] M . Wilm, M . Mann, Anal. Chem. 68 (1996) 1. [51] K. Vanhoutte, W. Van Dongen, E. L. Esmans, Rapid Commun. Mass Spectrom. 12 (1998) 15. [52] A . Schmidt, M . Karas, T. Dulcks, J. Am. Soc. Mass Spectrom. 14 (2003) 492. [53] Y . L i , R. B. Cole, Anal. Chem. 75 (2003) 5739. [54] W. M . A . Niessen, J. Chromatogr. A 794 (1998) 407. [55] W. M . A . Niessen, J. Chromatogr. A 1000 (2003) 413. [56] M . T. Davis, D. C. Stahl, S. A . Hefta, T. D. Lee, Anal. Chem. 67 (1995) 4549. [57] J. N . I. Alexander, G. A . Schultz, J. B. Poly, Rapid Commun. Mass Spectrom. 12 (1998) 1187. [58] A . J. Oosterkamp, E. Gelpi, J. Abian, J. Mass Spectrom. 33 (1998) 976. [59] K. B. Tomer, M . A . Moseley, L. J. Deterding, C. E. Parker, Mass Spectrom. Rev. 13(1994)431. [60] L. J. Licklider, C. C. Thoreen, J. Peng, S. P. Gygi, Anal. Chem. 74 (2002) 3076. [61] T. Natsume, Y . Yamauchi, H. Nakayama, T. Shinkawa, M . Yanagida, N . Takahashi, T. Isobe, Anal. Chem. 74 (2002) 4725. [62] 1.1. Stewart, L. Zhao, T. Le Bihan, B. Larsen, S. Scozzaro, D. Figeys, G. D. Mao, O. Ornatsky, M . Dharsee, C. Orsi, R. Ewing, T. Goh, Rapid Commun. Mass Spectrom. 18 (2004) 1697. 26 [63] C. L. Gatlin, G. R. Kleemann, L. G. Hays, A . J. Link, J. R. Yates, Anal. Biochem. 263 (1998) 93. [64] T. Le Bihan, H. S. Duewel, D. Figeys, J. Am. Soc. Mass Spectrom. 14 (2003) 713. [65] A . Amirkhani, M . Wetterhall, S. Nilsson, R. Danielsson, J. Bergquist, J. Chromatogr. A 1033 (2004) 257. [66] R. D. Voyksner, H. Lee, Anal. Chem. 71 (1999) 1441. [67] J. L. Bertsch, S. M . Fisher, K. D. Henry, E. M . Wong ; Hewlett-Packard Company: US5838003, 1998. [68] B. B. Schneider, D. J. Douglas, D. D. Y . Chen, J. Am. Soc. Mass Spectrom. 13 (2002) 906. [69] X . Xu , J. Zhai, W. Shui, G. Xu, P. Yang, Anal. Lett. 37 (2004) 2711. [70] W. J. Thompson, J. W. Eschelbach, R. T. Wilburn, J. W. Jorgenson, J. Am. Soc. Mass Spectrom. 16 (2005) 312. [71] T. Bantan, R. Milacic, B. Mitrovic ' , B. Pihlar, J. Anal. At. Spectrom. 14 (1999) 1743. [72] M . E. McComb, L. J. Donald, H. Perreault, Can. J. Chem. 77 (1999) 1752. [73] M . Holcapek, K. Volna, P. Jandera, L. Kolarova, K. Lemr, M . Exner, A . Cirkva, J. Mass Spectrom. 39 (2004) 43. [74] P. K. S. Blay, S. Brombacher, D. A . Volmer, Rapid Commun. Mass Spectrom. 17 (2003) 2153. [75] C. Y. Yang, Y. Wang, M . Splendore, R. A. Thakur,, ASMS; 2003. [76] M . P. Balogh, L C GC Europe 17 (2004) 24. [77] L. R. R. Ogorzalek, H. R. Udseth, R. D. Smith, J. Am. Soc. Mass Spectrom. 3 (1992)695. [78] E. R. Badman, P. A . Chrisman, S. A . McLuckey, Anal. Chem. 74 (2002) 6237. [79] J. M . Wells, P. A . Chrisman, S. A . McLuckey, J. Am. Soc. Mass Spectrom. 13 (2002). [80] A . J. Rulison, R. C. Flagan, Rev. Sci. Instrum. 64 (1993) 683. [81] R. Kostiainen, A. P. Bruins, Rapid Commun. Mass Spectrom. 8 (1994) 549. [82] L. Zeng, D. B. Kassell, Anal. Chem. 70 (1998) 4380. 27 [83] A . I. Nepomuceno, D. C. Muddiman, R. H. Bergen, III, J. R. Craighead, M . J. Burke, R E. Caskey, J. A . Allan, Anal. Chem. 75 (2003) 3411. [84] M . Moini, L. Jiang, Anal. Chem. 72 (2000) 20. [85] J. W. Flora, A . P. Null, D. C. Muddiman, Anal. Bioanal. Chem. 373 (2002) 538. [86] F. Zhou, W. Shui, Y . Lu, P. Yang, Y . Guo, Rapid Commun. Mass Spectrom. 16 (2002) 505. [87] T. Wang, L. Zeng, J. Cohen, D. B. Kassell, Comb. Chem. High Throughput Screen. 2 (1999) 327. [88] V. de Biasi, N . Haskins, A . Organ, R. Bateman, K. Giles, S. Jarvis, Rapid Commun. Mass Spectrom. 13 (1999) 1165. [89] B. Feng, A. H. Patel, P. M . Keller, R. J. Slemmon, Rapid Commun. Mass Spectrom. 15(2001)821. [90] B. Feng, S. McQueney, T. M . Mezzasalma, R. J. Slemmon, Anal. Chem. 73 (2001)5691. [91] D. Morrison, A . E. Davies, A . P. Watt, Anal. Chem. 74 (2002) 1896. [92] P. R. Tiller, L. A . Romanyshyn, U . D. Neue, Anal. Bioanal. Chem. 377 (2003) 788. [93] L. Yang, T. D. Mann, D. Little, N . Wu, R. P. Clement, P. J. Rudewicz, Anal. Chem. 73 (2001) 1740. [94] B. B. Schneider, D. J. Douglas, D. D. Y . Chen, Rapid Commun. Mass Spectrom. 16 (2002) 1982. [95] B. B. Schneider, D. J. Douglas, D. D. Y . Chen, Rapid Commun. Mass Spectrom. 15 (2001) 2168. [96] L. Zhou, B. Yue, D. V. Dearden, E. D. Lee, A . L. Rockwood, M . L. Lee, Anal. Chem. 75 (2003) 5978. [97] B. B. Schneider, V. I. Baranov, H. Javaheri, T. R. Covey, J. Am. Soc. Mass Spectrom. 14(2003) 1236. [98] B. B. Schneider, X . Guo, L. M . Fell, T. R. Covey, J. Am. Soc. Mass Spectrom. submitted April (2005). 28 Chapter 2. Atmospheric Pressure Ion Lens for ESI Sources in Mass Spectrometry 2.1. Early work involving atmospheric pressure focusing devices As a result of coulombic repulsion effects in the ESI plume and the low sampling efficiency characteristic of skimmer devices, the largest ion transmission lossesin' ESI/MS occur in the atmospheric pressure region. A number of groups have developed atmospheric pressure electrostatic devices to deal with this problem, but in general their function and benefit were not well understood [1.-4]. In 1984, Yamashita and Fenn incorporated a cylindrical electrode surrounding the ESI emitter (Fig. 1.3, [1]) but no details were given as to any advantages this design might have brought. Smith et al. installed a focusing ring downstream from the ESI emitter onto a CE/MS instrument [2]. The focusing ring seems to be equivalent to what is now commonly known as the curtain plate or the interface plate. Curtain gas flows between the focusing ring and the ion-sampling nozzle much as it does in more recent ESI source designs between the interface and orifice plates. Beavis et al. used a ring of fine wire located behind the ESI tip to help focus the spray plume into small spots onto a sample substrate [3]. The sample spots were subsequently analyzed by secondary ion mass spectrometry (SIMS). Franzen built a ring electrode on the inside wall of a heated capillary inlet that was meant to modify the electrical field such that ions would be more efficiently drawn into the capillary [4]. It was not specified if any signal increase was observed. Using an ESI-based levitation device for charged particles, Feng and Agnes integrated a series of four annular electrodes with decreasing diameters to guide the progeny droplets towards the sampling orifice of the vacuum chamber [5]. The authors observed improved ion currents obtained from the isolated droplets when the four-electrode arrangement was used compared to configurations lacking the auxiliary electrodes. 2.2. Previous work from our laboratory Schneider installed an oblong-shaped stainless steel ring electrode behind the tip of the ESI capillary and tested it at reduced flow rates as well as for nebulizer assisted electrospray with promising results [6,7]. The device was named atmospheric pressure 29 ion lens because its role was to change the direction of ion motion in the atmospheric pressure region, before the ions enter into the lower pressure region of the mass spectrometer. At reduced flow rates (200 nl/min), it was found that the total number of ions entering the mass spectrometer increased by a factor of 3.5 and the signal-to-noise ratio (S/N) increased by a factor of 3, compared to when no lens was being used. By changing the potential applied to the ion lens, different charge states of the analyte were favored. For example, higher charge states for sugars and proteins were generated when a high lens potential was applied. It was unclear, however, if the increase in magnitude of the higher charge states was due to increased focusing by the ion lens or due to a change in the mechanism of the ESI process/The stability of the signal was also found to increase using the ion lens. When used in combination with a nebulizer-assisted ESI source (1 - 5 uJ/min), the relative standard deviation (RSD) of the signal was reduced by approximately a factor of 2, while the signal was found to increase from 1.5 to 4 times. An additional advantage of using the ion lens was found to be increased flexibility in the position of the emitter. The ion lens was capable of maintaining the signal when the position of the ESI emitter was changed both vertically and horizontally without major loss in intensity. This is significant because in areas such as high throughput separation analysis interfaced to ESI/MS, optimizing the position of multiple sprayers is difficult to achieve. The ion lens could potentially become an inexpensive way to decrease the signal dependency on the sprayer position, thus improving the flexibility of incorporating various types of separation techniques interfaced with ESI/MS. In order to understand the effect of the ion lens on the ion trajectories, simulations of the equipotential lines around the ESI emitter were done using MacSIMION 2.0 software for the source geometries corresponding to reduced flow rate and nebulizer assisted ESI (Fig. 2.1). 3 0 tquipotentiai Lines l o n L e n s , 5100 V lon Trajectories Equipotential Lines Orifice Plate 190 V Sprayer 3000 V Curtain Plate 1000 V Housing 0 V a) Orifice Plate 190 V Sprayer .3500 V Orifice Curtain Plate 2000 V Housing 0 V tquipotentiai Lines b) Equ ipo ten t i a l L i n e s Orifice Plate 190 V Curtain Plate: 1000 V Orifice Plate 190 V Sprayer 5000 V Curtain Plate 1000 V Ion Trajectories Housing 0 V Ion Trajectories lon Lens 5000 V c) Sprayer 5000 V Housing 0V d) Figure 2 . 1 a) Schematic of a typical reduced flow rate ESI source demonstrating the defocusing nature of the equipotential lines near the tip of the sprayer; b) reduced flow rate ESI source with an ion lens around the tip of the sprayer, reducing the defocusing of the equipotential lines; c) schematic of equipotential lines generated for a standard ion spray source; d) equipotential lines generated for an ion spray source with an atmospheric pressure ion lens. The ion trajectories are qualitative. 31 In both cases, the lens has a flattening effect on the equipotential lines around the sprayer, which seems to indicate an overall focusing effect of the ions by reducing their tendency to spread out. Since the ion trajectory follows the electrical field, which is perpendicular to the equipotential lines, the ion motion is directed towards the sampling orifice rather than allowed to diverge freely and collide into the interface plate. 2.3. Other recent developments Agilent has commercialized an asymmetrical ion lens [8], as discussed in Chapter 1 (Fig. 1.7). It has also patented a number of other atmospheric pressure electrostatic devices including a circular auxiliary electrode located behind the ESI emitter tip [9]. The operation of this modified ESI source involves applying a field enhancing potential to the auxiliary electrode for the purpose of increasing the electric field gradient between the capillary tip and the counter electrode. The field enhancing potential may be alternated with a focusing potential applied to the auxiliary electrode, which is meant to converge the electrospray plume towards the sampling orifice. Finally, in a recent publication, Thompson et al. investigated the effects of a hemispherically shaped electrostatic lens at atmospheric pressure for nanoelectrospray [10]. By monitoring the current density inside the electrospray plume as well as the ion transmission inside the mass spectrometer, it was determined that the average current inside the plume was increased by approximately three times as a result of compression of the space-charge. Electrical filed simulations showed flattening of the equipotential lines around the sprayer tip confirming Schneider's observations. Thompson et al. also propose that lens functioning is dependent on the spray mode. Whereas the lens may be effective at focusing ions for certain spray modes such as the "cone-jet" or "cusp-jet" modes, as defined by Juraschek and Rollgen [11], it may have no effect on other modes. This implies that maintaining the spray stability is critical during the operation of the ESI source involving the ion lens. 2.4. Objectives of the present project Although Schneider et al. showed a number of advantages of using an atmospheric pressure ion lens, work is still required to understand more clearly the effect 32 and mechanism of the lens. While a simulation program is helpful and will be used, it is difficult to accurately simulate the ions' trajectory at atmospheric pressure because of space charge effects and collisions with neutral gas molecules. Experimental work is thus still necessary in order to better understand the capabilities of the atmospheric pressure ion lens. One important question, for example, is i f the effect of ion lens is universal. We want to find out how well the ion lens works under various conditions such as using different ESI source geometries. This particular project looks at both ion spray and traditional nanoelectrospray geometries each operating on a different type of mass spectrometer. We also want to understand what the optimum working conditions of the lens are, and how easy it is to achieve these conditions. This also involves testing different ion lens sizes. Another important issue is to understand the impact of the lens on different analytes. In this work, we will study a number of biological molecules with various experimental conditions. The changes in the charge states of each analyte will be monitored. Finally, the stability of the spray and the S/N ratio of the signal will also be studied. 2.4. References [11 M . Yamashita, J.B. Fenn, J. Phys. Chem. 88 (1984) 4671. |2 | R.D. Smith, C J . Barinaga, H.R. Udseth, Anal. Chem. 60 (1988) 1948. [3] R.C. Beavis, W. Ens, D.E. Main, K . G . Standing, Anal. Chem. 62 (1990) 1259. [4] J. Franzen, in, Bruker Franzen Analytik G M B H , US5736740, 1998. [5] X . Feng, G.R. Agnes, J. Am. Soc. Mass. Spectrom. 11 (2000) 393. [6] B.B. Schneider, D.J. Douglas, D.D.Y. Chen, Rapid Commun. Mass Spectrom. 15 (2001) 2168. [7] B.B. Schneider, D.J. Douglas, D.D.Y. Chen, J. Am. Soc. Mass Spectrom. 13 (2002) 906. |"8] J.L. Bertsch, S.M. Fisher, K D . Henry, E . M . Wong, in, Hewlett-Packard Company, US5838003, 1998. [9] G. L i , H. Yin , in, Agilent Technologies, Inc., US 6462337, 2000. 33 [10] W.J. Thompson, J.W. Eschelbach, R.T. Wilburn, J.W. Jorgenson, J. Am. Soc. Mass Spectrom. 16 (2005) 312. [11] R. Juraschek, F.W. Rollgen, Int. J. Mass Spectrom. 177 (1998) 1. 34 Chapter 3. Instrumentation, Experimental Procedures and Sample Preparation 3.1. Instrumentation Two types of mass spectrometers were used for this project: one was a single quadrupole mass spectrometer equipped with an ion spray source, while the other was a triple quadrupole mass spectrometer equipped with a conventional off-line nanoelectrospray source. 3.1.1. Operation principles of quadrupole mass filters The quadrupole mass analyzer consists of four cylindrical rods that serve as electrodes. Opposite rods are electrically connected, one pair being coupled to the positive end and the other to the negative terminal of a variable dc source. A variable radio-frequency (rf) voltage is also superimposed to each pair as shown in Fig. 3.1. U-Vcos(cDt ) Figure 3. 1 Schematic of a quadrupole mass filter 35 The potential at any point inside the quadrupole is given by the following equation: <Hx,y) = (3.1) where r0 is the field radius (distance from the centre of the quadrupole to any rod) and Q?0 is defined as: O 0 =(£/-Vcos(ft)/)) (3.2) where U is the pole to ground dc voltage component, V is the zero to peak rf component, and co is the angular frequency of the rf voltage. The motion of ions in the x and y directions is independent in the quadrupole field and is governed by the following equations: ^ + ^ ( c / - V c o s ( f t ) / ) ) = 0 dt mrn (3.3) ^--^(U-Vcos(cot)) = 0 dt mr0 where e is the electron charge and m is the ion mass. These equations can be used to obtain the Mathieu equation, shown below: d2u -.1 + (a-2qcos(2%))u = 0 (3.4) cot where £ = — , u = xory and SeU mr0 CO 4eV Q* = ~% = — mr0 co (3.5) The solution to the Mathieu equation is complex [1], but it is sufficient to say that the trajectories calculated from it are classified as being either stable or unstable. For stable motions, the amplitude of the ion oscillation is finite and the ion is able to pass through the quadrupoles. In the case of unstable motion, the oscillations grow exponentially until the ions eventually hit the rods and become neutralized, or they exit laterally from the 36 field. Since the stability of the ions depends only on the a and q parameters, the a-q plane will consist of regions of stability and instability. Overlapping the stability regions obtained for the x and y directions (which differ by a factor of-1) produces several regions in which ion motion is stable within the quadrupole field. The first region of stability is shown in Fig. 3.2. a 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Figure 3. 2 The first stability region of a quadrupole field. m„ m 2, and m 3 represent ions with different masses on an operating line. m 2 is within the stability region, while m, and m 3 are not. When r0, oo, U, and Vare fixed, ions of the same m/e will have the same operating point (a, q) on the stability diagram. Since alq is equal to 2U/V and is independent of mass, all masses will lie on the same operating (or mass scan) line of constant alq value. When the U/V ratio is increased, the operating line starts approaching the tip of the stability region allowing only a narrow range of ions with stable trajectories to be transmitted. The closer the mass scan line is to the tip of the stability region, the higher the resolution. By varying the magnitudes of U and V while maintaining their ratio constant (to maintain constant mass resolution), ions with different masses will enter the stability region and 37 will thus be transmitted. This process is called mass scanning and is used to obtain the mass spectra. 3.1.2. The Toby single quadrupole mass spectrometer Toby is a p prototype instrument very similar to the A P I 100 single quadrupole mass spectrometer model from MDS SC1EX. The main components of the instrument are shown in Fig. 3.3: ESI E m i t t e r C u r t a i n G a s E n t r y R o d s (Q0) R F R o d s Interface Plate O r i f i c e P l a t e T u r b o D e f l e c t o r Figure 3. 3 Schematic of the Toby prototype mass spectrometer 3.1.2.1. The ion source Bruins initially developed the ion spray source design in 1987 [2] and its fundamentals are described in the introduction part (Chapter 1) of this thesis. The optimization of the ion source parameters is critical for obtaining a stable signal. This involves adjusting the sprayer voltage and position, the nebulizer gas pressure, the capillary diameter and position, and the solvent composition of the sample. In addition, the interface and orifice plate voltages, the curtain gas pressure, as well as the 38 skimmer ring voltage must also be carefully adjusted. For this project, these parameters have been optimized for each analyte. It is important to note that some settings (such as sprayer voltage and distance from the interface) required frequent re-adjustment. 3.1.2.2. Features of the Toby prototype mass spectrometer Ions formed by the ion spray source pass through a dry nitrogen curtain gas, a sampling orifice (0.25 mm diameter), and expand into a region of approximately 5 Torr. The core of the expansion is then sampled by a skimmer (0.75 mm orifice diameter) into an RF only quadrupole ion guide (Q0) operating at approximately 7 x 10" Torr. In this region, ions are collisionally cooled to an energy of approximately 1 eV. The short rf rods help focus the ions into the mass filter quadrupole (Ql). The pressure in this chamber is approximately 7 x 10"6 Torr. Using a rotary pump for the interface region and turbo pumps to evacuate the Q0 and Q1 chambers, this apparatus has a three-stage differential pumping system. 3.1.2.3. Ion detection Ions were detected with a Channeltron™ channel electron multiplier (CEM) operated in ion counting mode. The C E M is a continuous dynode device shaped like a horn and capable of detecting both positive and negative ions. When an ion strikes the surface near the entrance, some electrons are ejected and then bounce along the surface of the detector. Each time an electron hits the surface, more electrons are ejected creating a "cascade" effect resulting in an amplification of the current pulse. For this project, the instrument was used in positive ion mode only, therefore the input of the detector was held at high negative potential and the output was at ground. The data recorded represents the total number of ions on a pre-set range of m/z ratios. 3.1.3. The PE SCIEX API III triple quadrupole mass spectrometer The API HI is a relatively old mass spectrometer model from MDS SCIEX, being introduced on the market in 1989. A schematic of the API III is shown in Fig. 3.4: 39 Curtain G a s Interface Col l is ion (CID) G a s Orifice Plate Nanoelectrospray emitter _ Faraday Plate Interface Plate L e n s E lement C r y o P u m p Figure 3. 4 Schematic of the API III triple quadrupole mass spectrometer 3.1.3.1. The ion source The ion source used with this mass spectrometer was a traditional off-line nanoelectrospray source as developed by Wilm and Mann [3] and described in the introduction part of this thesis. A schematic of the nanoelectrospray source set-up is shown in Fig. 3.5: 40 Figure 3. 5 a) schematic of the off-line nanoESI interface; b) image of the emitter positioned in front of the interface orifice as seen through the microscope The nanoelectrospray set-up consists of a needle pressurizing device (a plastic syringe), a positioning micromanipulator in the x, y, and z directions, and a microscope used to control the position of the needle in front of the vacuum orifice. The needle is filled with the sample solution, mounted on a holder, and perfectly aligned in front of the interface orifice at a distance of 1-2 mm. If the application of sprayer voltage is not enough to overcome flow resistance in the needle, the needle opening can be gently pressurized using the syringe in order to start the spray. A common problem with this type of source arises when the sample precipitates at the tip of the needle, blocking the flow. If pressurizing the device is not effective, lowering the spray voltage and gently touching or scraping the tip of the needle on the surface near the interface orifice may be an effective way of opening up the tip. If this does not work, the sprayer voltage may be increased, and the process repeated until the spray is re-established. However, great care must be taken operating the nanoelectrospray source, as breaking the fine needle tip can easily occur. For this project, metal coated (Au/Pd), borosilicate nanoelectrospray needles were purchased from Proxeon Biosystems in medium and long tip sizes. The flow rate 41 obtained with these tips is typically between 10 nl/min - 40 nl/min (the longer tips cover a lower flow rate range compared to the medium tips). These tips allow easy solution loading, and can maintain a stable spray for several hours without plugging. 3.1.3.2. Features of the API III mass spectrometer In contrast with the API 100, a single cryogenic pump operates to create the vacuum inside the entire API 111 instrument (i.e. it is not differentially pumped). Another common feature that is lacking in the API III is the presence of a skimmer. Ions produced by the nanoelectrospray source thus enter the sampling orifice after passing through heated nitrogen curtain gas and enter Q0 directly. Next, the ions are guided into the first quadrupole mass filter (Ql). The API III is a tandem mass spectrometer containing two quadrupoles that can act as mass filters (Ql and Q3) and an rf-only quadrupole (Q2) that can be used as a collision cell for ion fragmentation. In our case, however, we only operate the instrument in normal MS mode, where one quadrupole (Ql or Q3) is scanned over a certain mass range while the other quadrupole is kept open. Q2 acts as an ion guide only. 3.1.3.3. Ion detection As for the API 100, the ion detection was achieved using a C E M in ion counting mode. 3.2. Sample Preparation Three biological molecules were chosen for testing for this particular project: cytochrome c, insulin, and bradykinin. A l l three molecules have been previously characterized by ESI/MS, therefore the solvent compositions compatible with ESI analysis were known. However, insulin was found to be quite insoluble with the solvent composition suggested in literature (1:1 methanol and deionized water, and 1% acetic acid) [4], therefore acetonitrile was added to the composition and sonication was used to aid sample dissolution. A l l stock solutions were stored at 4°C. Sample preparation is summarized in Table 3.1 and the solvents used for sample preparation are listed in Table 3.2: 42 Table 3. 1 Summary of the solvent compositions and sample concentrations used for this project Analyte Molecular Weight Source Solution Composition Concentrations Cytochrome c from horse heart (95% purity) 12,318 Da Sigma 90% H 2 0 10% C H 3 O H ~ l x l 0 " 6 M Insulin from bovine pancreas (80-89% purity) 5733.5 Da Sigma 33% H 2 0 33% CH3OH 33% CH3CN 1 % CH3COOH ~ l x l 0 " 6 M Bradykinin* (acetate salt, 99% purity) 1062.2 Da Sigma 60% H 2 0 40% CH3OH 0 .1%CH 3 COOH ~ l x l O ' M a n d l x l 0 " 6 M *Bradykinin amino acid sequence: Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg Table 3. 2 Solvents used for sample preparation Solvent Type Source Water* HPLC Grade Fisher Scientific Methanol* HPLC Grade Fisher Scientific Acetonitrile HPLC Grade Fisher Scientific Acetic Acid Glacial, ACS Grade Fisher Scientific *Solvent was distilled in-lab due to contamination problems 3.3. Experimental set-up and procedures 3.3A. Ion spray source equipped with ion lens Figure 3.6 shows the ion spray source fitted with the atmospheric pressure ion lens [5]: 43 6 4 1 X 12 Figure 3. 6 Schematic of the ion spray source with an ion lens. The ion spray mount (1) is attached to the mass spectrometer with a stud inserted through the mounting hole (2). The electrospray potential is applied to a stainless steel tee (3) through the conductive mount (1). Two concentric stainless steel capillaries (4) hold the inner glass capillary and allow the nebulizer gas flow from the nebulizer gas line (5). The sample is introduced through the fused silica capillary (6) to the inner stainless steel tube (7). The ion lens (8) was located near the tip of the inner stainless steel tube (7). The ion lens had a mounting bracket (9) and an adjustable arm (10). Pivots (11 and 12) allowed the lens to be positioned in different locations. The length of protrusion of the inner stainless steel tube from the outer tube is labeled " X " . The sample was delivered to the sprayer through a fused silica capillary (Polymicro Technologies) with a 150 u,m outer diameter and a 50 |j,m inner diameter, at a flow rate usually set at 1 ul/min. A Harvard Apparatus syringe pump was used for sample infusion. The fused silica capillary passed through two concentric stainless steel tubes (Small Parts Inc.), with standard wall thickness of 21 and 27 gauges and protruded approximately 0.5 mm (length " X " is optimized for each experiment). A stainless steel tee containing the nebulizer gas flow line (Valco Instruments) was used to hold the sprayer in place. The nebulizer was either compressed air (breathing grade, Praxair) or nitrogen (high purity, Praxair). The electrospray potential was applied through the mountain bracket to the stainless steel tee. 44 t In addition to the original oblong shaped stainless steel ion lens used by Schneider, a number of lenses of various diameters were tested. These lenses were circular, as shown for comparison in Fig. 3.7: v _ J a) b) Figure 3. 7 Front view of a) an oblong shaped lens and b) a circular lens (8); the figure also demonstrates the location of the stainless steel sprayer tube (7) within the lens. The various lens sizes that were tested are summarized in Table 3.3: Table 3. 3 Diameter of lenses tested on the ion spray source Ion Lenses' dimensions (diameters ) 11 x 15 mm* 15 mm 12 mm 10 mm 8 mm 7 mm 6.4 mm 5 mm 3 mm *original lens used by Schneider 45 The lenses were positioned perpendicularly to the axis of the stainless steel sprayer and approximately 1-2 mm behind the sprayer tip. An adjustable arm on the mounting bracket was used to adjust the position of the ion lens relative to the sprayer as seen in Fig. 3.6. The high voltage to the lens was applied using a Spellman C Z E 1000R power supply. High purity nitrogen (Praxair) was used as a curtain gas. 3.3.2. Ion lens for nanoelectrospray source Figure 3.8 shows the nanoelectrospray source fitted with an ion lens: Figure 3 . 8 The nanoelectrospray source fitted with an adjustable ion lens Five to ten microliters of solution were loaded into a nanoelectrospray capillary using a gel-loading tip. The capillary was then inserted into the stainless steel holder, which in turn was tightened into the Teflon mount. A ring of brass surrounded the capillary holder 46 inside the Teflon mount, and was electrically connected to a screw placed on top of the mount. The sprayer voltage was applied by hooking the power supply cable to this screw (cable not shown). The ion lens voltage was applied directly to the lens arm, by hooking the cable to one of the positioning screws and tightening it in place (this cable also not shown). The emitter was then brought close to the interface (less than 1 mm distance from the interface plate) and centered at the orifice. The ion lens, arm, and positioning parts were machined from stainless steel. The lens was circular, and measured 6 mm inner diameter, 15 mm outer diameter, and 2 mm thickness. When the ion lens was not being used, it was pushed backwards as to not interfere with the nanoelectrospray process. When the lens was being used, it was positioned behind the needle tip, at approximately 1 mm distance from the interface plate. Fig. 3.9 shows a simulated image of the relative positions of the lens and the tip to the interface plate using Simion 7.0, (Idaho National Engineering and Environmental Laboratory, Idaho Falls). Figure 3. 9 Two-dimensional and three-dimensional views of the nanoelectrospray source and lens positioned in front of the mass spectrometer orifice The curtain gas was high purity nitrogen (Praxair). 47 3.3.3. Instrument Parameters As mentioned earlier, ion source parameters were often changed either for re-optimization or to test the effect on ion transmission. However, operation of a mass spectrometer requires the optimization of a number of other important parameters. These parameters were adjusted for each analyte, and are summarized in Table 3.4 for the Toby and in Table 3.5 for the API III: Table 3. 4 Summary of parameters optimized for each analyte on the Toby mass spectrometer Instrument Component Parameter Optimized for Bradykinin Parameter Optimized for Insulin Parameter Optimized for Cytochrome c Ion Spray Voltage varied Voltage varied Voltage varied Interface Plate 1000 V 1000 V 1000 V Orifice Plate 30 V 100 V 100 V Skimmer ring 0 V 30 V 200 V Entry rods voltage -5 V - 5 V - 4 V Inter quadrupole -7 V -7 V -5 V lens Focus rods voltage -15 V -22 V - 8 V Ql rod offset -4.5 V -3.5 V -3.5 V voltage C E M -5000 V -4500 V -6000 V Q l resolution 134 129 129 Q l delta mass 0.18 0.08 0.09 48 Table 3. 5 Summary of parameters optimized for bradykinin on the API III mass spectrometer Instrument Component Parameter Optimized for Bradykinin Nanospray capillary Voltage varied Interface plate 100 V Orifice plate 20 V QO rod offset voltage 0 V Q l resolution 132 Q l delta mass 0.26 Q l rod offset voltage 0 V Lens element 7 -40 V Q2 rod offset voltage -50 V Q3 resolution 116 Q3 delta mass 0.12 Q3 rod offset voltage - 100 V Lens element 9 -250 V Faraday plate -250 V C E M -5000 V 3.4. References [1] P.H. Dawson, Quadrupole Mass Spectrometry and its Applications, Elsevier Scientific Publishing Company, Amsterdam, 1976. [2] A.P. Bruins, T.R. Covey, J.D. Henion, Anal. Chem. 59 (1987) 2642. [3] M . Wilm, M . Mann, Anal. Chem. 68 (1996) 1. [4] K . W . Y . Fong, D. Chan, T. -W, J. Am. Soc. Mass. Spectrom. 10 (1999) 72. [5] B.B. Schneider, D.J. Douglas, D.D.Y. Chen, J. Am. Soc. Mass Spectrom. 13 (2002)906. 49 Chapter 4. Effects of an Atmospheric Pressure Ion Lens for ESI Sources 4.1. Impact of the ion lens on the charge states of gas-phase molecules The ion spray source equipped with Schneider's original atmospheric pressure ion lens was used to ionize solutions of cytochrome c, insulin and bradykinin. Each of the three analytes differs with respect to size, structure, and the number of charge states it is capable of bearing in gas-phase. Our interest was to monitor changes in the charge states of each analyte in order to determine the effective range of the ion lens. In other words, we want to know if there are any regions in the studied range that are discriminated or favoured with the use of the ion lens. The mass spectra of the three analytes are shown in Fig. 4.1. The analytes used cover a charge state range that is extensive enough for us to make reasonable conclusions. Various parameters can lead to shifts in the analyte charge state. The affecting parameters include solvent composition, pH, analyte concentration, instrument geometry, curtain gas pressure, and orifice-skimmer voltage difference [1]. Fig. 4.1 a) and b) represent the mass spectra of cytochrome c obtained with the same instrumental parameters, but acquired before and after electronic repairs to the instrument. In order to obtain meaningful results, we attempted to maintain all instrumental parameters constant once they were optimized, while varying only the parameter of interest. 4.1.1. Ion lens vs. original source configuration, and the effect on charge state As mentioned in Chapter 2, Schneider observed that the sensitivity gains with the ion lens were greater for higher charge states. We verified this observation through some experiments that were done to compare the effect of using an ion lens versus using the original source configuration on different charge states of cytochrome c. The improvement in peak intensity when using the ion lens at an optimum voltage was found to be indeed greater for higher charge states compared to the same peaks obtained using the original ion source at its optimum settings. The results from this experiment are summarized in Table 4.1. The intensity appears to increase as the charge state increases. 50 Table 4. 1 Improvements in cytochrome c peaks intensity using the ion lens Cytochrome c Charge State Intensity Improvement Factor Ion Lens /Ion Spray Optimum Voltage Combination +7 2 x 500 V/4000 V +8 5 x 250 V/4000 V +9 10 x 250 V/4250 V + 10 29 x 750 V/5000 V + 11 45 x 750 V/5000 V Using the ion lens at an optimum voltage also increased the spray stability, reflected in smaller RSD values calculated for multiple scans for the various charge states. Experiments have also shown that using the ion lens allows operation of the ion spray at a lower voltage. For example, analyzing cytochrome c would normally require an optimum ion spray voltage around 5000 V. Using the ion lens, however, allows the ion spray to be used starting at about 3500 V, as will be shown later. This may be one reason why using the lens promotes transmission of the higher charge states. It is possible that a high voltage difference between the emitter and the orifice plate normally leads to the higher charge states being particularly unstable due to high kinetic energy and collisions with ambient gas. Since the ion lens allows a lower ion spray voltage to be applied while still maintaining a stable spray, the higher charge states may be less energetic, they do not loose their charge through collisions, and more ions are transmitted. 51 a) 6.0E+06 5.0E+06 (A 4-1 § 4.0E+06 o o c 3.0E+06 o jS 2.0E+06 o I-1.0E+06 O.OE+00 500 Spectrum of Cytochrome C +9 +11 +12 1000 +10 +8 +7 +6 +5 +4 1500 m/z 2000 2500 b) 4.5E+05 4.0E+05 3.5E+05 1 3.0E+05 O 2.5E+05 O 2.0E+05 •§ 1.5E+05 -j *~ 1.0E+05 5.0E+04 0.0E+00 500 Spectrum of Cytochrome C TIT +13 +1.1 + 1.2 +1.4| + 15 +9 1000 +8 1500 m/z 2000 2500 Figure 4. 1 Mass spectra of the analytes tested for this project: a) horse heart cytochrome c; b) horse heart cytochrome c (at a different time period); continued on pg. 53. 52 c) O.OE+00 500 S p e c t r u m o f I n s u l i n 700 900 1100 1300 m/z 1500 1700 1900 2100 d) Spec t rum of Bradyk in in 1.4E+07 1.2E+07 *j 1.0E+07 3 O 8.0E+06 c O 6.0E+06 rc O 4.0E+06 H 2.0E+06 0.0E+00 +2 +i 200 400 600 800 m/z 1000 1200 1400 Figure 4.1 c) bovine pancreas insulin; d) bradykinin; continued from page 52. 53 4.1.2. Variation of ion spray and ion lens voltages, and the effects on cytochrome c charge states 4.1.2.1. Results for cytochrome c Trends in charge states intensities (+7 to +15) were monitored for cytochrome c by varying the ion spray voltage while maintaining the ion lens voltage at fixed values. These trends from one experiment are shown in Fig. 4.2. 54 1.5E+04 Cytochrome C +7 Charge State Trends at Various lon Spray and lon Lens Voltage Combinations lon Lens & c 3 O o c o 1.0E+04 5.0E+03 0.0E+00 3800 4300 4800 5300 5800 Ion Spray Voltage 6300 Voltage: —•—0 V - • - 2 5 0 V 500 V 750 V —*—1000 V —#—1250 V —I—1500 V ——1750V 2000 V 2250 V 2500 V 1.5E+05 # 1.0E+05 c 3 O o O 5.0E+04 Cytochrome C +8 Charge State Trends 0.0E+00 >>'''' ^ ^ ^ ^ 3800 4300 4800 5300 5800 6300 Ion Spray Voltage lon Lens Voltage: —•—6V —•—250 V 500 V 750 V -*—1000 V —•—1250 V —+—1500 V 1750 V 2000 V 2250 V 2500 V 5.0E+05 Cytochrome C +9 Charge State Trends lon Lens Voltage: 0.0E+00 3800 4300 4800 5300 5800 Ion Spray Voltage Figure 4. 2 Trends in the intensity of various charge states of cytochrome c obtained by varying the ion spray voltage while holding the ion lens at fixed voltages; continued, pg. 56. 55 8.0E+05 6.0E+05 CO +•» c 3 O o c o 4.0E+05 2.0E+05 0.0E+00 Cytochrome C +10 Charge State Trends 3800 4300 4800 5300 5800 I o n S p r a y V o l t a g e 6300 Ion Lens Voltage: - 4 - 0 V —•—250 V 500 V 750 V -*—1000 V —•—1250 V —f— 1500V 1750V —2000 V 2250 V 2500 V Cytochrome C +11 Charge State Trends 3.0E+05 i 2.0E+05 c 3 o o § 1.0E+05 0.0E+00 3500 4000 4500 5000 5500 6000 65| I o n S p r a y V o l t a g e Ion Lens Voltage: -0V 250 V 500 V 750 V -1000 V •—1250V -1500 V •1750 V 2000 V 2250 V 72800 Cytochrome C +12 Charge State Trends 3.0E+05 CO C 2.0E+05 3 O O C O 1.0E+05 0.0E+00 Ion Lens Voltage: 3800 4300 4800 5300 5800 6300 I o n S p r a y V o l t a g e s o v 250 V 500 V 750 V 1000 V 1250 V 1500 V 1750 V 2000 V 2250 V 2500 V Figure. 4.2 continued from pg. 55. 4.0E+05 Cytochrome C +13 Charge State Trends to +-» c 3 O o c o 3.0E+05 2.0E+05 1.0E+05 0.0E+00 3800 4800 5300 5800 I o n S p r a y V o l t a g e Ion Lens Voltage: - • — o\7 250 V 500 V 750 V 1000 V 1250 V -1500 V 1750 V 2000 V 2250 V 2500 V Cytochrome C +14 Charge State Trends 1.4E+05 1.2E+05 £ 1.0E+05 c 3 O o c o 8.0E+04 6.0E+04 4.0E+04 2.0E+04 0.0E+00 Ion Lens Voltage: 250 V 500 V 750 V - * - 1 0 0 0 V — 1 2 5 0 V —t—1500 V 1750V -2000 V 2250 V 2500 V 3800 4300 4800 5300 5800 I o n S p r a y V o l t a g e 6300 5.0E+04 Cytochrome C +15 Charge State Trends 0.0E+00 3800 4300 4800 5300 5800 6300 I o n S p r a y V o l t a g e Ion Lens Voltage: 0 V ^ -•—250 V 500 V 750 V *—1000 V 1250 V 1500 V 1750 V 2000 V 2250 V 2500 V Figure 4.2 continued from pg. 56. With the exception of the curve obtained with the lens floating at 0 V, which remains relatively constant, it is clear that the effect of the ion lens is different for the various charge states. The optimum ion spray voltage shifts from ~ 4000 V for the +7 charge state to -5500 V for the +15 charge state. The optimum ion lens voltage is relatively low for the lower charge states (ex. -250 V for the +7 charge state) and increases with increasing charge state (ex. -1250V for the +15 charge state). Thus, in order to obtain the best signal for a particular charge state, we need the most favourable ion spray / ion lens voltage combination. For cytochrome c, it seems that the higher the charge state is, the higher the ion spray and the ion lens voltages required for the most improvement. 4.1.2.2. Discussion of results for cytochrome c Considering an unmodified ion spray source where the ion spray voltage is increased over a certain range, an optimum voltage is achieved where there is maximum ion transmission; as the voltage continues to rise, a decrease in transmission is usually observed. This can be explained in terms of changing spray modes related to capillary current pulsations, as mentioned in Chapter 1 [2]. When the ion spray source is equipped with an ion lens operating at the right voltage, the spray mode responsible for a stable spray (ex. cone-jet mode) may be achieved and maintained at much lower voltage, and even higher ion spray voltages, relative to the optimum voltage of the original ion source emitter. With the right combination of ion lens and ion spray voltages, a stable spray mode can be maintained over a wide voltage range, depending on the requirements. For example, a combination of relatively high ion spray and ion lens voltages would be chosen for analyzing ions of high charge states. An explanation for this observation is that a high voltage applied to the emitter promotes the formation of highly charged, highly energetic ions that would normally diverge due to severe space charge repulsion effects. The lens operated at an optimum focusing voltage reduces the spread and helps the charged ions converge towards the sampling orifice, improving their transmission. When the applied lens voltage is too low relative to the ion spray voltage, the effect on the peak intensity is detrimental. This happens because the lens starts to compete with the orifice plate as a counter electrode. Since the distance between the emitter tip and the lens edge is smaller than the distance between the tip to the orifice 58 plate, the ions will be attracted easier to the ion lens rather than to the orifice plate. Referring to Fig. 4.2, the intensity curve obtained when the ion lens was floating at 0 V is consistently low and of similar shape for all the charge states because at this voltage, the ion lens is equally damaging to all of the charge states. This situation was simulated, and is shown in comparison with an ion source used without the ion lens in Fig. 4.3 (a, b). As the voltage on the ion lens increases, the ions begin to be repelled and focused towards the orifice, resulting in improved transmission (Fig. 4.3 c)). However, i f the ion lens voltage is too high, the lens becomes defocusing, and the ions transmission decreases (Fig. 4.3 d)). 59 Figure 4. 3 Simulation of the ion spray source equipped with the atmospheric pressure ion lens; the electrospray voltage was set to 4000 V , the interface plate was set at 1000 V and the orifice plate at 100 V ; a) ion transmission without a lens; b) ion transmission with lens at 0 V ; continued on pg. 61. 60 Figure 4.3 c) ion transmission with optimized lens voltage (4500 V); d) ion transmission with a defocusing ion lens voltage (5500 V). 61 4.1.3. Variation in ion spray and ion lens voltages, and the effects on insulin charge states 4.1.3.1. Results for insulin Fig. 4.4 shows the changes in the intensity of various charge states of insulin (+3 to +6) as the ion spray voltage was varied while maintaining the ion lens at fixed values. 62 Insul in +3 C h a r g e State T rends at V a r i o u s lon S p r a y a n d lon L e n s Vo l tage C o m b i n a t i o n s lon Lens Voltage: - • - O V 2.0E+06 0.0E+00 3000 3500 4000 4500 5000 5500 6000 6500 lon Sp ray Vo l t age Insul in +4 C h a r g e State T rends 4.0E+06 3.5E+06 0) 3.0E+06 i 2.5E+06 o " 2.0E+06 - 1.5E+06 1.0E+06 5.0E+05 3000 3500 4000 4500 5000 5500 6000 6500 lon Sp ray Vo l tage Figure 4. 4 T r e n d s i n the i n t e n s i t y o f v a r i o u s c h a r g e states o f b o v i n e i n s u l i n o b t a i n e d b y v a r y i n g the i o n s p r a y v o l t a g e w h i l e h o l d i n g the i o n l e n s at f i x e d v o l t a g e s ; continued on pg. 64. 63 Insul in +5 C h a r g e State T rends 3000 4000 5000 lon Spray Vol tage 6000 Insul in + 6 Cha rge State T rends *-> c O 2.0E+06 1.5E+06 <S 1.0E+06 c o 5.0E+05 0.0E+00 7000 3000 3500 4000 4500 5000 5500 lon Sp ray Vo l tage 6000 6500 Figure 4.4 continued from pg. 63. 64 Similar to the cytochrome c experiment, the ion lens allows operation of the electrospray over a wider voltage range. The higher the ion lens voltage is, the higher the ion spray optimum value needed for maximum transmission. However, in the case of insulin there are a few combinations of ion spray and ion lens voltages that will produce similar peak intensities (there is no obvious best combination). There are also more sudden jumps in peak intensities with increasing ion spray voltage as the charge state of insulin increases. 4.1.3.2. Discussion of results for insulin The sudden increase in ion transmission as the insulin charge state increases is an indication of the ions' "response" to changes in electrical field strength as well as of the nature of the analyte. It also indicates a particular threshold voltage needed by each charge state in order to be efficiently transmitted. Higher charge states will need a smaller increase in potential in order to be accelerated towards the orifice plate, which is reflected in the more abrupt increase in intensity when the ion spray voltage is increased. 4.1.4. Variation in ion spray and ion lens voltages, and the effects on bradykinin charge states In the case of bradykinin, the singly and doubly charged states were monitored. With the original ion source, bradykinin usually requires approximately 3500 V applied on the ion spray to obtain optimum signal. Using the ion lens, a stable spray was maintained over a higher range of ion spray voltages, as seen in Fig. 4.5. Optimum ion intensity with the use of the ion lens can be obtained with more than one combination of ion spray / ion lens voltage values, as was also observed for insulin. This may be considered as an advantage, as parameter optimization can be simplified. 65 Bradyk in in +1 Charge State Trends at Var ious lon Spray and lon Lens Vol tage Combina t ions 3.0E+05 2.5E+05 | 2.0E+05 ° 1.5E+05 o 1.0E+05 5.0E+04 0.0E+00 Voltage: -« -o v -•-250 V 500 V 750 V -*-1000 V —-1250V -+-1500 V 3000 4000 5000 6000 lon Spray Vol tage 7000 Bradyk in in +2 Charge State Trends 2.0E+07 ,A 1.5E+07 C ° 1.0E+07 c o ~ 5.0E+06 0.0E+00 3000 lon Lens Voltage: ^ - o v -•-250 V i 1 — 500 V 750 V -*-1000 V -—1250 V -•-1500 V — 4000 5000 6000 lon Spray Vol tage 7000 Figure 4. 5 Trends in the intensity of two charge states of bradykinin obtained by varying the ion spray voltage while holding the ion lens at fixed voltages 66 4.2. The Effect of Ion Lens Size A series of experiments were done to determine the relationship between the ion lens diameter and the efficiency of the lens, as well as how the size of a lens affects the charge state distribution of a gas-phase molecule. Cytochrome c was an ideal analyte in this study because of its many charge states present in the gas-phase. Fig. 4.6 compares changes in peak intensities for various cytochrome c charge states from using the conventional ion source and the source equipped with lenses of three different diameters. The ion lens voltage was optimized as the ion spray voltage was varied. Multiple scans (20 in total) were acquired in order to determine if there would be an increase in spray stability with any particular lens. The 15 mm diameter lens produced excellent results in this case, with an exponential increase in intensity with increasing charge state. Note that the 4-11 charge state was not even detectable without the ion lens (signal was buried in the background noise). Although the spray stability was quite good to start with at optimum ion spray voltage, RSD decreased by approximately 50% when the 15 mm diameter lens was used at its optimum setting. An equivalent experiment was also done with lenses of smaller diameters (7, 5, and 3 mm). The 7 mm lens was the only lens that produced slight improvements in peak intensities (results not shown). No obvious improvement in spray stability was observed (RSD was approximately in the same range) with the smaller diameter lenses. To better understand the effects of the ion lens, some experiments were also done where the ion spray voltage remained fixed, but the ion lens voltage was varied. Fig. 4.7 shows the results from one experiment. Here, trends seem to indicate that lower charge states are better transmitted using a larger diameter lens combined with a lower ion lens voltage, while higher charge states can be well transmitted with more than one lens size/lens voltage combinations. The smaller the lens is, the higher the ion lens voltage required for maximum ion transmission. These observations make sense, since as the ion lens diameter decreases (with ion spray voltage being fixed) the ions begin to be attracted to the lens rather than the orifice plate, decreasing transmission. However when a high enough lens potential is applied, the ions are repelled back towards the sampling orifice and transmission increases. It must be noted that experimentally, the ion lens voltage has 67 always been lower than the ion spray voltage. Altogether, these experiments indicate that a larger lens diameter is preferable because it allows a wider range of charge states to be transmitted, without discriminating against higher or lower charge states in particular. 68 c o a ) = 2 O < Lens S ize Effect at Optimum Lens Voltage for 3 OE+05 ^ ^ c ^ - ° i n e C + 7 C n a r g e s t a t e „ 2.5E+05 2.0E+05 1.5E+05 1. OE+05 5.0E+04 0.0E+00 4 • — n o lens 15 mm 12 mm 8 mm w • 3000 3500 4000 4500 5000 Ion Spray Vol tage 5500 6000 2 QE+oepytochrome C + 8 Charge State Trends c 1.5E+06 o - w co 1.0E+06 ir o a> o ^ 5.0E+05 0.0E+00 • — n o lens •—15 mm 12 mm 8 mm 3000 3500 4000 4500 5000 Ion Spray Voltage 5500 6000 c 3 O o o O) ca > < 2.5E+06 2.0E+06 1.5E+06 1.0E+06 5.0E+05 Cytochrome C + 9 Charge State Trends 0.0E+00 - • — n o l ens * 15 mm 12 mm 8 mm — / — « ^ 3000 3500 4000 4500 5000 Ion Spray Voltage 5500 6000 Figure 4. 6 Changes in cytochrome c peaks intensity with different ion lens diameters; the ion lens voltage was optimized for each ion spray voltage; continued on pg. 70. 69 Cytochrome C +10 Charge State Trends i2 o o o CO o > < 9.0E+05 8.0E+05 7.0E+05 6.0E+05 5.0E+05 4 .0E+05 3.0E+05 2.OE+05 1.OE+05 0.0E+00 n o lens 15 m m 12 m m 8 m m 3000 3500 4000 4500 5000 5500 6000 Ion Spray Voltage 13 o o © CO CIS '_ o > < Cytochrome C +11 Charge State Trends - • — 1 5 m m 12 m m 8 m m 3000 3500 4000 4500 5000 Ion Spray Voltage 5500 6000 Figure 4.6 continued from pg. 69. 70 & c 3 O o c o o o c o Cytochrome C +6 Charge State: Effect of Various lon Lens Sizes at Different lon lens Voltages and Fixed lon Spray 1.8E+04 1.6E+04 1.4E+04 1.2E+04 1.0E+04 8.0E+03 6.0E+03 4.0E+03 2.0E+03 0.0E+00 Voltage (3500 V) 15 mm 12 mm 10 mm 8 mm 7 mm 6.4 mm 5 mm 3 mm 500 1000 lon Lens Voltage 1500 2000 Cytochrome C +7 Charge State; lon Spray: 3500 V 4.5E+05 4.0E+05 3.5E+05 3.0E+05 2.5E+05 2.0E+05 1.5E+05 1.0E+05 5.0E+04 0.0E+00 15 mm m 12 mm \ 10 mm \ . 8 mm \ -*- 7 mm • 6.4 mm 1 5 mm 3 mm / ^ ^ ^ ^ 500 1000 lon Lens Voltage 1500 2000 Cytochrome C +8 Charge State; lon Spray: 3500 V ^ 3.0E+05 - 2.0E+05 1.0E+05 0.0E+00 500 1000 lon Lens Voltage 1500 2000 Figure 4. 7 Trends in cytochrome c peak intensity for various ion lens diameters at fixed ion spray voltage and varying ion lens voltage; continued on pg. 72 71 c 3 o o 9 OE+05 y t o c n r o m e C + 9 C n a r 9 e State; Ion Spray: 3500 V 8.0E+05 7.0E+05 6.0E+05 5.0E+05 4.0E+05 3.0E+05 2.0E+05 1.OE+05 0.0E+00 500 1000 Ion Lens Voltage 1500 2000 c 3 o 1.4E+05 1.2E+05 1.OE+05 8.0E+04 Cytochrome C +10 Charge State; Ion Spray: 3500 V ^ 6.0E+04 | - 4.0E+04 2.0E+04 0.0E+00 500 1000 Ion Lens Voltage 1500 2000 Cytochrome C +11 Charge State; Ion Spray: 3500 V 3.0E+04 2.5E+04 O 2.0E+04 c a u (A in 0.0E+00 500 1000 Ion Lens Voltage 1500 2000 Figure 4.7. continued from pg. 71 4.3. Spray Stability and Signal-to-Noise Measurements The spray stability was evaluated by calculating the RSD for multiple scans obtained using the 15 mm diameter ion lens and comparing them to the RSD obtained using the original ion source configuration. The S/N ratio was measured by dividing the analyte signal by three times the standard deviation of the blank at the mass of interest. In the case of cytochrome c, the results were mixed. Repeat experiments showed considerable increase in S/N for some of the more intense peaks (ex. ~ 25 times for +9 charge state), however, for weaker peaks of the spectrum (ex. +11 charge state), the S/N decreased slightly. In general, the RSD obtained using the ion lens was either the same or higher than values obtained using the original configuration, indicating that the spray stability was not actually improved. Comparing RSD from day-to-day experiments, it was found that the ion lens does not have much effect on the day-to-day reproducibility either. Similar experiments were done for both insulin and bradykinin. It was found that if the spray stability and signal intensities were good in the first place, the lens did little to improve them. 4.4. Nanoelectrospray ion source equipped with an atmospheric pressure ion lens To test the performance of the nanoelectrospray ion source equipped with an ion lens, bradykinin was used as analyte. Bradykinin is a small, soluble peptide fragment, which helped reduce the chances of plugging the capillary tip through precipitation. It also has only three charge states. In our experiments, only the +2 charge state was monitored because the +1 charge state was undetectable and the +3 charge state was buried in a high background at lower m/z range, which is typical of this type of nanoelectrospray analysis. Increasing the curtain gas pressure can reduce this high background noise. This, however, also results in reduced sensitivity. The distance of the sprayer tip to the sampling orifice is also critical in nanoelectrospray analysis. Thus, a balance must be found between these parameters in order to get optimum sensitivity and minimize the background noise. Our purpose was to use the atmospheric pressure ion lens to help enhance the sensitivity as well as to lower the dependency of the signal on the aforementioned parameters. Fig. 4.8 shows simulations of ion trajectories for 73 nanoelectrospray a) without an ion lens, and b) with an ion lens as seen in the potential energy view generated with SIMION 7.0. The simulation illustrates how at an optimum voltage, the ion lens changes the potential energy in the electrostatic field around the emitter and is able to focus the ions into the mass spectrometer. 74 Figure 4. 8 Potential energy view of the a) conventional nanoelectrospray set-up with nanospray at 600 V , interface plate at 100 V and orifice plate at 20 V; b) nanoelectrospray equipped with the ion lens at 800 V 75 4.4.1. Results and Discussion 4.4.1.1. Signal intensity and nanoelectrospray stability Initial experiments indicated that the effect of the lens was not pronounced if the spray was already very stable. However, at lower nanoelectrospray voltages, where the ion transmission was not optimum in the first place, using the ion lens resulted in increased signal intensity. Further experiments confirmed our initial observations and the results from one such experiment are shown in Fig. 4.9. Bradyk in in +2 Charge State at Var ious lon Lens and Nanoelec t rospray Vol tage Comb ina t i ons 1.3E+06 1.1 E+06 c o 9.0E+05 c o n 7.0E+05 o 5.0E+05 3.0E+05 AT ^ J a n o s p r a y /o l t age : —•— boU V * 700 V - ^ • 7 5 0 V 800 V - * - 8 5 0 V 1 500 1000 lon Lens Vol tage 1500 Figure 4. 9 Changes in bradykinin +2 charge state peak intensity while varying the ion lens voltage at fixed nanospray voltages. Note that the " 0 V " point indicates "no ion lens" being used in this case. The intensity curves in Fig. 4.9 indicate sharp increases in intensity as the ion lens voltage is increased with the nanoelectrospray emitter being held at lower voltages. A s the nanoelectrospray voltage increases and the ion transmission improves, the ion lens produces only slight improvements, and the intensity remains relatively constant. The ion 76 lens can thus be used to maintain a constant signal over a more flexible range of nanoelectrospray voltages. One problem that was associated with the use of the ion lens was the evaporation of the solution inside the glass capillary. This is normally not an issue; however, in this case, the ion lens is so close to the hot interface plate that it also becomes hot and, in turn, it heats up the tip of the capillary. The tip of the capillary only contains a few microliters of the analyte solution, which, through evaporation, reduces the analysis time. The spray stability was also compared with and without the ion lens by calculating the RSD of multiple scans for a number of nanoelectrospray/ion lens voltage combinations. Blank solutions were also run under the same parameters. The results are shown in Table 4.2: Table 4. 2 Comparison of spray stability for the conventional nanoelectrospray source vs. using the ion lens at various voltage combinations ton Lens Voltage Nanospray Voltage RSD Bradykinin RSD Blank N / A 600 0.38 0.42 100 600 0.14 0.09 200 600 0.09 0.56 300 600 0.10 0.05 N / A 700 0.17 0.38 100 700 0.09 0.11 200 700 0.10 0.07 300 700 0.14 0.04 N / A 800 0.21 0.33 100 800 0.21 0.05 200 800 0.13 0.07 300 800 0.09 0.01 500 800 0.08 0.02 Generally, the ion lens greatly lowered the RSD of the bradykinin signal, indicating a stabilizing effect of the nanoelectrospray. The average absolute intensity of the bradykinin peak increased two to three times when using the optimum ion lens voltage compared to the conventional ion source set-up. For the blank, the lens had a similar effect: the average absolute intensity at the mass of interest increased, and the RSD decreased. This indicated that despite the lenses 77 stabilizing effect, it also enhanced the chemical background, and thus the S/N ratio is not actually improved. 4.4.1.2. Relationship between distance of the emitter to orifice and ion lens effect Distancing the emitter from the sampling orifice normally results in decreased sensitivity because less of the spray plume reaches the orifice. One advantage associated with having the emitter further back is that less solvent enters the mass spectrometer resulting in a cleaner background, especially at the low m/z range. However, distancing the emitter also results in less analyte ions entering the mass spectrometer, a problem we tried to address by using the ion lens to increase transmission. We found that while the ion lens was useful at increasing the ion transmission as the emitter was pulled away from the orifice (as shown in Fig. 4. 10), the background noise at the lower m/z range also increased, confirming the earlier observations. Experiments have thus shown that for nanoelectrospray the ion lens appears to focus not only the analyte ions, but also any other charged species that may be formed in the process, an effect most likely associated with the position of the lens and sprayer relative to the interface plate. Nevertheless, the ion lens may still be used to decrease the signal dependency on the capillary position, making spray optimization easier. Fig. 4.10 shows that as the capillary tip is pulled farther away from the orifice, the ion lens voltage can only be increased up to a certain value before losing the signal. This value decreases with increasing distance. It is possible that increasing the ion lens voltage beyond a certain point results in the ions being defocused, this effect being more pronounced when the distance between capillary tip and interface is increased. 78 Effect of Distancing Sprayer for Bradykinin +2 Charge State; Nanospray Held at 500V 1.E+06 T 0 m m 0 . 2 5 m m 0 .50 m m 0 . 7 5 m m 400 600 800 Ion Lens Voltage 1000 1200 Effect of Distancing Sprayer at 600 V 0 m m 0 . 2 5 m m 0 .5 m m 0 . 7 5 m m 1.0 m m 200 400 600 800 lon Lens Voltage 1000 1200 2.E+06 0.E+00 Effect of Distancing Sprayer at 700 V 0 m m 0 . 2 5 m m 0 .5 m m 0 . 7 5 m m 1 m m 1.25 m m 200 400 600 800 lon Lens Voltage 1000 1200 Figure 4. 10 Changes in bradykinin peak intensity with distancing of the nanoelectrospray emitter from the orifice combined with the use of the ion lens at various voltages. Distance increments of 0.25 mm were used. 79 4.5. References [1] G. Wang, R.B. Cole, in R.B. Cole (Editor), Electrospray Ionization Mass Spectrometry, John Wiley & Sons, Inc., New York, 1997. [2] R. Juraschek, F.W. Rollgen, Int. J. Mass Spectrom. 177 (1998) 1. 80 Chapter 5. Conclusions and Future Work Despite continuous instrumental problems interfering with our experiments, we were able to show that an atmospheric pressure ion lens has the potential to increase the spray stability and ion transmission in ESI/MS. The key to maximizing this potential is having well-controlled instrumental and environmental parameters, which in our case has not been trivial. Since the ion lens has a different effect on various analytes and charge states, optimization may be time consuming and thus impractical. The advantages and disadvantages of the ion lens must first be considered before using it for a particular application. Future work could involve writing a custom program for SIMION to accurately determine the most favorable ion lens voltage needed in various situations. As a result, the optimization time would be greatly reduced. This would not be a simple task, since space charge effects and neutral gas collisions that occur at atmospheric pressure also need to be taken into consideration. The lens could also be useful in stabilizing and enhancing the operation of multiple sprayers, an area of interest in the industry today due to its increasing orientation towards high-throughput analyses. More work in this area will be done in our laboratory. Clearly, the interest in using atmospheric pressure electrostatic devices to improve the performance of ESI/MS is on the rise, and the commercialization of Agilent's asymmetrical electrode is a proof to that. As more is understood about their mechanism and operation, these devices may prove to be indispensable in the future. 81 

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