Ascorbic acid for homogenous redox buffering in electrospray ionization–mass spectrometry

Electrospray ionization (ESI) involves the dispersion of a liquid containing analytes of interest into a fine aerosol by applying a high potential difference to the sample solution with respect to a counter electrode. Thus, from the electrochemical point of view, the ESI source represents a two-electrode controlled-current electrochemical flow cell. The electroactive compounds part of the solvent sprayed may be altered by occurring electrolysis (oxidation in positive ion mode and reduction in negative ion mode). These reactions can be troublesome in the context of unknown identification and quantification. In the search for a simple, inexpensive, and efficient way to suppress electrochemical oxidation in positive ESI, the usability of ascorbic acid, hydroquinone, and glutathione for homogenous redox buffering was tested. Performance of the antioxidants was assessed by analyzing pharmaceutical compounds covering a broad range of functional groups prone to oxidation. Different emitter setups were applied for continuous infusion, flow injection, and liquid chromatography/mass spectrometry experiments. Best performance was obtained with ascorbic acid. In comparison to hydroquinone and glutathione, ascorbic acid offered superior antioxidant activity, a relatively inert oxidation product, and hardly any negative effect on the ionization efficiency of analytes. Furthermore, ascorbic acid suppressed the formation of sodiated forms and was able to induce charge state reduction. Only in the very special case of analyzing a compound isobaric to ascorbic acid, interference with the low-abundant [ascorbic acid+H](+) signal may become a point of attention.     

A minimalist model for exploring conformational effects on the electrospray charge state distribution of proteins

The electrospray ionization (ESI) charge state distribution of proteins is highly sensitive to the protein structure in solution. Unfolded conformations generally form higher charge states than tightly folded structures. The current study employs a minimalist molecular dynamics model for simulating the final stages of the ESI process in order to gain insights into the physical reasons underlying this empirical relationship. The protein is described as a string of 27 beads ("residues"), 9 of which are negatively charged and represent possible protonation sites. The unfolded state of this bead string is a random coil, whereas the native conformation adopts a compact fold. The ESI process is simulated by placing the protein inside a solvent droplet with a 2.5 nm radius consisting of 1600 Lennard-Jones particles. In addition, the droplet contains 14 protons which are modeled as highly mobile point charges. Disintegration of the droplet rapidly releases the protein into the gas phase, resulting in average charge states of 4.8+ and 7.4+ for the folded and unfolded conformation, respectively. The protonation probabilities of individual residues in the folded state reveal a characteristic pattern, with values ranging from 0.2 to 0.8. In contrast, the protonation probabilities of the unfolded protein are more uniform and cover the range from 0.8 to 1.0. The origin of these differences can be traced back to a combination of steric and electrostatic effects. Residues exhibiting a small accessible surface area are less likely to capture a proton, an effect that is exacerbated by partial electrostatic shielding from nearby positive residues. Conversely, sites that are sterically exposed are associated with electrostatic funnels that greatly increase the likelihood of protonation. Unfolding enhances the steric and electrostatic exposure of protonation sites, thereby causing the protein to capture a greater number of protons during the droplet disintegration process.     

Influence of mobile phase,source parameters and source type on electrospray ionization efficiency in negative ion mode

Electrospray ionization (ESI) efficiency is known to be affected by the properties of the analytes, source design and source parameters. In this study, the ionization efficiency of 17 acidic compounds at various conditions in ESI negative ion mode was evaluated. Namely, the influence of organic solvent content in the mobile phase, ionization source parameters, ionization source geometry and functionality (conventional ESI, ESI with thermal focussing and with additional internal nebulizer gas) was studied. It was observed that the ionization efficiency in thermal focussing ESI is only marginally affected by the organic solvent composition, while for conventional ESI and ESI with internal nebulizer gas, the ionization efficiency increases significantly with increasing organic modifier content. For all ionization sources and mobile phase compositions, the ionization efficiency values between different setups showed good correlation. Copyright © 2016 John Wiley & Sons, Ltd.     

Continuous flow-extractive desorption electrospray ionization: Analysis from “non-electrospray ionization-friendly” solvents and related mechanism

Due to their low polarities and dielectric constants, analytes in solvents such as hexane, chloroform, and ethyl acetate exhibit poor electrospray ionization (ESI) efficiency. These are deemed to be “non-ESI-friendly” solvents. Continuous flow extractive desorption electrospray ionization (CF-EDESI) is a novel ambient ionization technique that was recently developed in our group to manipulate protein charge distributions. Here we demonstrate its potential for ionizing analytes from non-ESI-friendly solvents. This feature makes CF-EDESI attractive to the general analytical community due to its apparent potential in lipidomics, normal phase separations, and hyphenation of mass spectrometry with HPLC-NMR systems. In this context, interest was subsequently initiated to discern mechanistic aspects of CF-EDESI. To achieve this, mechanistic experiments associated with a seemingly similar ambient ionization technique, extractive electrospray ionization (EESI), were emulated to compare CF-EDESI and EESI. Analysis of a series of fatty acids in multiple solvents in the negative ionization mode revealed differences between the two techniques. Whereas EESI has been previously shown to operate via extraction of analytes into the spray solvent, data presented here for CF-EDESI point toward a liquid-liquid mixing process to facilitate ionization. Further, a partial factorial design experiment was performed to evaluate the effects of different experimental variables on signal intensity. Sample flow rate was confirmed to be among the most significant factors to affect sensitivity. As a whole, the work presented provides greater insight into a new ambient ionization process, which exhibits expanded capabilities over conventional ESI; in this case, for direct analysis from non-ESI-friendly solvents.     

Electrostatic field–induced tip‐electrospray ionization mass spectrometry for direct analysis of raw food materials

Rapid characterization of metabolites and risk compounds such as chemical residues and natural toxins in raw food materials such as vegetables, meats, and edible living plants and animals plays an important part in ensuing food quality and safety. To rapidly characterize the analytes in raw food materials, it is essential to develop in situ method for directly analyzing raw food materials. In this work, raw food materials including biological tissues and living samples were placed between an electrode and mass spectrometric (MS) inlet under a strong electrostatic field; analytes were rapidly induced to generate electrospray ionization (ESI) from the sample tip by adding a drop of solvent onto the sample. Therefore, the electrostatic field–induced tip‐ESI‐MS allows raw samples to avoid contacting high voltage, and thus this method has the advantage for in vivo analysis of food living plants and animals. Metabolite profiling, residues of pesticides and veterinary drugs, and natural toxins from raw food materials have been successfully detected. The analytical performances, including the linear ranges, sensitivity, and reproducibility, were investigated for direct sample analysis. The ionization mechanism of electrostatic field–induced tip‐ESI was also discussed in this work.     

On the mechanism of extractive electrospray ionization (EESI) in the dual-spray configuration

Dual-spray extractive electrospray ionization (EESI) mass spectrometry as a versatile analytical technique has attracted much interest due to its advantages over conventional electrospray ionization (ESI). The crucial difference between EESI and ESI is that in the EESI process, the analytes are introduced in nebulized form via a neutral spray and ionized by collisions with the charged droplets from an ESI source formed by spraying pure solvent. However, the mechanism of the droplet–droplet interactions in the EESI process is still not well understood. For example, it is unclear which type of droplet–droplet interaction is dominant: bounce, coalescence, disruption, or fragmentation? In this work, droplet–droplet interaction was investigated in detail based on a theoretical model. Phase Doppler anemometry (PDA) was employed to investigate the droplet behavior in the EESI plume and provide the experimental data (droplet size and velocity) necessary for theoretical analysis. Furthermore, numerical simulations were performed to clarify the influence of the sheath gas flow on the EESI process. No coalescence between the droplets in the ESI spray and the droplets in the sample spray was observed using various geometries and sample flow rates. Theoretical analysis, together with the PDA results, suggests that droplet fragmentation may be the dominant type of droplet–droplet interaction in the EESI. The interaction time between the ESI droplet and the sample droplet was estimated to be <5 μs. This work gives a clear picture of droplet–droplet interactions in the dual-spray EESI process and detailed information for the optimization of this method for future applications that require higher sensitivity.     

Alleviation of ion suppression effect in sonic spray ionization with induced alternating current voltage

In this study, alleviation of ion suppression effect in sonic spray ionization mass spectrometry (SSI‐MS) was investigated. Ion suppression effect was firstly compared between electrospray ionization (ESI) and conventional SSI, and more severe ion suppression effect was observed with SSI. Ion suppression effect of SSI was also found difficult to be alleviated by simply optimizing major parameters. Alternatively, we found that with the assistance of an alternating current (AC) voltage with low amplitude, the ion suppression effect was greatly alleviated (comparable with conventional ESI). That AC voltage was applied outside the SSI spray tip, and no direct contact between the electrode and spray solution was necessary. Besides the alleviation of the ion suppression effect, this newly‐developed method, termed as induced electrosonic spray ionization (IESSI), appeared to preserve similar charge state distribution with SSI for protonated cytochrome c, hemoglobin, and bradykinin. IESSI could also obtain significantly improved ion intensities (~1000‐fold over conventional SSI). In addition, tolerance of concentrated salts for IESSI‐MS was investigated through the analysis of cytochrome c in the presence of concentrated sodium chloride (NaCl) or ammonium acetate (NH₄OAc). Copyright © 2014 John Wiley & Sons, Ltd.     

Size exclusion chromatography coupled to electrospray ionization mass spectrometry for analysis and quantitative characterization of arsenic interactions with peptides and proteins

Arsenic‐binding proteins are of toxicological importance since enzymatic activities can be blocked by arsenic interactions. In the present work, a novel methodology based on size exclusion chromatography coupled to electrospray ionization mass spectrometry (SEC‐ESI‐MS) was developed with special emphasis to preserve the intact proteins and their arsenic bindings. The eluent composition of 25 mM Tris/HCl, pH 7.5, with the addition of 100‐mM NaCl optimized for SEC with UV detection provided the highest SEC separation efficiency, but was not compatible with the ESI‐MS because of the non‐volatility of the buffer substance and of the salt additive. In order to find the best compromise between chromatographic separation and ionization of the arsenic‐binding proteins, buffer type and concentration, pH value, portion of organic solvent in the SEC eluent as well as the flow rate were varied. In the optimized procedure five different arsenic‐binding peptides and proteins (glutathione, oxytocin, aprotinin, α‐lactalbumin, thioredoxin) covering a molar mass range of 0.3–14 kDa could be analyzed using 75% 10‐mM ammonium formate, pH 5.0/25% acetonitrile (v : v) as eluent and a turbo ion spray source operated at 300 °C and 5.5 kV. A complete differentiation of all peptides and proteins involved in the arsenic‐binding studies as well as of their arsenic‐bound forms has become feasible by means of the extracted ion chromatograms (XIC) of the mass spectrometric detection. The new method offered the possibility to estimate equilibrium constants for the reaction of phenylarsine oxide with different thiol‐containing biomolecules by means of the XIC peak areas of reactants and products. Limits of detection in the range of 2–10 µM were obtained by SEC‐ESI‐MS for the individual proteins. Copyright © 2009 John Wiley & Sons, Ltd.     

Minimizing analyte electrolysis in electrospray ionization mass spectrometry using a redox buffer coated emitter electrode

An emitter electrode with an electroactive poly(pyrrole) (PPy) polymer film coating was constructed for use in electrospray ionization mass spectrometry (ESI‐MS). The PPy film acted as a surface‐attached redox buffer limiting the interfacial potential of the emitter electrode. While extensive oxidation of selected analytes (reserpine and amodiaquine) was observed in positive ion mode ESI using a bare metal (gold) emitter electrode, the oxidation was suppressed for these same analytes when using the PPy‐coated electrode. A semi‐quantitative relationship between the rate of oxidation observed and the interfacial potential of the emitter electrode was shown. The redox buffer capacity, and therefore the lifetime of the redox buffering effect, correlated with the oxidation potential of the analyte and with the magnitude of the film charge capacity. Online reduction of the PPy polymer layer using negative ion mode ESI between analyte injections was shown to successfully restore the redox buffering capacity of the polymer film to its initial state. Published in 2010 by John Wiley & Sons, Ltd.     

Ion internal energy distributions validate the charge residue model for small molecule ion formation by spray methods

This paper reports a detailed study of the internal energy distribution of ions formed by four electrospray ionization (ESI)-related ionization methods, with particular emphasis on electrosonic spray ionization (ESSI). Substituted benzylpyridinium ions were used as thermometer ions to probe the internal energy distribution. The influence of different instrumental parameters was studied. Cone and skimmer voltages as well as the collision energy were found to strongly affect the ion internal energy distribution, whereas the distance between the emitter and the inlet of the mass spectrometer, the nebulizing gas pressure or the flow rate showed no influence. The internal energy distribution obtained with an ESSI source was compared with those obtained for electrospray (ESI), nanoelectrospray (nanoESI) and sonic spray ionization (SSI) on the same mass spectrometer with the same instrumental parameters. No clear differences were observed. As the charge residue model is the only ion formation mechanism possible for SSI, we conclude that benzylpyridinium ions are formed by the pathway suggested by this model.     

Atmospheric pressure thermospray ionization using a heated microchip nebulizer

When a standard atmospheric pressure chemical ionization (APCI) or atmospheric pressure photoionization (APPI) ion source is used without applying the corona discharge or photoirradiation, atmospheric pressure thermospray ionization (APTSI) of various compounds can be achieved. Although largely ignored, this phenomenon has recently gained interest as an alternative ionization technique. In this study, this technique is performed for the first time on a miniaturized scale using a microchip nebulizer. Sample ionization with the presented microchip‐APTSI (µAPTSI) is achieved by applying only heat and gas flow to a nebulizer chip, without any other methods to promote gas‐phase ionization. To evaluate the performance of the described µAPTSI setup, ionization efficiency for a set of test compounds was monitored as the microchip positioning, temperature, nebulizer gas flow rate, sample solution composition, and solvent flow rate were varied. The µAPTSI mass spectra of the test compounds were also compared to those obtained with ESI and APCI. The µAPTSI produces ESI‐like spectra with low background noise, favoring the formation of protonated or deprotonated molecules of compounds that are ionizable in solution. Multiple charging of peptides without in‐source fragmentation was also observed. Unlike ESI, however, the µAPTSI source can tolerate the presence of mobile phase additives like trifluoroacetic acid (TFA) without significant ion suppression. The µAPTSI source can be used with standard mass spectrometer ion source hardware, being a unique alternative to the present interfacing techniques. Copyright © 2009 John Wiley & Sons, Ltd.     

Polarization induced electrospray ionization mass spectrometry for the analysis of liquid,viscous and solid samples

In this study, a polarization‐induced electrospray ionization mass spectrometry (ESI‐MS) was developed. A micro‐sized sample droplet was deposited on a naturally available dielectric substrate such as a fruit or a stone, and then placed close to (~2 mm) the orifice of a mass spectrometer applied with a high voltage. Taylor cone was observed from the sample droplet, and a spray emitted from the cone apex was generated. The analyte ion signals derived from the droplet were obtained by the mass spectrometer. The ionization process is similar to that in ESI although no direct electric contact was applied on the sample site. The sample droplet polarized by the high electric field provided by the mass spectrometer initiated the ionization process. The dielectric sample loading substrate facilitated further the polarization process, resulting in the formation of Taylor cone. The mass spectral profiles obtained via this approach resembled those obtained using ESI‐MS. Multiply charged ions dominated the mass spectra of peptides and proteins, whereas singly charged ions dominated the mass spectra of small molecules such as amino acids and small organic molecules. In addition to liquid samples, this approach can be used for the analysis of solid and viscous samples. A small droplet containing suitable solvent (5–10 µl) was directly deposited on the surface of the solid (or viscous) sample, placed close the orifice of mass spectrometer applied with a high voltage. Taylor cone derived from the droplet was immediately formed followed by electrospray processes to generate gas‐phase ions for MS analysis. Analyte ions derived from the main ingredients of pharmaceutical tablets and viscous ointment can be extracted into the solvent droplet in situ and observed using a mass spectrometer. Copyright © 2015 John Wiley & Sons, Ltd.     

The Role of Nebulizer Gas Flow in Electrosonic Spray Ionization (ESSI)

In this work, we investigated the role of the nebulizer gas flow in electrosonic spray ionization (ESSI), by systematically studying the relation between the flow and the ion signals of proteins, such as cytochrome c and holomyoglobin using ESSI-mass spectrometry (MS). When a neutral solution was delivered with a small sample flow rate (≤5 μL/min), no obvious transition from electrospray ionization (ESI) to ESSI was found as the gas velocity varies from subsonic to supersonic speed. Droplets mostly experienced acceleration instead of breakup by the high-speed nebulizer gas. On the contrary, using particular experimental conditions, such as an acidic solution or high sample flow rate (≥200 μL/min), more folded protein ions appear to be kept in droplets of diminishing size due to breakup by the high-speed nebulizer gas in ESSI compared with ESI. Theoretical analyses and numerical simulations were also performed to explain the observed phenomena. These systematic studies clarify the ionization mechanism of ESSI and provide valuable insight for optimizing ESSI and other popular pneumatically assisted electrospray ionization methods for future applications.     

Electric modeling and characterization of pulsed high‐voltage nanoelectrospray ionization sources by a miniature ion trap mass spectrometer

A better understanding of nanoelectrospray ionization (nano‐ESI) would be beneficial in further improving the performances of nano‐ESI. In this work, the pulsed high‐voltage (HV) nano‐ESI has been electrically modeled and then systematically characterized by both voltage‐current and mass spectrometry measurements. First, the equivalent resistance of a nano‐ESI source changes with respect to both emitter tip diameter and the HV applied. Increased voltage could improve both spray current and ionization efficiency of the pulsed HV nano‐ESI. Compared with conventional DC HV method, a pulsed HV has less heating effect on the capillary tip and thus allowing the application of a much higher voltage onto a nano‐ESI source. As a result, a pulsed HV nano‐ESI could further boost the ionization efficiency of nano‐ESI by employing even higher voltages than conventional DC nano‐ESI sources.     

Non‐covalent dimers of the lysine containing protonated peptide ions in gaseous state: electrospray ionization mass spectrometric study

Study of the non‐covalent molecular complexes in gas phase by electrospray ionization mass spectrometry (ESI‐MS) represents a promising strategy to probe the intrinsic nature of these complexes. ESI‐MS investigation of a series of synthetic octapeptides containing six alanine and two lysine residues differing only by their positions showed the formation of non‐covalent dimers, which were preserved in the gas phase. Unlike the monomers, the dimers were found to show only singly protonated state. The decrease in the solvent polarity from water to alcohol showed enhanced propensity of formation of the dimer indicating that the electrostatic interaction plays a crucial role to stabilize the dimer. Selective functionalization studies showed that ε‐NH₂ of lysine and C‐terminal amide (? CONH₂) facilitate the dimerization through intermolecular hydrogen bonding network. Copyright © 2010 John Wiley & Sons, Ltd.     

Study of the noncovalent interactions of ginsenosides and amyloid‐β‐peptide by CSI‐MS and molecular docking

Noncovalent interactions between drugs and proteins play significant roles for drug metabolisms and drug discoveries. Mass spectrometry has been a commonly used method for studying noncovalent interactions. However, the harsh ionization process in electrospray ionization mass spectrometry (ESI‐MS) is not conducive to the preservation of noncovalent and unstable biomolecular complexes compared with the cold spray ionization mass spectrometry (CSI‐MS). A cold spray ionization providing a stable solvation‐ionization at low temperature is milder than ESI, which was more suitable for studying noncovalent drug‐protein complexes with exact stoichiometries. In this paper, we apply CSI‐MS to explore the interactions of ginsenosides toward amyloid‐β‐peptide (Aβ) and clarify the therapeutic effect of ginsenosides on Alzheimer's disease (AD) at the molecular level for the first time. The interactions of ginsenosides with Aβ were performed by CSI‐MS and ESI‐MS, respectively. The ginsenosides Rg1 bounded to Aβ at the stoichiometries of 1:1 to 5:1 could be characterized by CSI‐MS, while dehydration products are more readily available by ESI‐MS. The binding force depends on the number of glycosyls and the type of ginsenosides. The relative binding affinities were sorted in order as follows: Rg1 ≈ Re > Rd ≈ Rg2 > Rh2, protopanaxatriol by competition experiments, which were supported by molecular docking experiment. CSI‐MS is expected to be a more appropriate approach to determine the weak but specific interactions of proteins with other natural products especially polyhydroxy compounds.     

In ESI‐source H/D exchange under atmospheric pressure for peptides and proteins of different molecular weights from 1 to 66 kDa: the role of the temperature of the desolvating capillary on H/D exchange

Transition of proteins from the solution to the gas phase during electrospray ionization remains a challenging problem despite the large amount of attention it has received during the past few decades. One of the major questions relates to the extent to which proteins in the gas phase retain their condensed phase structures. We have used in‐electrospray source hydrogen/deuterium exchange to determine the number of deuterium incorporations as a function of protein mass, charge state and temperature of the desolvating capillary where the reaction occurs. All experiments were performed on a Thermo LTQ FT Ultra equipped with a 7‐T superconducting magnet. Ions were generated by an IonMax Electrospray ion source operated in the positive ESI mode. Deuterium exchange was performed by introducing a droplet of D₂O beneath the ESI capillary. We systematically investigated gas phase hydrogen/deuterium (H/D) exchange under atmospheric pressure for peptides and proteins of different molecular weights from 1 to 66 kDa. We observed that almost all proteins demonstrate similar exchange rates for all charge states and that these rates increase exponentially with the temperature of the desolvating capillary. We did not observe any clear correlation of the number of H/D exchanges with the value of the cross section for a corresponding charge state. We have demonstrated the possibility of performing in‐ESI source H/D exchange of large proteins under atmospheric pressure. The simplicity of the experimental setup makes it a useful experimental technique that can be applied for the investigation of gas phase conformations of proteins. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of the atmospheric pressure ionization mass spectrometric process obtained using a fused-silica emitter with the high voltage applied upstream

The atmospheric pressure ionization process obtained when a mixture of methanol and water (90:10, v/v) also containing 50 microM sodium hydroxide is dispersed from a fused-silica emitter was studied. A combination of a high electric field and a nebulizer gas with the high voltage applied upstream in the liquid flow was utilized to facilitate the spray process. By comparing the dependences of the spray current and ion signals on the spray potential, it was found that electrical corona discharges were obtained for potentials higher than about 2.6 kV, which resulted in a mixed electrospray and chemical ionization process. By introducing vapour from a solvent, such as benzene or toluene, with a low ionization energy into the nebulizing gas, it was found that the appearance of the corresponding molecular ion was correlated with a change in the slope of the spray current-potential curve. This indicates that the breakpoints in the spray current-potential curves observed were correlated with the onsets of corona discharges. It was shown that the mixed ionization process gives rise to increased amounts of protonated solvent molecules and assists in the formation of sodiated adduct ions from an uncharged fatty acid methyl ester.     

Basic Vapor Exposure for Tuning the Charge State Distribution of Proteins in Negative Electrospray Ionization: Elucidation of Mechanisms by Fluorescence Spectroscopy

Manipulation for simplifying or increasing the observed charge state distributions of proteins can be highly desirable in mass spectrometry experiments. In the present work, we implemented a vapor introduction technique to an Agilent Jet Stream ESI (Agilent Technologies, Santa Clara, CA, USA) source. An apparatus was designed to allow for the enrichment of the nitrogen sheath gas with basic vapors. An optical setup, using laser-induced fluorescence and a pH-chromic dye, permits the pH profiling of the droplets as they evaporate in the electrospray plume. Mechanisms of pH droplet modification and its effect on the protein charging phenomenon are elucidated. An important finding is that the enrichment with basic vapors of the nitrogen sheath gas, which surrounds the nebulizer spray, leads to an increase in the spray current. This is attributed to an increase in the electrical conductivity of water-amine enriched solvent at the tip exit. Here, the increased current results in a generation of additional electrolytically produced OH(-) ions and a corresponding increase in the pH at the tip exit. Along the electrospray plume, the pH of the droplets increases due to both droplet evaporation and exposure to basic vapors from the seeded sheath gas. The pH evolution in the ESI plume obtained using pure and basic seeded sheath gas was correlated with the evolution of the charge state distribution observed in mass spectra of proteins, in the negative ion mode. Taking advantage of the Agilent Jet Stream source geometry, similar protein charge state distributions and ion intensities obtained with basic initial solutions, can be obtained using native solution conditions by seeding the heated sheath gas with basic vapors.     

Characterization of charge separation in the array of Micromachined UltraSonic Electrospray (AMUSE) ion source for mass spectrometry

An initial investigation into the effects of charge separation in the Array of Micromachined UltraSonic Electrospray (AMUSE) ion source is reported to gain understanding of ionization mechanisms and to improve analyte ionization efficiency and operation stability. In RF-only mode, AMUSE ejects, on average, an equal number of slightly positive and slightly negative charged droplets due to random charge fluctuations, providing inefficient analyte ionization. Charge separation at the nozzle orifice is achieved by the application of an external electric field. By bringing the counter electrode close to the nozzle array, strong electric fields can be applied at relatively low DC potentials. It has been demonstrated, through a number of electrode/electrical potential configurations, that increasing charge separation leads to improvement in signal abundance, signal-to-noise ratio, and signal stability.