Corona discharge neutralizer for electrospray aerosols used with condensation nucleation light-scattering detection

To obviate the use of radioactive materials, a simple unipolar electrical neutralizer based on a corona discharge was developed to neutralize electrospray aerosols used when coupling capillary liquid chromatography (CLC) and capillary electrophoresis (CE) to condensation nucleation light-scattering detection (CNLSD). The electrical neutralizer could be operated with either a positive or negative corona to generate ions to neutralize oppositely charged aerosols generated by electrospray and allow sensitive detection with CNLSD. The device could further be operated with organic solvents as well as aqueous buffer. The parameters affecting the corona discharge and neutralization process were studied and optimized. Compared to a CNLSD system using a radioactive neutralizer, the system with the electrical neutralizer showed higher signal and signal-to-noise ratio for test compounds, which indicated better neutralization efficiency of the charged aerosol. Highly reproducible results were obtained with the robust and durable electrical neutralizer with both CLC- and CE-electrospray-CNLSD systems.     

Charge reduction electrospray mass spectrometry

A new mass spectrometric technique, charge reduction electrospray mass spectrometry (CREMS), allowing the analysis of complex mixtures of biological molecules is described. The charge state of ions produced by electrospray ionization may be reduced in a controlled manner to yield predominantly singly charged ions through reactions with bipolar (i.e., both positively and negatively charged) ions generated using a 210Po alpha particle source. The electrospray-generated multiply charged ions undergo charge reduction in a "neutralization chamber" positioned before the entrance nozzle to the mass spectrometer. The ions are detected using a commercial orthogonal electrospray time-of-flight mass spectrometer, although the neutralization chamber can be adapted to virtually any mass analyzer. The CREMS results obtained exhibit a signal intensity drop-off with increasing oligonucleotide size similar to that observed with matrix-assisted laser desorption/ionization mass spectrometry. Proton-transfer reactions were found to be responsible for reducing charge on proteins and oligonucleotides in both positive and negative ion mode.     

A quadrupole ion trap mass spectrometer with three independent ion sources for the study of gas-phase ion/ion reactions

An instrument for the study of gas-phase ion/ion reactions in which three independent sources of ions, namely, two electrospray ionization sources and one atmospheric sampling glow discharge ionization source, are interfaced to a quadrupole ion trap mass analyzer is described. This instrument expands the scope of gas-phase ion/ion reaction studies by allowing for manipulation of the charge states of multiply charged reactant and product ions. Examples are provided involving the formation of protein-protein complexes in the gas phase. Complexes with charge states that cannot be formed from reactant ion charge states present in the normal electrospray charge state distributions can be formed in the new apparatus. Strategies that rely on both reactant ion charge state manipulation and product ion charge state manipulation are demonstrated. In addition, simplification of product ion spectra generated from dissociation of complexes formed via ion/ion reactions can be effected by using the discharge source to reduce the charge state of the product ions to primarily 1+.     

Dual needle corona discharge to generate stable bipolar ion for neutralizing electrosprayed nanoparticles

The performances of dual needle corona discharge (NCD) as bipolar ion source to neutralize the electrospray (ES) particles were characterized and optimized. The NCD was constructed from a tungsten needle and grounded mesh electrode in the needle-to-plane configuration. The dual NCD created a bipolar ion environment by mixing the balanced concentration of positive and negative ions produced by each NCD. The dual NCD was placed in an electrospray aerosol generator (EAG) apparatus to reduce the charge state of the ES particles. Polystyrene latex (PSL) suspensions having the particle size range of 96–256 nm were used as the precursor solution for the electrospray process. Some characterizations to the NCD were carried out to obtain optimum operating voltage and air flow rate. The size distribution and charge fraction of the electrospray PSL (ES-PSL) particles exiting the EAG were also investigated. The result showed the dual NCD could generate stable bipolar ions by mixing positive and negative ions with balanced concentration. The bipolar ions from the dual NCD were capable of neutralizing and reducing the charge state of highly charged ES-PSL particles larger than 100 nm. The EAG, equipped with the dual NCD, could generate ES-PSL particles with stable concentration and consistent size distribution. The charge fraction calculation of the ES-PSL particles showed that more than 80% of the particles exiting the EAG were positively charged.     

Analysis of solids, liquids, and biological tissues using solids probe introduction at atmospheric pressure on commercial LC/MS instruments

Direct analysis of samples using atmospheric pressure ionization (API) provides a more rapid method for analysis of volatile and semivolatile compounds than vacuum solids probe methods and can be accomplished on commercial API mass spectrometers. With only a simple modification to either an electrospray (ESI) or atmospheric pressure chemical ionization (APCI) source, solid as well as liquid samples can be analyzed in seconds. The method acts as a fast solids/liquid probe introduction as well as an alternative to the new direct analysis in real time (DART) and desorption electrospray ionization (DESI) methods for many compound types. Vaporization of materials occurs in the hot nitrogen gas stream flowing from an ESI or APCI probe. Ionization of the thermally induced vapors occurs by corona discharge under standard APCI conditions. Accurate mass and mass-selected fragmentation are demonstrated as is the ability to obtain ions from biological tissue, currency, and other objects placed in the path of the hot nitrogen stream.     

Charge ratio analysis method: approach for the deconvolution of electrospray mass spectra

A new method to interpret electrospray mass spectral data based on calculating the ratio of mass-to-charge (m/z) values of multiply charged ions is described. The mass-to-charge ratios of any two multiply charged ions corresponding to a single compound are unique numbers that enable the charge states for each ion to be unequivocally identified. The multiply charged ions in electrospray mass spectra originate from the addition or abstraction of protons, cations, or anions to and from a compound under analysis. In contrast to existing deconvolution processes, the charge ratio analysis method (CRAM), identifies the charge states of multiply charged ions without any prior knowledge of the nature of the charge-carrying species. In the case of high-resolution electrospray mass spectral data, in which multiply charged ions are resolved to their isotopic components, the CRAM is capable of correlating the isotope peaks of different multiply charged ions that share the same isotopic composition. This relative ratio method is illustrated here for electrospray mass spectral data of lysozyme and oxidized ubiquitin recorded at low- to high-mass resolution on quadrupole ion trap and Fourier transform ion cyclotron mass spectrometers, and theoretical data for the protein calmodulin based upon a reported spectrum recorded on the latter.     

Supercharged protein and peptide ions formed by electrospray ionization

The multiple charging of large molecules in electrospray ionization provides key advantages for obtaining accurate molecular weights by mass spectrometry and for obtaining structural information by tandem mass spectrometry and MS(n) experiments. Addition of glycerol or m-nitrobenzyl alcohol into the electrospray solutions dramatically increases both the maximum observed charge state and the abundances of the high charge states of protein and peptide ions. Adding glycerol to acidified aqueous solutions of cytochrome c shifts the most abundant charge state from 17+ to 21+, shifts the maximum charge state from 20+ to 23+, and shifts the average charge state from 16.6+ to 20.9+. Much less m-nitrobenzyl alcohol (<1%) is required to produce similar results. With just 0.7% m-nitrobenzyl alcohol, even the 24+ charge state of cytochrome c is readily observed. Similar results are obtained with myoglobin and (Lys)4. For the latter molecule, the 5+ charge state is observed in the electrospray mass spectrum obtained from solutions containing 6.7% m-nitrobenzyl alcohol. This charge state corresponds to protonation of all basic sites in this peptide. Although the mechanism for enhanced charging is unclear, it does not appear to be a consequence of conformational changes of the analyte molecules. This method of producing highly charged protein ions should be useful for improving the performance of mass measurements on mass spectrometers with performances that decrease with increasing m/z. This should also be particularly useful for tandem mass spectrometry experiments, such as electron capture dissociation, for which highly charged ions are desired.     

Mass distribution measurement of water-insoluble polymers by charge-reduced electrospray mobility analysis

1-Methyl-2-pyrrolidone (NMP) seeded with 5% trifluoroacetic acid is identified as a singular buffer, polar enough to produce fine electrospray drops, yet having excellent solubility for many industrial polymers such as polystyrene (PSR) and poly(methyl methacrylate) (PMMA). Four PSR mass standards (M = 9.2, 34.5, 68, and 170 kDa) with narrow mass distributions are electrosprayed from their solutions in this buffer. The high charge on the resulting ions is reduced to unity with a radioactive source, whereby their electrical mobility distributions, determined by a differential mobility analyzer, yield unambiguously their size distribution. Each standard produces (at high solution concentration) several mobility peaks associated with the formation of particles containing from one to six polymer molecules, used to establish a relation Z(M) between electrical mobility Z and polymer mass. Within the indeterminacy given by inaccuracies in the nominal masses of the standards, this relation indicates that the polymers form spherical balls with a density close to the bulk density of polystyrene, as seen previously with poly(ethylene glycol) chains. Good mobility spectra from the same buffer are also obtained for PMMA (M = 49 kDa). Because NMP is less conductive and contains more involatile impurities than common aqueous buffers, the electrospray ions formed tend to carry a small contaminant crust, which distorts the inferred mass distribution unless a high spray quality is achieved.     

Comparison between an unipolar corona charger and a polonium-based bipolar neutralizer for the analysis of nanosized particles and biopolymers

In this work we present results on the charging efficiency of nanoparticles by means of a corona based unipolar charging unit. This device was designed to replace a Po210 bipolar charger unit in a commercial electrospray aerosol generator (TSI Mod 3480). The charging efficiency has been investigated for negative and positive charged particles of various chemical composition in the size range between 5 and 18 nm. The corona current has been found to be the most influential operation parameter on the charging efficiency. With a positive electrospray droplet charge and a negatively-biased corona needle, a rapidly decreasing yield of singly positively charged aerosol particles with increasing corona current was found. An increasing yield of negatively charged particles was observed with increasing current of the corona process. Providing appropriate corona settings nanoparticles with charge levels similar to these obtained with a Po210 charger were found. At optimal corona settings the yield of singly charged particles was found to be two to four times higher for negative and positive particles compared to bipolar charging. This gain in the charging efficiency increases directly the sensitivity of analysis and enhances all measurement and manipulation processes of airborne nanoparticles for which electrical charging is required.     

Parallel ion parking of protein mixtures

The multiple charging phenomenon resulting from electrospray ionization of proteins, while useful for the ability to make several mass measurements on a single component, can lead to highly complex spectra when mixtures are analyzed, as each component can generate multiple ions of distinct mass-to-charge ratio. Ion/ion proton-transfer reactions can overcome this problem by reduction of all components to the +1 charge state, but this typically requires the ability to extend the mass range of the instrument well beyond that available in most commercial instruments. Furthermore, reduction of protein charge to +1 also results in a reduction in detector response. Here it is shown that application of a relatively high amplitude, low-frequency auxiliary ac signal to the end cap electrodes of a 3-D ion trap during an ion/ion reaction can slow the ion/ion reaction rates of ions over a broad m/z range, in a process termed HALF parallel ion parking. Adjustment of the frequency and amplitude of the applied voltage allows the mass range into which the initial ion signal is moved to be controlled, allowing for the simplification of multicomponent mixtures within a mass range that is more commonly available on commercial systems. In addition to decreasing spectral complexity, this is advantageous for mixtures with low-abundance components, as there is less compromise with detector response than in reduction to the +1 charge state. Preliminary evidence also suggests that the ion collision cross section may play an important role in determining which charge states are most significantly inhibited from further ion/ion reactions under a given set of ion parking conditions.     

Electrospray ionization high-field asymmetric waveform ion mobility spectrometry-mass spectrometry

High-field asymmetric waveform ion mobility spectrometry (FAIMS) is a new technique that separates gas-phase ions at atmospheric pressure (760 Torr) and room temperature. A FAIMS instrument acts as an ion filter and can be set to continuously transmit one type of ion. Despite the stringent requirement for a flow of clean, dry gas in the FAIMS analyzer region, a method of coupling electrospray to FAIMS has been developed. The identity of the electrospray ions separated by FAIMS was determined using mass spectrometry (FAIMS-MS). The theory of FAIMS is discussed, and electrospray FAIMS-MS spectra of several compounds in modes P1, P2, N1, and N2 are presented. Ions appearing in P1 and N1 modes tend to have mobilities that increase as a function of increasing electric field strength, whereas ions appearing in P2 and N2 modes tend to have mobilities that decrease. In general, low-mass ions are focused in P1 and N1 modes, whereas larger ions (e.g., proteins) are focused in P2 and N2 modes. Short-chain peptides, (Gly)(n) where n = 1-6, are shown to cross over from P1 mode into P2 mode as the chain length increases. The removal of the low-mass solvent cluster ions, combined with a reduction of the background noise in electrospray FAIMS-MS, results in an improved signal-to-noise ratio for mass spectra of larger ions (e.g., cyctochrome c) when compared with conventional electrospray-MS. Preliminary results also suggest that various charge states of cytochrome c can be distinguished by FAIMS, implying that the ion mobility of these species at high electric field strength is sensitive to the structure of the protein ion. The linearity of response of electrospray FAIMS-MS was investigated using leucine enkephalin and shows the calibration curve to be linear for ～3 orders of magnitude.     

Identification of metal cations, metal complexes, and anions by electrospray mass spectrometry in the negative ion mode

Pneumatically assisted electrospray mass spectrometry (ES-MS) is used in the negative ion mode for aqueous metal (M) solutions in an excess of hydrochloric or nitric acid, where the major anion X = Cl- or NO3-. A collision energy of approximately 20 eV removes anion-solvent clusters for most elements and leaves negative complex ions of the general form (Mn+Xn+1)-. Complexation with anions prevents charge reduction reactions at least to n = 3, even in cases where the third ionization energy of M greatly exceeds the first ionization energy of the solvent. These negative ions thus preserve the charge state of the metal cation from the solution and allow identification of both cations and anions in a single set of electrospray conditions. Cations such as Fe3+ or Cu2+ that have a lower oxidation state in solution produce a distribution of negative ions, each with a single negative charge overall; e.g., an Fe3+ solution produces both Fe(III)X4- and Fe(III)X3-. This distribution of FeIII and FeII species is attributed to electrochemical reduction of Fe3+ at the negatively charged ES needle. "Native" anions such as perrhenate or molybdate produce singly charged analogues such as ReO4- or HMoO4-. Metal-EDTA complexes are seen as M(III)Y- or M(II)HY-. The sensitivity for these "native" anions is suppressed by competition with the excess chloride or nitrate used to produce the metal-containing complex ions.     

Electrospray-atmospheric sampling glow discharge ionization source for the direct analysis of liquid samples

Coupling electrospray with atmospheric sampling glow discharge ionization for the direct analysis of liquid-phase samples is demonstrated. Electrospray is utilized for nebulizing and transporting intact sample molecules into the glow discharge where ionization occurs through various pathways, including electron ionization and ion-molecule reactions with reagent ions. Reagent ions are formed through ionization of air molecules in an area of reduced pressure. The effects of discharge current, electrospray voltage, and solution flow rate on the absolute and relative ion intensities observed in the mass spectra are discussed. This technique is applicable to compounds containing various functional groups and encompassing a range of volatility. Analysis of organic compounds with varying volatility and polarity is discussed to illustrate the utility of this ionization technique.     

Mass spectrometry of acoustically levitated droplets

Containerless sample handling techniques such as acoustic levitation offer potential advantages for mass spectrometry, by eliminating surfaces where undesired adsorption/desorption processes can occur. In addition, they provide a unique opportunity to study fundamental aspects of the ionization process as well as phenomena occurring at the air-droplet interface. Realizing these advantages is contingent, however, upon being able to effectively interface levitated droplets with a mass spectrometer, a challenging task that is addressed in this report. We have employed a newly developed charge and matrix-assisted laser desorption/ionization (CALDI) technique to obtain mass spectra from a 5-microL acoustically levitated droplet containing peptides and an ionic matrix. A four-ring electrostatic lens is used in conjunction with a corona needle to produce bursts of corona ions and to direct those ions toward the droplet, resulting in droplet charging. Analyte ions are produced from the droplet by a 337-nm laser pulse and detected by an atmospheric sampling mass spectrometer. The ion generation and extraction cycle is repeated at 20 Hz, the maximum operating frequency of the laser employed. It is shown in delayed ion extraction experiments that both positive and negative ions are produced, behavior similar to that observed for atmospheric pressure matrix-assisted laser absorption/ionization. No ion signal is observed in the absence of droplet charging. It is likely, although not yet proven, that the role of the droplet charging is to increase the strength of the electric field at the surface of the droplet, reducing charge recombination after ion desorption.     

Model of corona discharge from a thin point in a space-charge-limited current regime

Exact self-similar solutions for the problem of electric field and charge density distribution in the external region of a unipolar corona discharge from a thin point emitter have been found. The obtained solutions are valid outside the framework of the traditional Deutsch approximation based on the small influence of a volume charge on the shape of equipotential surfaces. The value of a saturated discharge current is estimated assuming that the corona discharge is generated by a certain part of the point.     

Evaporation and discharge dynamics of highly charged droplets of heptane,octane, and p-xylene generated by electrospray ionization

We report studies of the evaporation and discharge dynamics of highly charged droplets generated by electrospray ionization from n-heptane, n-octane, and p-xylene doped with Stadis-450, a conductivity-enhancing agent. A phase Doppler anemometer (PDA) characterizes individual droplets moving through the uniform electric field within an ion mobility cell according to size, velocity, and charge. Repeated reversal of the electric field allows multiple PDA measurements on selected droplets with diameters ranging from 3 to 60 microm and up to 10(7) elementary positive charges. This "ping-pong" technique provides individual droplet histories from which we determine the dynamics of solvent evaporation and charge loss. On average, n-heptane discharges at 101% of the Rayleigh limit of charge, while n-octane and p-xylene droplets discharge at 87% and 89% of their respective limits. Discharge events release an average of 19% of the charge in n-heptane and 17% of the charge in both n-octane and p-xylene. Within the limits of the measurements, no detectable change in droplet diameter accompanies observed discharge events, indicating the loss of a relatively small fraction of the total volume. We compare these results to previous experiments, theoretical models for droplet evaporation and discharge, and predictions from the Rayleigh model. We report both Stadis-450 and triethylamine mass spectra in octane and discuss issues regarding the use of hydrocarbon solvents in electrospray mass spectrometry.     

A numerical algorithm for simulation of the electric corona discharge in the triode system

A numerical algorithm is described to calculate the charge density, electric field and corona current distribution in the corona triode. The algorithm employs a hybrid technique based on the Boundary and Finite Element Methods (FEM). FEM is used to determine the electric field because of free space charge produced by the corona discharge. The Boundary Element Method (BEM) is applied for calculating the other component of electric filed as a result of the voltage applied to the electrodes. The Method of Characteristics (MOC) is used to update the space charge density distribution. The characteristic lines are traced backwards from points of the analysed domain to the corona wire. The current density, electric field and space charge density distributions can be controlled by changing the configuration of the system. Results of calculations in a few different cases show the influence of different parameters on the work of the corona triode.     

Effect of reducing disulfide-containing proteins on electrospray ionization mass spectra

Electrospray ionization produces multiply charged molecular ions for biomolecules with molecular weights in excess of 100,000. This allows mass spectrometers with limited mass-to-charge range to extend their molecular weight range by a factor equal to the number of charges. The maximum number of observed charges for peptides and smaller proteins correlates well with the number of basic amino acid residues (Arg, Lys, His), except for disulfide-containing molecules, such as lysozyme and bovine albumin. However, reduction of disulfide linkages with 1,4-dithiothreitol (Cleland's reagent) may allow the protein to be in an extended conformation and make "buried" basic residues available for protonation to yield higher charged molecular ions by the electrospray ionization process. For larger proteins reduction of disulfide bridges greatly increases the maximum charge state, but charging of basic amino acid residues remains less efficient than for smaller proteins.     

Atmospheric pressure laser-induced acoustic desorption chemical ionization fourier transform ion cyclotron resonance mass spectrometry for the analysis of complex mixtures

We present a novel nonresonant laser-based matrix-free atmospheric pressure ionization technique, atmospheric pressure laser-induced acoustic desorption chemical ionization (AP/LIAD-CI). The technique decouples analyte desorption from subsequent ionization by reagent ions generated from a corona discharge initiated in ambient air or in the presence of vaporized toluene as a CI dopant at room temperature. Analyte desorption is initiated by a shock wave induced in a titanium foil coated with electrosprayed sample, irradiated from the rear side by high-energy laser pulses. The technique enables facile and independent optimization of the analyte desorption, ionization, and sampling events, for coupling to any mass analyzer with an AP interface. Moreover, the generated analyte ions are efficiently thermalized by collisions with atmospheric gases, thereby reducing fragmentation. We have coupled AP/LIAD-CI to ultrahigh-resolution FT-ICR MS to generate predominantly [M + H](+) or M(+?) ions to resolve and identify thousands of elemental compositions from organic mixtures as complex as petroleum crude oil distillates. Finally, we have optimized the AP/LIAD CI process and investigated ionization mechanisms by systematic variation of placement of the sample, placement of the corona discharge needle, discharge current, gas flow rate, and inclusion of toluene as a dopant.     

Electrospray ionization of protein mixtures at low pH

Solutions composed of single proteins and mixtures of proteins are subjected to electrospray ionization to study the influence of protein components on the responses of one another. Protein matrix effects in electrospray ionization are particularly relevant to the development of top-down protein identification methodologies involving protein mixtures, whereby whole protein ions are subjected to tandem mass spectrometry. Emphasis is placed largely on solutions composed of equal parts methanol and water and 1% acetic acid. The results, therefore, are relevant to low-pH solutions with significant organic content, a commonly used set of conditions in electrospray ionization mass spectrometry that tends to denature proteins. Under these conditions, very similar response curves are measured for a variety of proteins after charge normalization. That is, when the data are plotted in terms of the concentration of charge sites, rather than in terms of the concentration of protein molecules, the slopes of the response curves as well as the point at which response becomes less than linear with concentration are similar. Charge normalization is made on the basis of the weighted average charge of a protein, as reflected in the electrospray ionization mass spectrum. When proteins can be regarded as a collection of equivalent charge sites, the signal response from one protein can be used to predict the responses for other proteins. Furthermore, it is also possible to predict the dependence of the signal response for a particular protein in a mixture on the concentration of other proteins in the mixture. Examining signal response on a weighted average charge basis appears to be an effective means for identifying situations in which the protein does not behave as a collection of equivalent charge sites.