Observation of gas-phase molecular dications formed from neutral organics in solution via the controlled-current electrolytic process inherent to electrospray

This article reports the first electrospray (ES) mass spectrometry observation of molecular dications that were formed in solution by sequential one-electron oxidation of the neutral molecules [viz., nickel(II) and cobalt(II) octaethylporphyrin] via the controlled-current electrolytic (CCE) process inherent to electrospray. Dication formation was found to require (1) the addition of electrolyte to the sample solution, which increased the magnitude of the ES current and, therefore, increased the extent of analyte electrolysis in the ES capillary, (2) a relatively low solution flow rate, which increased the electrolysis time (i.e., the time the analyte remained in the capillary), thereby providing more time for the analytes to diffuse to the metal-solution interface and react, and (3) the use of a platinum ES capillary, which, because it is difficult to oxidize, increased the proportion of the faradaic current that might be provided by electrolysis of solution species compared to that proportion available when the typical stainless steel capillary is used. These interpretations of the data are made on the basis of the known characteristics of the CCE process inherent to ES, supplementary data obtained from direct solution-phase observation of the metalloporphyrin redox products formed within the different metal ES capillaries by means of a novel ES ion source, and off-line cyclic voltammetry studies of the metalloporphyrins performed by using platinum and stainless steel working electrodes.     

Insights into analyte electrolysis in an electrospray emitter from chronopotentiometry experiments and mass transport calculations

Insights into the electrolysis of analytes at the electrode surface of an electrospray (ES) emitter capillary are realized through an examination of the results from off-line chronopotentiometry experiments and from mass transport calculations for flow through tubular electrodes. The expected magnitudes and trends in the interfacial potential in an ES emitter under different solution conditions and current densities, using different metal electrodes, are revealed by the chronopotentiometry data. The mass transport calculations reveal the electrode area required for complete analyte electrolysis at a given volumetric flow rate. On the basis of these two pieces of information, the design of ES emitters that may maximize and those that may minimize analyte electrolysis during ES mass spectrometry are discussed.     

Ionization and transmission efficiency in an electrospray ionization—mass spectrometry interface

The ionization and transmission efficiencies of an electrospray ionization (ESI) interface were investigated to advance the understanding of how these factors affect mass spectrometry (MS) sensitivity. In addition, the effects of the ES emitter distance to the inlet, solution flow rate, and inlet temperature were characterized. Quantitative measurements of ES current loss throughout the ESI interface were accomplished by electrically isolating the front surface of the interface from the inner wall of the heated inlet capillary, enabling losses on the two surfaces to be distinguished. In addition, the ES current lost to the front surface of the ESI interface was spatially profiled with a linear array of 340-microm-diameter electrodes placed adjacent to the inlet capillary entrance. Current transmitted as gas-phase ions was differentiated from charged droplets and solvent clusters by measuring sensitivity with a single quadrupole mass spectrometer. The study revealed a large sampling efficiency into the inlet capillary (>90% at an emitter distance of 1 mm), a global rather than a local gas dynamic effect on the shape of the ES plume resulting from the gas flow conductance limit of the inlet capillary, a large (>80%) loss of analyte ions after transmission through the inlet arising from incomplete desolvation at a solution flow rate of 1.0 microL/min, and a decrease in analyte ions peak intensity at lower temperatures, despite a large increase in ES current transmission efficiency.     

Redox buffering in an electrospray ion source using a copper capillary emitter.

An electrospray ion source used in electrospray mass spectrometry is a two-electrode, controlled-current electrochemical flow cell. Electrochemical reactions at the emitter electrode (oxidation and reduction in positive and negative ion modes respectively) provide the excess charge necessary for the quasi-continuous production of charged droplets and ultimately gas-phase ions with this device. We demonstrate here that a copper capillary emitter, in place of the more commonly used stainless-steel capillary emitter, can be utilized as a redox buffer in positive ion mode. Anodic corrosion of the copper capillary during normal operation liberates copper ions to solution and in so doing maintains the interfacial potential at this electrode near the equilibrium potential for the copper corrosion process [E degrees = 0.34 V versus standard hydrogen electrode (SHE)]. Fixing the interfacial potential at the emitter electrode provides control over the electrochemical reactions that take place at this electrode. It is shown that the oxidation of N-phenyl-1,4-phenylenediamine to N-phenyl-1,4-phenylenediimine (E(p/2) = 0.48 V versus SHE) can be completely avoided using the copper emitter, whereas this analyte is completely oxidized with a stainless-steel capillary emitter under the same conditions. Moreover, using N-phenyl-1,4-phenylenediimine, we demonstrate that reduction reactions can occur at the copper emitter electrode in positive ion mode. Emitter corrosion, in addition to redox buffering, provides a convenient means to introduce metal ions into solution for analytical use in electrospray mass spectrometry.     

Liquid phase separation of proteins based on electrophoretic effects in an electrospray setup during sample introduction into a gas-phase electrophoretic mobility molecular analyzer (CE–GEMMA/CE–ES–DMA)

Nanoparticle characterization is gaining importance in food technology, biotechnology, medicine, and pharmaceutical industry. An instrument to determine particle electrophoretic mobility (EM) diameters in the single-digit to double-digit nanometer range receiving increased attention is the gas-phase electrophoretic mobility molecular analyzer (GEMMA) separating electrophoretically single charged analytes in the gas-phase at ambient pressure. A fused-silica capillary is used for analyte transfer to the gas-phase by means of a nano electrospray (ES) unit. The potential of this capillary to separate analytes electrophoretically in the liquid phase due to different mobilities is, at measurement conditions recommended by the manufacturer, eliminated due to elevated pressure applied for sample introduction. Measurements are carried out upon constant feeding of analytes to the system. Under these conditions, aggregate formation is observed for samples including high amounts of non-volatile components or complex samples. This makes the EM determination of individual species sometimes difficult, if not impossible. With the current study we demonstrate that liquid phase electrophoretic separation of proteins (as exemplary analytes) occurs in the capillary (capillary zone electrophoresis, CE) of the nano ES unit of the GEMMA. This finding was consecutively applied for on-line desalting allowing EM diameter determination of analytes despite a high salt concentration within samples. The present study is to our knowledge the first report on the use of the GEMMA to determine EM diameters of analytes solubilized in the ES incompatible electrolyte solutions by the intended use of electrophoresis (in the liquid phase) during sample delivery. Results demonstrate the proof of concept of such an approach and additionally illustrate the high potential of a future on-line coupling of a capillary electrophoresis to a GEMMA instrument.     

Current measurements within the electrospray emitter

A movable disc-like wire probe electrode placed inside the electrospray (ES) capillary was used to measure currents flowing within the ES device for the first time. Currents were measured between the wire probe and the ES capillary. Current maps revealing measured current versus wire probe position were generated for a variety of solution conditions in the positive and negative ion modes and are compared to potential maps. The electrospray device was found to subsist on highly stable total currents; this current regulator aspect of the ES device showed remarkable resiliency regardless of the proportion of current produced at the wire probe electrode versus the ES capillary. However, kinks observed in the current and potential maps are attributed to adsorbed air participating in electrochemical reactions, and turbulence in solution flow in the region of the Taylor cone. From differential electrospray emitter potential (DEEP) maps, current maps, and cyclic voltammetry experiments performed at different wire probe locations, evidence is provided for separate regimes of current flow in the bulk solution and in the thin "skin" of highly conductive electrolyte constituting the outer surface (air interface) of the Taylor cone. Current maps reveal that current is drawn more evenly along the length of the ES capillary when solutions are highly conductive, in agreement with previous results for DEEP maps. In less conductive solutions, the area close to the capillary exit contributes more heavily to current production. Evidence that contaminant participation in electrochemical processes occurring within the electrospray device can be largely responsible for production of the excess charge in ES droplets is also provided. These investigations complement previous DEEP mapping studies to further elucidate the details of the electrochemical processes occurring within the electrospray device.     

Relative importance of basicity in the gas phase and in solution for determining selectivity in electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry is a critically important technique for the determination of small molecules, but its application for this purpose is complicated by its selectivity. For positive ion ESI-MS analysis of basic analytes, several investigators have pointed to the importance of analyte basicity as a source of selectivity. Currently, however, it is not known whether basicity in the gas phase or in solution is ultimately most important in determining responsiveness. The objective of these studies was to investigate the relative importance of basicity in solution and in the gas phase as factors that predict selectivity in positive ion ESI-MS analysis. ESI-MS response was compared for a diverse series of protonatable analytes in two different solvents, neat methanol and methanol with 0.5% acetic acid. A correlation was observed between analyte pK(b) and electrospray response. However, the response for the analytes with very high pK(b) values was significantly higher than would be expected based on concentration of the protonated form or the analyte in solution, and this higher response did not appear to result from gas-phase proton transfer reactions. Although all of the analytes investigated had higher gas-phase basicities than the solvent, their relative responses were not dictated by gas-phase basicity. Higher response was observed for all of the analytes studied in acidified methanol compared with neat methanol, and this higher response was most pronounced for weakly basic analytes. These findings support the use of analyte pK(b) for rational method development in ESI-MS analysis of small molecules.     

Pulsed dual electrospray ionization for In/In reactions

A pulsed dual electrospray ionization source has been developed to generate positive and negative ions for subsequent ion/ion reaction experiments. The two sprayers, typically a nano-electrospray emitter for analytes and an electrospray emitter for reagents, are positioned in a parallel fashion close to the sampling orifice of a triple quadrupole/linear ion trap tandem mass spectrometer (Sciex Q TRAP). The potentials applied to each sprayer are alternately pulsed so that ions of opposite polarity are generated separately in time. Ion/ion reactions take place after ions of each polarity are sequentially injected into a high-pressure linear ion trap, where axial trapping is effected by applying an auxiliary radio frequency voltage to the end lenses. The pulsed dual electrospray source allows optimization of each sprayer and can be readily coupled to any spray interface with no need for instrument modifications, provided the potentials required to transmit the ion polarity of interest can be alternated in synchrony with the emitter potentials. Ion/ion reaction examples such as charge reduction of multiply charged protein ions, charge inversion of peptides ions, and protein-protein complex formation are given to illustrate capabilities of the pulsed dual electrospray source in the study of gas-phase ion/ion chemistry.     

Electrolytic deposition of metals on to the high-voltage contact in an electrospray emitter: implications for gas-phase ion formation

The electrospray ion source is an electrolytic flow cell. Electrolytic reactions in the electrospray emitter maintain the production of charged droplets by this ion source that contain an excess of ions of one polarity. These redox reactions necessarily change the composition of the solution that initially enters the emitter. As a result, the ions ultimately observed in the gas phase by electrospray mass spectrometry (ESMS) may be substantially influenced by both the nature and extent of these electrochemical reactions. It is demonstrated in this paper that Ag(+), Cu(2+) and Hg(2+) ions in solution can be electrolytically reduced and deposited as the respective metals on to the surface of the high-voltage contact in the electrospray emitter in negative ion mode electrospray. The deposited metals are shown to be liberated from the surface by switching the electrospray high-voltage polarity to operate in the positive ion mode. The deposited metals are oxidized in positive ion mode, releasing the metal ions back into solution where they are detected in the electrospray mass spectrum. In a semi-quantitative analysis, it was found that up to 50% of the Ag(+) in a 2.5 microM solution was deposited on the high-voltage contact of the emitter as the solution flowed through the emitter. Deposition of Cu(2+) and Hg(2+) was less efficient. These data illustrate that in the analysis of metals by ESMS, one must be aware that both the concentration and form of the metals may be altered by electrochemical processes in the emitter. Hence reduction or oxidation of metals in the electrospray emitter, which may remove ions from solution, or change metal valence, would be expected to impact both quantitative metal determinations and metal speciation attempts using ESMS.     

Some thoughts on electrospray ionization mechanisms

Electrospray ionization (ESI) mechanisms are highly complex, due to a series of physical and chemical phenomena taking place on a complex system, as a solution is. In fact, even if the solution of an analyte in a protic medium can be considered at first sight to be a two-component system, the presence of solvent dissociation equilibria and the possible interactions solvent-solvent dissociation products, solvent dissociation products-analyte make this system highly complex, also for the presence of possible ionic compounds (for example, Na(+), K(+)) which strongly affect the above equilibria. A high number of research articles have been published, mainly devoted to charged droplet production and to gas-phase ion generation. They all show the high complexity of the processes affecting electrospray measurements related to either the chemical equilibria present in the condensed phase and to electrolysis processes at the emitter tip or to the processes occurring in the sprayed droplets. As a result, the chemical composition inside the small droplets from which the analyte ions are generated can be significantly different from those in sprayed solution. In this review, after a short survey of the proposed ESI mechanisms, some experiments are described. They were performed to examine if ion mobility in solution, before the formation of the sprayed charged droplets, can affect the ESI results. The data, obtained by studying both inorganic and organic analytes, indicate that the ESI spectra are dependent on the analyte dimension and charge state which, as a consequence, affect their ion mobility in solution.     

Effects of ground loop currents on signal intensities in electrospray mass spectrometry

The occurrence of electrochemical processes during the operation of an electrospray ionization (ESI) source is well established. In the positive ion mode, electrons are drawn from the ESI metal capillary to a high voltage power supply. These electrons are the product of charge-balancing oxidation reactions taking place at the liquid/metal interface of the ion source. In a recent study, (Anal. Chem.2001, 73, 4836-4844), our group has shown that the introduction of a ground loop can dramatically enhance the rate of these oxidation processes. Such a ground loop can be introduced by connecting the sample infusion syringe (or the liquid chromatography column, in the case of LC-MS studies) to ground. The magnitude of the ground loop current can be controlled by the electrolyte concentration in the analyte solution, and by the dimensions of the capillary connecting the syringe needle and the ESI source. Using ferrocene as a model system, it is demonstrated that the introduction of such a ground loop can significantly enhance the signal intensity of analytes that form electrochemically ionized species during ESI. However, analytes that form protonated molecular ions, such as reserpine, also show higher signal intensities when a ground loop is introduced into the system. This latter observation is attributed to the occurrence of electrolytic solvent (acetonitrile and/or water) oxidation processes. These reactions generate protons within the ion source, and thus facilitate the formation of [M + nH](n+) ions. Overall, this work provides an example of how the careful control of electrochemical parameters can be exploited to optimize signal intensities in ESI-MS.     

A glass capillary ultramicroelectrode with an electrokinetic sampling ability.

A glass capillary ultramicroelectrode (tip diameter approximately 1.2 microm) having an electrokinetic sampling ability is described. It is composed of a pulled glass capillary filled with an inner solution and three internal electrodes (Pt working and counter electrodes and an Ag/AgCl reference electrode). The voltammetric response of the capillary electrode is based on electrokinetic transport of analyte ions from the sample solution into the inner solution across the conical tip. It was found that the electrophoretic migration of analytes at the conical tip is faster than electroosmotic flow, enabling electrokinetic transport of analyte ions into the inner solution of the electrode. By using [Fe(CN)6]4- and (ferrocenylmethyl)trimethylammonium (FcTMA+) ions as model analytes, differential pulse voltammetric responses of the capillary electrode were investigated in terms of tip diameter of the capillary, sampling voltage, sampling time, detection limit and selectivity. The magnitude of the response depends on the size and charge of analyte ions. With a capillary electrode having a approximately 1.2-microm tip diameter, which minimizes non-selective diffusional entry of analytes, the response after 1 h sampling at +1.7 V is linearly related to [Fe(CN)6]4- concentration in the range of 0.50-5.0 mM with the detection limit of 30 microM. Application of a potential of the same sign as that of the analyte ion forces the analyte to move out from the electrode to the solution, enabling reuse of the same capillary electrode. The charge-selective detection of analytes with the capillary electrode is demonstrated for [Fe(CN)6]4- in the presence of FcTMA+.     

Electrochemical processes in a wire-in-a-capillary bulk-loaded, nano-electrospray emitter

Experiments are described that illustrate solvent oxidation, emitter electrode corrosion, and analyte oxidation in positive ion mode nano-electrospray mass spectrometry using a wire-in-a-capillary, bulk-loaded nano-electrospray emitter geometry. Time-lapsed color photography of pH and metal specific indicator solutions within operating nano-electrospray emitters, as well as temporal changes in the ions observed in the nano-electrospray mass spectra, are used to probe these reactions, judge their magnitude, and study the time dependent changes in solution composition and gas-phase ion signal brought about as a result of these electrochemical reactions. The significance of these observations for analytical applications of nano-electrospray mass spectrometry are discussed.     

Negative ion formation in electrospray mass spectrometry

Analytical and Chemical Sciences, Research Triangle Institute, Research Triangle Park, North Carolina, USA Negative ion electrospray (ES) operating on a single quadrupole mass spectrometer for the detection of low-molecular-weight molecules is discussed. The ES interface was operated at a positive cylindrical electrode potential to produce negative ions, and the results obtained were compared to the positive ion mode. As in the case of operation in the more common positive mode, negative ions with varying degrees of solvation and structurally relevant fragments can be obtained from a variety of solute species, including β-lactam antibiotics, aminoglycosides, aminocyclitols, tetracyclines, sulfonamides, nucleotides, peptides, and explosives. No fragmentation of parent species, except those from some labile explosives, was provided because low potential differences are applied between the capillary and the first skimmer, and electrical discharge is avoided in the gas phase. An increase in the capillary voltage resulted in collision-induced decomposition to produce structurally relevant fragment ions in both operation modes. An evaluation of representative chromatographic solvents indicated that 2-propanol added with oxygen in the ES bath gas is best suited to suppress electrical (corona) discharge phenomena in negative ion operation, whereas it aids in solution nebulization, desolvation, and transfer of ions in solution to the gas phase. For positive ion mode, no such precaution was necessary. Conditions that promote the formation of ions in solution usually improve ES response. Therefore, an increase in the solvent pH can increase the sensitivity in negative ion ES ionization. Negative ion ES offers the advantage of providing complementary structural information to help in the characterization of an unknown compound or to confirm a certain tentatively proposed structure. Nucleotides and explosives were best characterized in negative ion mode owing to the ease with which they form anions in solution, and they could be detected down to the l-pg /gML level.     

Ultrasonication-assisted spray ionization mass spectrometry for the analysis of biomolecules in solution

In this paper, we describe a novel technique—ultrasonication-assisted spray ionization (UASI)—for the generation of singly charged and multiply charged gas-phase ions of biomolecules (e.g., amino acids, peptides, and proteins) from solution; this method employs a low-frequency ultrasonicator (ca. 40 kHz) in place of the high electric field required for electrospray ionization. When a capillary inlet is immersed into a sample solution within a vial subjected to ultrasonication, the solution is continually directed to the capillary outlet as a result of ultrasonication-assisted capillary action; an ultrasonic spray of the sample solution is emitted at the outlet of the tapered capillary, leading to the ready generation of gas-phase ions. Using an ion trap mass spectrometer, we found that singly charged amino acid and multiply charged peptides/proteins ions were generated through this single-step operation, which is both straightforward and extremely simple to perform. The setup is uncomplicated: only a low-frequency ultrasonicator and a tapered capillary are required to perform UASI. The mass spectra of the multiply charged peptides and proteins obtained from sample solutions subjected to UASI resemble those observed in ESI mass spectra.     

Expanded use of a battery-powered two-electrode emitter cell for electrospray mass spectrometry

A battery-powered, controlled-current, two-electrode electrochemical cell containing a porous flow-through working electrode with high surface area and multiple auxiliary electrodes with small total surface area was incorporated into the electrospray emitter circuit to control the electrochemical reactions of analytes in the electrospray emitter. This cell system provided the ability to control the extent of analyte oxidation in positive ion mode in the electrospray emitter by simply setting the magnitude and polarity of the current at the working electrode. In addition, this cell provided the ability to effectively reduce analytes in positive ion mode and oxidize analytes in negative ion mode. The small size, economics, and ease of use of such a battery-powered controlled-current emitter cell was demonstrated by powering a single resistor and switch circuit with a small-size, 3 V watch battery, all of which might be incorporated on the emitter cell.     

Electrospray mass spectrometry of methanol and water solutions suppression of electric discharge with SF6 gas

An equation by D. P. H. Smith predicts the capillary voltage required for the onset of electrospray (ES). For different solvents the voltage increases with the square root of the surface tension. Water requires a potential that is 1.8 times higher than that for methanol. This is verified experimentally. The higher potential required for water leads to ES in the presence of corona electric discharge. For low total ES plus corona currents, the electrosprayed analyte ion intensity is not adversely affected by the presence of discharge. At high total currents, there is a large decrease of analyte sensitivity. The sensitivity decrease is probably due to adverse space charge effect at high currents. The discharge can be suppressed by adding sulfur hexafluoride to the ambient gas. Both sensitivity and signal stability are improved. However, the sensitivity still remains lower by a factor of ≈ 4 relative to that observed with methanol. This is attributed to lower efficiency of gas-phase ion formation from charged water, relative to methanol, droplets.     

On-line microwave-induced helium plasma atomic emission detection for capillary zone electrophoresis

We report a feasibility study on using a microwave-induced helium plasma atomic emission detector (MIP-AED) as an on-line detector in capillary zone electrophoresis (CZE). To couple CZE to MIP-AED, we used an ion exchange membrane capillary to connect the separation capillary to the interfacing capillary. The outlet end of the interfacing capillary was placed directly in the discharge tube of the MIP-AED system. The electroosmotic flow generated in the separation capillary carried the analytes and the electrolyte buffer solution through the interfacing capillary into the MIP-AED discharge tube where the analytes were detected. The performance of the CZE/MIP-AED system was evaluated with trimethyltin chloride, dimethyltin dichloride, n-propanol, and 2-butanone. The preliminary results indicate that the MIP-AED can be used in CZE to provide element-specific detection for target analytes.     

Analysis of ion-association reaction in aqueous solution and its utilization by capillary zone electrophoresis.

Electrophoretic migration of analytes in capillary zone electrophoresis (CZE) reflects the dissolved status of analytes in solution, and the electrophoretic mobility is controlled to develop the resolution among analytes by adding a "modifier" to the migrating solution. Such addition of modifier is essentially the utilization of molecular interactions. Precise measurement of electrophoretic mobility by CZE allows analyzing molecular interactions, and CZE apparatus is very useful for physicochemical measurements. This review focuses on the advantages on using CZE to analyze equilibrium reaction; the capillary electrophoretic method and mathematical analyses that apply acid dissociation and complex formation reactions are also validated. Ion association reactions are deeply related to analytical chemistry and separation science, and CZE has been used for the investigation of ion-ion interactions. Various types of interactions have been clarified through the CZE measurements: contributions of hydrophobicity, probability, and aromatic-aromatic interaction were quantitatively evaluated. Ion association reaction in aqueous solution also elucidates the stepwise reactions of liquid-liquid distribution of ion associates. Development and applications of ion association reaction in CZE analysis are also introduced.