Controlling analyte electrochemistry in an electrospray ion source with a three-electrode emitter cell

The inherent electrochemistry occurring at the emitter electrode of an electrospray ion source was effectively controlled by incorporating a three-electrode controlled-potential electrochemical cell into the controlled-current electrospray emitter circuit. Two different basic cell designs were investigated to accomplish this control, namely, a planar flow-by working electrode and a porous flow-through working electrode design, each operated with a potentiostat floated at the electrospray high voltage. Control of the analyte electrochemistry was tested using the indole alkaloid reserpine, which is often used to test the specifications of electrospray mass spectrometry instrumentation. Reserpine was relatively easy to oxidize (E(p) = 0.73 V vs Ag/AgCl) in the acidic electrospray medium (acetonitrile/water 1:1 v/v, 5.0 mM ammonium acetate, 0.75 vol % acetic acid) and was oxidized when the conventional electrospray emitter was used at low solution flow rate. With the proper cell auxiliary electrode configuration and adjustment of the working electrode potential, it was found that reserpine oxidation could be "turned off" at flow rates as low as 2.5 microL/min as well as at flow rates as high as 30-40 microL/min. Just as important, it was also possible to "turn on" essentially 100% oxidation of reserpine in this flow rate range. The area of the auxiliary electrode along with flow rate, which affect mass transport of analytes to this electrode, were found to be critical in controlling the electrochemical reactions in the emitter cell. Such control over analyte electrochemical reactions in the emitter has been difficult or impossible to achieve with a conventional electrospray emitter. This control is paramount in obtaining experimental results free from electrochemically generated artifacts of the analyte or in exploiting electrochemical reactions involving the analyte to analytical advantage.     

Mapping of potential gradients within the electrospray emitter

A novel electrochemical probe has been designed, built, and used to characterize the distribution in solution potential within the metal capillary and Taylor cone of the electrospray (ES) device. The measurement system consists of three electrodes-a counter electrode held at highly negative potential that serves as the cathode, and two anodes consisting of a disk-shaped, mobile, internal (working) electrode, and the internal surface of the surrounding ES capillary (auxiliary electrode, held at ground potential). One-dimensional differential electrospray emitter potential (DEEP) maps detailing solution potential gradients within the electrospray emitter and in the region of the Taylor cone are constructed by measuring the potential at the working electrode vs the ES capillary, as a function of working electrode position along the emitter axis. Results show that the measured potential difference increases as the internal probe travels toward the ES capillary exit, with values rising sharply as the base of the Taylor cone is penetrated. Higher conductivity solutions exhibit potentials of higher magnitude at longer distances away from the counter electrode, but these same solutions show lower potentials near the ES capillary exit. Removal of easily oxidizable species from the solution causes the measured potential difference to have nonzero values at distances further within the capillary, and the values measured at all points are raised. Results are consistent with the characterization of the electrospray system as a controlled-current electrolytic flow cell. Elucidation of the electrochemical details of the electrospray process can lead to mass spectrometric signal enhancement of certain species present in the spraying liquid and also allow the detection of molecules that are usually not observable due to their low ionization efficiencies.     

Study and application of a controlled-potential electrochemistry-electrospray emitter for electrospray mass spectrometry

This paper discusses continued studies and new analytical applications of a recently developed three-electrode controlled-potential electrochemical cell incorporated into an electrospray ion source (Van Berkel, G. J.; Asano, K. G.; Granger, M. C. Anal. Chem. 2004, 76, 1493-1499.). This cell contains a porous flow-through working electrode (i.e., the emitter electrode) with high surface area and auxiliary electrodes with small total surface area that are incorporated into the emitter electrode circuit to control the electrochemical reactions of analytes in the electrospray emitter. The current at the working and auxiliary electrodes, and current at the grounding points upstream and downstream of the emitter in the electrospray circuit, were recorded in this study, along with the respective mass spectra of model compound reserpine, under various operating conditions to better understand the electrochemical and electrospray operation of this emitter cell. In addition to the ability to control analyte oxidation in positive ion mode (or reduction in negative ion mode) in the electrospray emitter, this emitter cell system was shown to provide the ability to efficiently reduce analytes in positive ion mode and oxidize analytes in negative ion mode. This was demonstrated by the reduction of methylene blue in positive ion mode and oxidation of 3,4-dihydroxybenzoic acid in negative ion mode. Also, the ability to control electrochemical reactions via potential control was used to selectively ionize (oxidize) analytes with different standard electrochemical potentials within mixtures to different charge states to overcome overlapping molecular ion isotopic clusters. The analytical benefit of this ability was illustrated using a mixture of nickel and cobalt octaethylporphyrin.     

Electrochemically modulated preconcentration and matrix elimination for organic analytes coupled on-line with electrospray mass spectrometry

Demonstrated for the first time is the use of electrochemically modulated preconcentration and sample matrix elimination combined on-line with electrospray mass spectrometry (EMPM/ES-MS) for the enhanced analysis of organics by ES-MS. EMPM is similar to adsorptive stripping analysis. Accumulation of the targeted analytes at the working electrode of an on-line electrochemical flow cell is accomplished via a nonelectrolytic adsorption process that is controlled through the proper combination of the solvent system, the working electrode material, and applied potential. Once on the electrode, the analyte may be washed free of sample matrix components detrimental to mass spectrometric detection. The potential applied to the electrode during the detection step is chosen to release or strip the analytes unaltered back into the solvent stream for mass spectrometric detection rather than to oxidize or reduce them as would be the case for electrochemical detection. Thus, retention and elution of a target analyte with EMPM are controlled by switching the working electrode potential, rather than via a switch in mobile-phase composition, as is done in more traditional preconcentration and cleanup schemes used on-line with ES-MS. The proof-of-principle studies described here use the breast cancer drug tamoxifen and a metabolite, 4-hydroxytamoxifen, as the target analytes. A thin-layer, flow-by electrode cell with a glassy carbon working electrode is used as the preconcentration device. The nature of the working electrode, the solvent systems, and the electrode potentials necessary to accumulate and strip tamoxifen and 4-hydroxytamoxifen are discussed. Calibration curves were fitted using the Langmuir isotherm. Detection limits (DLs) using a 5.0 min preconcentration period with selected reaction monitoring for tamoxifen (m/z 372 --> 72) were bracketed as 0.010 nM < DL < 0.025 nM. The ability to simultaneously detect low nanomolar levels of both tamoxifen and 4-hydroxytamoxifen in pristine solution and 1/10 diluted urine is also demonstrated.     

Study of electrochemical reactions using nanospray desorption electrospray ionization mass spectrometry

The combination of electrochemistry (EC) and mass spectrometry (MS) is a powerful analytical tool for studying mechanisms of redox reactions, identification of products and intermediates, and online derivatization/recognition of analytes. This work reports a new coupling interface for EC/MS by employing nanospray desorption electrospray ionization, a recently developed ambient ionization method. We demonstrate online coupling of nanospray desorption electrospray ionization MS with a traditional electrochemical flow cell, in which the electrolyzed solution emanating from the cell is ionized by nanospray desorption electrospray ionization for MS analysis. Furthermore, we show first coupling of nanospray desorption electrospray ionization MS with an interdigitated array (IDA) electrode enabling chemical analysis of electrolyzed samples directly from electrode surfaces. Because of its inherent sensitivity, nanospray desorption electrospray ionization enables chemical analysis of small volumes and concentrations of sample solution. Specifically, good-quality signal of dopamine and its oxidized form, dopamine o-quinone, was obtained using 10 μL of 1 μM solution of dopamine on the IDA. Oxidation of dopamine, reduction of benzodiazepines, and electrochemical derivatization of thiol groups were used to demonstrate the performance of the technique. Our results show the potential of nanospray desorption electrospray ionization as a novel interface for electrochemical mass spectrometry research.     

On-line probe for fast electrochemistry/electrospray mass spectrometry. Investigation of polycyclic aromatic hydrocarbons

A newly invented probe accessory for fast electrochemistry/electrospray mass spectrometry (EC/ESMS) is presented and evaluated. The device features a low-volume, three-electrode electrochemical cell which has been designed with a minimum distance between the working electrode and the "Taylor cone" inherent to the electrospray process. This configuration limits the time between electrochemical generation of ions and mass spectrometric analysis to an absolute minimum. A fused-silica layer insulates the microcylinder working electrode from the sample solution until immediately prior to the electrospray region, postponing electrode processes until the last moment. The same fused-silica layer insulates the working electrode from the surrounding auxiliary electrode, a stainless steel capillary that also serves as the electrospray capillary. The performance and capabilities of the novel electrochemistry/electrospray mass spectrometry system have been evaluated using polycyclic aromatic hydrocarbons (PAHs) as test analytes. In the positive ion EC/ESMS mode, oxidized forms (one-electron removal) of PAHs are produced in high yield. The ability to analyze reaction products appearing subsequent to the initial oxidation is also demonstrated.     

Enhancement of ionization efficiency by electrochemical reaction products in on-line electrochemistry/electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

A miniaturized two-electrode electrochemical (EC) cell was developed and was coupled on-line with an electrospray ionization Fourier transform ion cyclotron resonance mass spectrometer (ESI-FTICR MS). Electrochemistry on-line with mass spectrometry, EC/ESI-FTICR MS, of triphenylamine (TPA), which undergoes one-electron oxidation to form a radical cation (TPA*+), demonstrates a significant sensitivity enhancement compared to ESI-FTICR MS. The on-line EC cell configuration with a stainless steel ES needle as the working electrode produces the highest sensitivity in EC/ESI-MS. The results provide evidence that, during the ES ionization, electrolytic reactions occur mainly in the ES tip region, as previously predicted. The results demonstrate that ESI-MS signal suppression by tetrabutylammonium perchlorate electrolyte, which can be a problem, is minimized in EC/ESI-MS. TPA*+ dimer tetraphenylbenzidine (TPB) can be detected by EC/ESI-MS, together with TPA*+, as TPB*+ and TPB2+. The high mass resolving power of FTICR MS was exploited to identify TPB2+ dication in the presence of [TPA*+ - H*]+ ions of the same m/z, from their respective isotopic distributions. The dimer dication TPB2+ can be detected only in EC/ESI-MS.     

Enhanced study and control of analyte oxidation in electrospray using a thin-channel,planar electrode emitter

A thin-channel, planar electrode emitter device is described and utilized for the study and control of electrochemical oxidation of analytes at the emitter electrode in an electrospray ion source. For analytes that are not particularly susceptible to oxidation, the planar electrode device functions analytically in a manner similar to emitter systems that utilize the more common stainless steel tubular electrodes. For more easily oxidized analytes, the device provides the means to achieve near 100% oxidation efficiency or to completely eliminate analyte oxidation through simple and rapid changes in electrode material, electrode area, electrode covering, channel height above the electrode, or solution flow rate. Compared to the use of tubular electrodes, the planar electrode emitter system provides improved flexibility in altering the nature of the electrode area and material, as well as altering analyte mass transport to the electrode surface. Each of these parameters is critical in the control of electrochemical reactions and can be easily studied or exploited with this emitter electrode configuration.     

Nanoporous organosilicas as preconcentration materials for the electrochemical detection of trinitrotoluene

We describe the use of nanoporous organosilicas for rapid preconcentration and extraction of trinitrotoluene (TNT) for electrochemical analysis and demonstrate the effect of template-directed molecular imprinting on TNT adsorption. The relative effects of the benzene (BENZ)- and diethylbenzene (DEB)-bridged organic-inorganic polymers, having narrow or broad pore size distributions, respectively, on electrochemical response and desorption behavior were examined. Sample volumes of 0.5-10 mL containing 5-1000 ppb TNT in a phosphate-buffered saline buffer were preconcentrated in-line before the detector using a microcolumn containing 10 mg of imprinted BENZ or DEB. Square-wave voltammetry was used to detect the first reduction peak of TNT in an electrochemical flow cell using a carbon working electrode and a Ag/AgCl reference electrode. Imprinted BENZ released TNT faster than imprinted DEB with considerably less peak tailing and displayed enhanced sensitivity and an improvement in the limit of detection (LOD) owing to more rapid elution of TNT from that material with increasing signal amplitude. For imprinted BENZ, the slope of signal versus concentration scaled linearly with increasing preconcentration volume, and for preconcentrating 10 mL of sample, the LOD for TNT was estimated to be 5 ppb. Template-directed molecularly imprinted DEB (TDMI-DEB) was 7-fold more efficient in adsorption of TNT from aqueous contaminated soil extract than nonimprinted DEB.     

Ambient gas influence on electrospray potential as revealed by potential mapping within the electrospray capillary

The effect of ambient gas on potentials inside the electrospray (ES) capillary was investigated. Potential measurements and differential electrospray emitter potential (DEEP) maps were obtained with the help of a small, movable, disklike platinum wire electrode inserted into the ES capillary. Typical solvents used for electrospray mass spectrometry such as methanol and mixtures of methanol-water and chloroform/methanol have been tested. It was found that oxygen is readily adsorbed from the surrounding ambient gas into the spraying liquid. Following adsorption, it resides in, or near to, the Taylor cone, thereby affecting the electrochemical potential near the ES capillary exit, as well as the character of the inherent electrochemical reactions occurring during the ES process. The potentials measured in an air environment with reactive oxygen present are contrasted against those obtained in an inert nitrogen environment. The kinetics of oxygen admission have been found to be quite fast, i.e., occurring in a matter of seconds, but it takes far longer to purge the system of oxygen by changing the ambient atmosphere to nitrogen. The oxygen effect is present in negative and positive ion modes of ES, but the total ES current is not affected by the change of ambient gas. The magnitude of the oxygen effect owing to ambient air was compared to the effect caused by initially dissolving oxygen in the solution prior to the start of ES; it was found that the presence of oxygen in the ambient gas has a far greater consequence. These results indicate that the presence of reactive gases, such as molecular oxygen, in the region of the ES emitter may have unintended secondary effects on the ES process prior to mass spectrometric analysis.     

Expanded electrochemical capabilities of the electrospray ion source using porous flow-through electrodes as the upstream ground and emitter high-voltage contact

Use of a porous flow-through electrode at the upstream ground contact or at both the upstream ground contact and the high-voltage emitter contact in an electrospray ion source was shown to provide for new types of electrochemical experiments utilizing only the electrochemistry inherent to electrospray. The normal stainless steel bore-through union serving as the upstream grounding point in a floated electrospray emitter system was replaced with a high surface area porous flow-through electrode assembly to achieve effective electrochemical reduction of analytes at this point in positive ion mode, and effective electrochemical oxidation of analytes in negative ion mode. This was demonstrated by the oxidation of 3,4-dihydroxybenzoic acid and reserpine in negative ion mode and by the reduction of thionine in positive ion mode. In the case of reversible oxidation (3,4-dihydroxybenzoic acid) and reduction (thionine) processes, partial rereduction and reoxidation of the products due to reaction with products generated by cathodic and anodic processes at the emitter were observed, respectively. By implementing two high surface area porous flow-through electrodes in the system, one as the upstream grounding point and the other as the emitter electrode, a multiple-step reaction scheme was achieved that included consecutive electrochemical reduction and oxidation reactions and a following chemical reaction as demonstrated by the hydroquinone tagging of an initially disulfide-linked peptide.     

Control of electrochemical reactions at the capillary electrophoresis outlet/electrospray emitter electrode under CE/ESI-MS through the application of redox buffers

It was found that combining capillary electrophoresis (CE) and electrospray ionization mass spectrometry (ESI-MS) overlays two controlled current techniques to form a three-electrode system (CE inlet, CE outlet/ES emitter, and MS inlet electrodes) in which the CE outlet electrode and the ES emitter electrode were shared between the CE and the ESI-MS circuits. Depending on the polarities and magnitudes of the voltages at the CE inlet, CE outlet/ES emitter, and MS inlet electrodes, the nature of the two redox reactions at the shared electrode was the same or different (both reduction, both oxidation, or one oxidation and the other reduction). Several redox buffers were introduced for controlling electrochemical reactions at the shared electrode. By reacting at this electrode, redox buffers were able to maintain electrode potentials below the onset of water electrolysis, thereby eliminating gas bubble formation and/or pH drift. The volume of the gas generated due to water electrolysis was used to quantitate water oxidation or reduction at this electrode. Two types of redox buffers were used. A reactive electrode with an oxidation potential below that of water was used as the electrode under anodic conditions. Also, a reactive compound with a redox potential below that of water was added to the CE and/or ESI running buffer. When the shared electrode was the anode of both CE and ESI-MS circuits, the use of iron or etched and sanded stainless steel (ss) wire, instead of platinum wire, suppressed bubble formation at the shared electrode. Under these conditions, corrosion of the Fe wire and formation of Fe2+ replaced oxidation of water, eliminating O2 gas bubble and H+ formation. When mixtures of peptides were analyzed, iron adducts of peptides were observed. For a fresh wire, however, the intensities of adduct ions were less than 3% of the protonated molecules. After a few days of operation, the intensities of the adduct ions increased to approximately 50%, due to rust formation on the Fe wire. On-column rinsing with a 40% solution of citric acid rejuvenated the Fe wire and reduced the adduct peak intensities to less than 3%. Unmodified ss wire did not quench bubble formation, which was attributed to its passivated surface. When Fe, ss, and Pt wires were used as the shared electrode under forward polarity CE and positive ESI mode, where the shared electrode acted as a cathode with respect to CE inlet and as an anode with respect to MS inlet, reduction of water at the cathodic end of the electrode and, in the case of ss and Pt wires, oxidation of water at the anodic end of the shared electrode produced a significant amount of bubbles. Under these conditions, however, a buffer containing 50 mM p-benzoquinone completely suppressed both cathodic reduction and anodic oxidation of water for CE currents up to 4 microA. Reduction of p-benzoquinone at the cathodic end of the shared electrode to hydroquinone, and oxidation of this hydroquinone at the anodic end of the electrode, replaced reduction and oxidation of water, eliminating bubble formation. A 0.1% acetic acid solution saturated with I2 was also found to suppress bubble formation at the cathode for CE currents up to 3 microA; however, strong iodine adduct ions were observed under CE/ESI-MS when a mixture of peptides was analyzed. The application of iron as an in-capillary electrode for the analysis of a peptide mixture and a protein digest demonstrated a high separation efficiency similar to when hydroquinone was used as a redox buffer.     

Electrospray ionization with a pointed carbon fiber emitter

A new type of electrospray ionization emitter employing a pointed carbon fiber has been developed for interfacing nanoliquid sampling techniques to mass spectrometry. The pointed carbon fiber protruding from an orifice with a surrounding hydrophobic surface confines a small Taylor cone at the tip, which generates a stable electrospray at the tip point. The small Taylor cone improves the electrospray efficiency thereby enhancing the detection limit. This emitter is rugged and able to generate stable electrospray over a wide range of flow rate, ESI voltage, and surface tension variation. Using a solution of angiotensin I, the carbon fiber emitter in 75-microm-i.d. fused-silica tubing was shown to give ion current comparable to that from a commercial 8 microm orifice nanospray emitter. Use of the emitter for ESI-MS/MS analysis of peptides was examined by infusing a mixture of cytochrome c and myoglobin tryptic digest peptides. Protein identification was demonstrated at the level of less than 1 fmol of the peptide consumed. The use of the carbon fiber emitter for interfacing monolithic capillary HPLC to MS was also demonstrated.     

Recommended Practices and Benchmark Activity for Hydrogen and Oxygen Electrocatalysis in Water Splitting and Fuel Cells

Electrochemical energy storage by making H₂ an energy carrier from water splitting relies on four elementary reactions, i.e., the hydrogen evolution reaction (HER), hydrogen oxidation reaction (HOR), oxygen evolution reaction (OER), and oxygen reduction reaction (ORR). Herein, the central objective is to recommend systematic protocols for activity measurements of these four reactions and benchmark activities for comparison, which is critical to facilitate the research and development of catalysts with high activity and stability. Details for the electrochemical cell setup, measurements, and data analysis used to quantify the kinetics of the HER, HOR, OER, and ORR in acidic and basic solutions are provided, and examples of state‐of‐the‐art specific and mass activity of catalysts to date are given. First, the experimental setup is discussed to provide common guidelines for these reactions, including the cell design, reference electrode selection, counter electrode concerns, and working electrode preparation. Second, experimental protocols, including data collection and processing such as ohmic‐ and background‐correction and catalyst surface area estimation, and practice for testing and comparing different classes of catalysts are recommended. Lastly, the specific and mass activity activities of some state‐of‐the‐art catalysts are benchmarked to facilitate the comparison of catalyst activity for these four reactions across different laboratories.     

Scanning capillary microscopy/mass spectrometry for mapping spatial electrochemical activity of electrodes

A new technique for microscopic imaging of electrochemically active surfaces is introduced. The technique combines concepts of probe microscopy and advances in mass spectrometry. The technique is based on a miniature electrochemical flow cell scanner. A liquid feed stream containing a redox component is introduced to the vicinity of the examined location through the annulus of a coaxial capillary set. The incoming reagent interacts with the target location, and the generated product stream is transferred through the inner capillary to an electrospray mass spectrometer, ESI-MS. Thus, a multicomponent, potential-dependent image of the products' distribution versus the location on the electrode is generated. The use of the technique is demonstrated by scanning the electrochemical heterogeneity of model electrodes.     

Sheathless capillary electrophoresis/electrospray ionization mass spectrometry using a pulled bare fused-silica capillary as the electrospray emitter

It has always been assumed that electrical contact at the capillary outlet is a necessary requirement when coupling capillary electrophoresis (CE) with electrospray ionization mass spectrometry (ESI-MS). In this study, we used a pulled bare-capillary tip as the ESI emitter, but neither was it coated with any electrically conductive materials nor was a high external voltage applied on its outlet. In this paper, we demonstrate that this straightforward approach may be used to generate multiply charged ions of proteins and peptides through electrospray ionization. Our results indicate that peptides and proteins, including bradykinin, cytochrome c, myoglobin, and tryptic digest products that elute from a pulled bare-capillary tip can be detected directly by ESI-MS using the tapered bare-capillary interface. Thus, we have demonstrated that CE and ESI-MS may be combined successfully without the need to modify the outlet of the capillary tip with an electrically contacting material.     

Label free screening of enzyme inhibitors at femtomole scale using segmented flow electrospray ionization mass spectrometry

Droplet-based microfluidics is an attractive platform for screening and optimizing chemical reactions. Using this approach, it is possible to reliably manipulate nanoliter volume samples and perform operations such as reagent addition with high precision, automation, and throughput. Most studies using droplet microfluidics have relied on optical techniques to detect the reaction; however, this requires engineering color or fluorescence change into the reaction being studied. In this work, we couple electrospray ionization mass spectrometry (ESI-MS) to nanoliter scale segmented flow reactions to enable direct (label-free) analysis of reaction products. The system is applied to a screen of inhibitors for cathepsin B. In this approach, solutions of test compounds (including three known inhibitors) are arranged as an array of nanoliter droplets in a tube segmented by perfluorodecalin. The samples are pumped through a series of tees to add enzyme, substrate (peptides), and quenchant. The resulting reaction mixtures are then infused into a metal-coated, fused silica ESI emitter for MS analysis. The system has potential for high-throughput as reagent addition steps are performed at 0.7 s per sample and ESI-MS at up to 1.2 s per sample. Carryover is inconsequential in the ESI emitter and between 2 and 9% per reagent addition depending on the tee utilized. The assay was reliable with a Z-factor of ~0.8. The method required 0.8 pmol of test compound, 1.6 pmol of substrate, and 5 fmol of enzyme per reaction. Segmented flow ESI-MS allows direct, label free screening of reactions at good throughput and ultralow sample consumption.     

Pt‐Based Nanocrystal for Electrocatalytic Oxygen Reduction

Currently, Pt‐based electrocatalysts are adopted in the practical proton exchange membrane fuel cell (PEMFC), which converts the energy stored in hydrogen and oxygen into electrical power. However, the broad implementation of the PEMFC, like replacing the internal combustion engine in the present automobile fleet, sets a requirement for less Pt loading compared to current devices. In principle, the requirement needs the Pt‐based catalyst to be more active and stable. Two main strategies, engineering of the electronic (d‐band) structure (including controlling surface facet, tuning surface composition, and engineering surface strain) and optimizing the reactant adsorption sites are discussed and categorized based on the fundamental working principle. In addition, general routes for improving the electrochemical surface area, which improves activity normalized by the unit mass of precious group metal/platinum group metal, and stability of the electrocatalyst are also discussed. Furthermore, the recent progress of full fuel cell tests of novel electrocatalysts is summarized. It is suggested that a better understanding of the reactant/intermediate adsorption, electron transfer, and desorption occurring at the electrolyte–electrode interface is necessary to fully comprehend these electrified surface reactions, and standardized membrane electrode assembly (MEA) testing protocols should be practiced, and data with full parameters detailed, for reliable evaluation of catalyst functions in devices.     

Ultrarapid desalting of protein solutions for electrospray mass spectrometry in a microchannel laminar flow device

The adverse effects of nonvolatile salts on the electrospray (ESI) mass spectra of proteins and other biological analytes are a major obstacle for a wide range of applications. Numerous sample cleanup approaches have been devised to facilitate ESI-MS analyses. Recently developed microdialysis techniques can shorten desalting times down to several minutes, the bottleneck being diffusion of the contaminant through a semipermeable membrane. This work introduces an approach that allows the on-line desalting of macromolecule solutions within tens of milliseconds. The device does not employ a membrane; instead, it uses a two-layered laminar flow geometry that exploits the differential diffusion of macromolecular analytes and low molecular weight contaminants. To maximize desalting efficiency, diffusive exchange between the flow layers is permitted only for such a time as to allow full exchange of salt, while incurring minimal macromolecule exchange. Computer simulations and optical studies show that the device can reduce the salt concentration by roughly 1 order of magnitude, while retaining approximately 70% of the original protein concentration. Application of this approach to the on-line purification of salt-contaminated protein solutions in ESI-MS results in dramatic improvements of both the signal-to-noise ratio and the absolute signal intensity. However, efficient desalting requires the diffusion coefficients of salt and analyte to differ by roughly 1 order of magnitude or more. This technique has potential to facilitate high-throughput analyses of biological macromolecules directly from complex matrixes. In addition, it may become a valuable tool for process monitoring and for on-line kinetic studies on biological systems.