Preparation of Durable Emitter of Electrospray Mass Spectrometry by Covalently Coating the Fused‐Silica Capillary Tip with Carbon‐Nanotube Sol‐Gel Composite Material

A simple approach for the preparation of emitter of electrospray ionization mass spectrometry by covalently coating the fused‐silica capillary tip with the conductive carbon‐nanotube sol‐gel composite material (CNTSCM) is described. The CNTSCM was prepared by dispersing single‐walled carbon nanotubes in the sol composed of a mixture of 3‐glycidoxypropyltrimethoxysilane and 3‐aminopropyltrimethoxysilane, and ethanol. The long‐term stability of the prepared ESI emitters was demonstrated by at least 180 h of continuous use. Signal intensity obtained by the prepared emitter was mass‐flux sensitive when the flow rate was lower than 500 nL/min, while the signal intensity performed a concentration dependence when the flow rate was in the range of 500–800 nL/min. The usefulness of such a prepared emitter was demonstrated by the analysis of various types of samples such as organic small molecular drugs, oligosaccharide, peptide, and protein.     

A sheath-flow nanospray interface for capillary electrophoresis/mass spectrometry

We have developed a novel sheath-flow interface for low-flow electrospray ionization mass spectrometry (ESI-MS) and capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS). The interface is composed of two capillaries. One is a tapered fused-silica ESI emitter suitable for microliter and nanoliter flow rate electrospray and the other is a tail-end gold-coated CE separation column that is inserted into the emitter. A sheath liquid is supplied between the column and the emitter capillaries. The gold coating and the sheath liquid are used as the conducting media for ESI and the CE circuit. This novel design was initially evaluated by an infusion ESI-MS analysis of the most common antiretroviral dideoxynucleosides, followed by CE/MS coupling analysis of several antidepressant drugs. With infusion studies, the effects of the sheath liquid and the sample flow rates on detection sensitivity and signal stability were investigated. For an emitter with an internal diameter of 30 microm, the optimum flow rates for the sheath and the sample were 200 and 300 nL/min, respectively. The main improvement of this approach in comparison with conventional sheath liquid approaches using an ionspray interface is the gain in sensitivity. Sensitivities were three times better for dideoxynucleosides analyzed by infusion and 12 times higher for antidepressant drugs analyzed by CE/MS with this interface compared with ionspray. The emitter is durable, disposable, and simple to fabricate.     

Micro-electrospray with stainless steel emitters

The physical processes underlying micro-electrospray (micro-ES) performance were investigated using a stainless steel (SS) emitter with a blunt tip. Sheathless micro-ES could be generated at a blunt SS tip without any tapering or sanding if ESI conditions were optimized. The Taylor cone was found to shrink around the inner diameter of the SS tubing, which permitted a low flow rate of 150 nL/min for sheathless microspray on the blunt tip (100 microm i.d. x 400 microm o.d.). It is believed that the wettability and/or hydrophobicity of SS tips are responsible for their micro-ES performance. The outlet orifice was further nipped to reduce the size of the spray cone and limit the flow rate to 50-150 nL/min, resulting in peptide detection down to attomole quantities consumed per spectrum. The SS emitter was also integrated into a polymethylmethacrylate microchip and demonstrated satisfactory performance in the analysis and identification of a myoglobin digest.     

A robust micro-electrospray ionization technique for high-throughput liquid chromatography/mass spectrometry proteomics using a sanded metal needle as an emitter

A 60 microm internal diameter (i.d.) stainless-steel needle was adapted to the orthogonal ESI probe ( microESI) of a commercial ion trap mass spectrometer, and used for capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) protein identification experiments. The modification allows for the use of nitrogen sheath gas which helps in the nebulization at LC flow rates exceeding 500 nL/min and eliminates problems caused by liquid junctions commonly used to initiate nanospray ionization (NSI). A comparison is made between the performance of a 75 microm i.d. column with a 15 microm pulled glass tip using a liquid junction, and that of a 150 microm i.d. column using the new microESI device. The combination of the 150 microm i.d. column and microESI gave sensitivity close to that of NSI (250 attomoles horse heart myoglobin digest on column), and proved to be more robust than the standard pulled glass tips of similar i.d. No evidence of metal needle catalyzed oxidation of methionine was observed during analysis of the tetrapeptide MRFA under a range of test conditions. Phosphorylated peptides in a beta-casein tryptic digest were also successfully identified using the microESI interface with a steel needle. In addition it was found that a mild sanding of the metal needle tip improved spray performance.     

Sheathless capillary electrophoresis electrospray ionization‐mass spectrometry interface based on poly(dimethylsiloxane) membrane emitter and thin conducting liquid film

This study develops a sheathless CE‐MS interface using a robust PDMS membrane emitter and liquid‐film electric conduction. A 3D mold was constructed for casting the device by using a one‐step casting procedure. The interface consisted of a 125 μm‐thick triangular emitter with a 50 μm‐diameter microchannel, a conducting reservoir, and a 375 μm‐diameter channel for assembling the separation capillary. The separation capillary was inserted into the 375 μm channel and connected to the emitter through the conducting reservoir. The electric contact for the CE outlet was established through a conductive liquid film in the space between the capillary terminus and the 375 μm channel. The one‐step casting procedure and using a membrane emitter instead of a tapered emitter produced an easily fabricated and robust interface. A stable electrospray was obtained from 30 to 350 nL/min. Analyzing a five‐peptide mixture in low‐EOF (60 nL/min) and high‐EOF (210 nL/min) conditions demonstrated the utility of the interface.     

Porous polymer monolith assisted electrospray from a glass microdevice

The coupling of a lab-on-a-chip microfluidic device to a nanoelectrospray ionization mass spectrometer has the potential to automate many routine analytical procedures and produce a powerful analytical tool. However, past coupling strategies have relied on complex manufacturing steps including drilling and etching the device to attach a capillary or building a nanospray emitter directly into the device. This study shows that a nanospray emitter can be easily fabricated using a porous polymer monolith (PPM) at the end of a glass microdevice. These devices are able to obtain a stable electrospray at a variety of flow rates (50-500 nL/min) but optimal results are obtained at lower flow rates (50-100 nL/min) compatible with electroosmotic flow processes. The PPM is photo-patterned so that it can be placed in any position within the channel of the device with no dead volume. The porous character and the hydrophobic nature of the PPM both aid in development of a stable electrospray process. Total ion current traces for the constant infusion of leucine-enkephalin and PPG show relative standard errors as low as 4%, and produce mass spectra with good signal-to-noise (S/N 43) from only 2 fmol of material. In addition, multiple experiments in a given day show good repeatability with variability as low as 13%, and the multiple flow paths inherent in the PPM limit sprayer clogging.     

Electrospray sample deposition for matrix-assisted laser desorption/ionization (MALDI) and atmospheric pressure MALDI mass spectrometry with attomole detection limits

Electrospray sample deposition was explored for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). In this method, nanoliter volumes of matrix/analyte mixture were electrosprayed from a high voltage biased (1-2 kV) fused-silica capillary onto a grounded MALDI plate mounted 100-500 microm from the capillary outlet. Electrospray deposition with these conditions produced sample spots 200-300 microm in diameter thus matching the laser spot size. Varying spray voltage and distance resulted in different crystal sizes and volatilization rates for alpha-cyano-4-hydroxycinnamic acid matrix. Best results were obtained when the sample was deposited as wet droplets as opposed to deposition as dried solid. Under 'wet-spray' conditions, 2-4 microm diameter crystals were formed and detection limits for several neuropeptides were 0.7-25 amol. Samples could be pre-concentrated on the plate by spraying continuously and allowing sample to evaporate in a small spot. Sample volumes as large as 580 nL were deposited yielding a detection limit of 35 pM for neurotensin 1-11. Electrospray sample deposition yielded similar results when using atmospheric pressure-MALDI coupled with a quadrupole ion trap mass spectrometer, except that the sensitivity was approximately seven-fold worse.     

Flow-rate characterization of microfabricated polymer microspray emitters

Microfabricated polymer microspray emitters are characterized in terms of applicable flow rates, temporal and spectral signal-to-noise ratios (SNRs), and solution composition. First, microspray emitters can be operated with 50% methanol/49% water/1% acetic acid from 250 nL/min up to 7 microL/min, with better SNRs above 1 microL/min. Interestingly, even at the lowest flow rates tested, they compare well with nanospray capillaries in terms of mass spectral performances. Secondly, they can be operated with acetonitrile from 10 up to 99% (v/v), with flow rates from 250 nL/min up to 4 microL/min. Even if the mass spectral performances (especially the spectral SNRs) vary with the acetonitrile content, this study validates such microfabricated microspray emitters as interfaces between liquid-phase separations and electrospray mass spectrometers.     

Negative mode sheathless capillary electrophoresis electrospray ionization-mass spectrometry for metabolite analysis of prokaryotes

Capillary electrophoresis (CE) was coupled to negative mode electrospray ionisation-mass spectrometry (MS) for separation and detection of phosphorylated and acidic metabolites in extracts of prokaryotes. Unlike previous CE-MS systems for metabolite analysis, a sheathless interface was used to improve sensitivity. To accomplish this, the separation capillary was modified by creating a porous junction near the outlet where the electrospray voltage and cathodic voltage for CE were applied. The outlet of the capillary was pulled to a 5 microm inner diameter to form an electrospray emitter and had a frit fabricated near the exit to prevent clogging. During analysis pressure was applied at the inlet of the separation column to create sufficient flow towards the detector. Limits of detection for 19 metabolites in full scan mode ranged from 20 nM for ADP ribose to 2.5 microM for alpha-ketoglutarate for 40 nL injections. Extracts of Escherichia coli, strain DH5-alpha, were analyzed using this system. In full scan mode, 118 different metabolites were detected. Tandem mass spectrometry was also employed to attempt identification. Reproducible fragmentation of 19 parent peaks was found and 10 of these produced spectra that were consistent with identification obtained from matching to compounds in the MetaCyc database. These results demonstrate the utility of a sensitive CE-MS system for large scale metabolite detection in biological samples.     

Microfabricated PDMS multichannel emitter for electrospray ionization mass spectrometry

A novel microfabricated multichannel emitter for electrospray ionization mass spectrometry (ESI-MS) was implemented with polydimethylsiloxane (PDMS) using a soft lithography technique. The emitters are formed as electrospray tips along a thin membrane on the edge of the device with channels of 100 microm x 30 microm dimensions. The electrospray performance of the PDMS emitters for a single channel device and a four channel device interfaced with a time-of-flight mass spectrometer was evaluated for detecting the molecular weight of reference peptides (angiotensin I and bradykinin). The emitters were durable at the flow rate of 1-20 microL min(-1) for more than 30 h of continuous electrospray with limit of detection of 1 microM (S/N 18). This microfabrication method for a PDMS multichannel emitter as an integral part of a microfluidic device will facilitate development of more complex microfluidic analysis systems using ESI-MS.     

A Study of Electrospray Ionization Emitters with Differing Geometries with Respect to Flow Rate and Electrospray Voltage

The performance of several electrospray ionization emitters with different orifice inside diameters (i.d.s), geometries, and materials are compared. The sample solution is delivered by pressure driven flow, and the electrospray ionization voltage and flow rate are varied systematically for each emitter investigated, while the signal intensity of a standard is measured. The emitters investigated include a series of emitters with a tapered outside diameters (o.d.) and unaltered i.d.s, a series of emitters with tapered o.d.s and i.d.s, an emitter with a monolithic frit and a tapered o.d., and an emitter fabricated from polypropylene. The results show that for the externally etched emitters, signal was nearly independent of i.d. and better ion utilization was achieved at lower flow rates. Furthermore, emitters with a 50 μm i.d. and an etched o.d. produced about 1.5 times more signal than etched emitters with smaller i.d.s and about 3.5 times more signal than emitters with tapered inner and outer dimensions. Additionally, the work presented here has important implications for applications in which maximizing signal intensity and reducing frictional resistance to flow are necessary. Overall, the work provides an initial assessment of the critical parameters that contribute to maximizing the signal for electrospray ionization sources interfaced with pressure driven flows.     

Micro-magnetic particles frit for capillary electrochromatography

This paper presents a new method for making frit using soft-ferrite-based micro-magnetic particles (MMPs) in a micro-space, such as in a capillary tube. The MMPs-frit was made by injecting an aliquot of 10 microm (outer diameter; o.d.)-MMPs-suspension in methanol (ca. 1mg/ml) into a capillary tube (75 microm inner diameter (i.d.) x 375 microm o.d. x ca. 35 cm length) that was already sandwiched between a pair of cylindrical Neodium (Nd-Fe-B) magnets (1.5 mm o.d. x 1.5 mm height, 280 mT) at a position where the frit was made. The MMPs were trapped in the capillary tube as a frit due to the attraction of the magnets placed at surface on the capillary tube. With regard to durability, the frit was stable for methanol flow with a flow rate of 400 microl/min at room temperature. Using such a frit, a capillary column (20 cm long) was prepared by injecting a 5 microm (o.d.)-ODS-particle suspension in methanol (ca. 0.4 mg/microl) into the capillary tube. The MMPs-frits-ODS-packed column was stable for methanol for a flow pressure less than 20MPa. When comparing the present column with a conventional sintered-frits-ODS-packed column for the purposes of separating five kinds of biogenic amines by means of an on-column derivatization capillary electrochromatography (CEC), the performance of the MMPs-frits capillary column was almost equivalent to that of the sintered-frits-ODS-packed column.     

An evaluation of the performance of porous polymer monolith assisted electrospray

A novel electrospray interface, which has distinct advantages over conventional pulled silica emitters, has been developed. This novel interface can be easily fabricated by forming a porous polymer monolith (PPM) at the end of a fused-silica capillary that facilitates a stable electrospray over a wide range of flow rates with only a modest increase in back-pressure. A comparison was made between the PPM-assisted electrospray and a commercial nanosprayer in terms of sensitivity, stability and robustness. A PPM-filled electrospray tip produced a day-to-day signal variation of 23% relative standard deviation (RSD) over a 3-day period when spraying a 1.0 microM test peptide solution. Furthermore, three different capillaries fabricated by the same process produced a signal variation of 17% RSD, indicating that the fabrication process shows good reproducibility. The multiple flow paths of the PPM function to split the flow and reduce clogging. Even following the accumulation of debris after prolonged use, a stable spray could still be generated with the PPM-filled capillary while the commercial nanosprayer ceased to function properly. In terms of sensitivity, PPM-assisted electrospray showed an enhancement in sensitivity at infusion flow rates between 100 to 1000 nL/min while commercial nanosprayers performed slightly better at flow rates below 100 nL/min. A sample purification step can be combined with the PPM-assisted sprayer, using the PPM as a stationary phase to desalt and preconcentrate samples prior to mass spectrometric detection.     

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.     

Picoelectrospray Ionization Mass Spectrometry Using Narrow-Bore Chemically Etched Emitters

Electrospray ionization mass spectrometry (ESI-MS) at flow rates below ~10 nL/min has been only sporadically explored because of difficulty in reproducibly fabricating emitters that can operate at lower flow rates. Here we demonstrate narrow orifice chemically etched emitters for stable electrospray at flow rates as low as 400 pL/min. Depending on the analyte concentration, we observe two types of MS signal response as a function of flow rate. At low concentrations, an optimum flow rate is observed slightly above 1 nL/min, whereas the signal decreases monotonically with decreasing flow rates at higher concentrations. For example, consumption of 500 zmol of sample yielded signal-to-noise ratios ~10 for some peptides. In spite of lower MS signal, the ion utilization efficiency increases exponentially with decreasing flow rate in all cases. Significant variations in ionization efficiency were observed within this flow rate range for an equimolar mixture of peptide, indicating that ionization efficiency is an analyte-dependent characteristic for the present experimental conditions. Mass-limited samples benefit strongly from the use of low flow rates and avoiding unnecessary sample dilution. These findings have important implications for the analysis of trace biological samples.

Capillary high-performance liquid chromatography/electrospray ion trap time-of-flight mass spectrometry using a novel nanoflow gradient generator

A type of high-performance liquid chromatography (HPLC) based on a novel nanoflow gradient generator (Asymptotic-Trace-10-Port-Valve (AT10PV) nanoGR generator) was developed and coupled with an electrospray ion trap time-of-flight mass spectrometer (ESI-IT-TOF MS). Stability of the nanoflow GR HPLC system was tested at flow rates of 20 and 50 nL/min by using a nanoflow meter. Average flow rates in a 2-h run were 51.2 nL/min with RSD 0.7% and 21.0 nL/min with RSD 1.8%. Repeatability of analysis of the nanoHPLC/ESI-IT-TOF MS system was also tested by injecting 1.0 microL of trypsin digested bovine serum albumin (BSA) (100 fmol) into a monolithic silica-ODS column (30 microm i.d., 150 mm in length) through a packed silica-ODS trapping column (particle size 5 microm, 150 microm i.d., 10 mm in length). At a flow rate of 50 nL/min, the result demonstrated a reasonably good repeatability of peak retention times (RSD: 0.32-1.1%) and base-ion peak areas (RSD: 4.4-6.6%).     

Characterization of microstructured fibre emitters: in pursuit of improved nano electrospray ionization performance

Full-dimensional computational fluid dynamics (CFD) simulations are presented for nano electrospray ionization (ESI) with various emitter designs. Our CFD electrohydrodynamic simulations are based on the Taylor-Melcher leaky-dielectric model, and the volume of fluid technique for tracking the fast-changing liquid-gas interface. The numerical method is first validated for a conventional 20 μm inner diameter capillary emitter. The impact of ESI voltage, flow rate, emitter tapering, surface hydrophobicity, and fluid conductivity on the nano-ESI behavior are thoroughly investigated and compared with experiments. Multi-electrospray is further simulated with 2-hole and 3-hole emitters with the latter having a linear or triangular hole arrangement. The simulations predict multi-electrospray behavior in good agreement with laboratory observations.     

Design, optimisation, and evaluation of a sheath flow interface for automated capillary electrophoresis-electrospray-mass spectrometry

A sheath-flow capillary electrophoresis-mass spectrometry (CE-MS) system utilizing a fully integrated large-bore stainless-steel emitter electrode tapered at the end for micro-ionspray operation has been developed and evaluated. A separation capillary with an outer diameter of up to 360 microm was inserted into the electrode thus forming a void volume of less than 15 nL between the capillary end and the electrospray ionisation (ESI) tip. The sheath liquid, usually methanol-water (80:20) with 0.1% formic acid for positive ion mode or methanol for negative ion mode, was delivered at 0.5-1.0 microL/min. Unlike previously reported CE-MS interfaces, the CE-MS probe was incorporated directly onto an Applied Biosystems/MDS SCIEX orthogonal-spray Turbo "V" ion source for ease of use and automatic operation. This integration enables fast and facile coupling and replacement of the separation capillary without interrupting the ion source configuration, and the sheath liquid supply. The reusable electrospray electrode was precisely fabricated and aligned with the length of the nebulizing gas tube for improved reproducibility. Automation was achieved through software control of both CE and tandem MS (MS/MS) for unattended batch sample analysis. The system was evaluated for attomole- to low femtomole-level profiling of model peptides and protein mixtures, bisphosphates, as well as antiviral nucleosidic drugs in cellular extracts.     

Gold-coated fused-silica sheathless electrospray emitters based on vapor-deposited titanium adhesion layers

Gold-coated fused-silica electrospray (ES) emitters based on vapor-deposited adhesion layers of titanium have been manufactured to investigate the possibilities of producing durable ES emitters applicable in chip-based analytical devices. The stabilities of the emitters were studied by both electrospray and electrochemical experiments and a marked increase in the emitter lifetime, compared to that for Cr/Au coated emitters, was found for the Ti/Au emitters in the ES durability tests. This indicates that Ti (rather than Cr) adhesion layers should be used in association with large-scale fabrication of ES emitters by vapor-deposition techniques. The lifetime of about 500-700 hours also allowed the Ti/Au-coated emitter to be used as an integrated part of a capillary liquid chromatography column coupled to a mass spectrometer in a series of LC/MS experiments. The Ti/Au coating was further studied by electrochemical techniques and scanning electron microscopy in conjunction with X-ray spectroscopy. It is shown that the eventual failure of the Ti/Au emitters in ES experiments was due to an almost complete detachment of the gold layer. Experimental evidence suggests that the detachment of the gold coating was due to a reduced adhesion to the titanium layer during oxidation in positive electrospray. Most likely, this was caused by the formation of an oxide layer on the titanium film. It is thus shown that unlimited emitter stabilities are not automatically obtained even if the metallic adhesion layer is stabilized by an oxide formation under positive electrospray conditions.     

Capillary pumps for autonomous capillary systems

Autonomous capillary systems (CSs), where liquids are displaced by means of capillarity, are efficient, fast and convenient platforms for many bioanalytical applications. The proper functioning of these microfluidic devices requires displacing accurate volumes of liquids with precise flow rates. In this work, we show how to design capillary pumps for controlling the flow properties of CSs. The capillary pumps comprise microstructures of various shapes with dimensions from 15-250 microm, which are positioned in the capillary pumps to encode a desired capillary pressure. The capillary pumps are designed to have a small flow resistance and are preceded by a constricted microchannel, which acts as a flow resistance. Therefore, both the capillary pump and the flow resistance define the flow rate in the CS, and flow rates from 0.2-3.7 nL s(-1) were achieved. The placement and the shape of the microstructures in the capillary pumps are used to tailor the filling front of liquids in the capillary pumps to obtain a reliable filling behaviour and to minimize the risk of entrapping air. The filling front can, for example, be oriented vertically or tilted to the main axis of the capillary pump. We also show how capillary pumps having different hydrodynamic properties can be connected to program a sequence of slow and fast flow rates in a CS.