Sheathless Interface Research Articles

A sheathless CE/ESI‐MS interface with an ionophore membrane‐packed electro‐conduction channel

A robust and convenient sheathless CE/ESI-MS interface realized with an ionophore membrane-packed electro-conduction channel is described. Sheathless interfaces that may provide higher sensitivity for MS detection than sheath flow-supported interfaces generally show instability and short lifetimes due to their imperfection in making an electrical contact with the emitter tip. In this work, we designed a sheathless interface based on an ionophore membrane-packed electro-conduction channel. At the joining point of the CE capillary and the emitter capillary, the conduction channel was implemented toward the exterior of the interface body, where a platinum wire electrode was placed. The conduction channel transferred the electric field from the external Pt electrode to the joining point, but prevented the effluent of CE from leaking. The interface body was designed to have receptacles for standard capillary tubing with finger-tight fittings, which allowed easy replacement of capillary tubing. Stable electrospray was observed for an extended time period without any signs of bubbling or damage to the emitter tip. No significant increment of dead-volume at the interface was observed for well-aligned capillaries. Sensitive and stable CE-MS detection of the model compound of creatinine and uric acid was demonstrated.

Sheathless capillary electrophoresis‐mass spectrometry using a pulsed electrospray ionization source

A sheathless interface has been developed for coupling CE with electrospray IT mass spectrometer. This interface utilized a pulsed ESI source. The use of a pulsed electrospray source allows the use of a sprayer with larger orifice, and thus alleviates the problem of column clogging during conductive coating and CE analysis. A pulsed ESI source operated at 20 Hz and 20% duty cycle was found to produce the optimal signals. For better signals, the maximum ion injection time in the IT mass spectrometer has to be set to a value close to the actual spraying time (10 ms). Using a sprayer with 50 microm od, more stable and enhanced signals were obtained in comparison with continuous CE-ESI-MS under the same flow rate (150 nL/min). The utility of this design is demonstrated with the analysis of synthetic drugs by CE-MS.

Development of a Nebulizer for a Sheathless Interfacing of NanoHPLC and ICPMS

A novel nebulizer (nDS-200) working at sample uptake rates of less than 500 nL min(-1) was developed for a sheathless interfacing of nanoHPLC (75-microm column i.d.) with ICPMS. It was based on a hollow fused-silica needle of which the tip (i.d. 10 microm, o.d. 20 microm) centered in a 254-microm-i.d. sapphire orifice. The nebulizer, equipped with a 3-cm(3) drain-free vaporization chamber, enabled a stable introduction into an ICP of aqueous mobile phases containing up to 95% acetonitrile at eluent flow rates between 50 and 450 nL min(-1). The low dead volume of the interface resulted in a peak width of 1.3 s (at half-height) and the entirely preserved chromatographic resolution. An example application of the coupling to the analysis of a tryptic digest of a SIP18 protein containing two to nine selenomethionine residues was described. The absolute detection limit was 25 fg (80Se), which allowed the detection of low-abundant selenopeptides at the femtomole level. In contrast to electrospray MS, the ICPMS detection in nanoHPLC is unaffected by the coeluting matrix and concomitant compounds and offers an elegant method for the detection and quantification of minor heteroelement-containing species prior to or in parallel with ESI MS analysis.

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 μm 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 μM for α-ketoglutarate for 40 nL injections. Extracts of Escherichia coli, strain DH5-α, 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.

Comparison of sheathless and sheath‐flow electrospray interfaces for the capillary electrophoresis‐electrospray ionization‐mass spectrometry analysis of peptides

Capillary electrophoresis coupled to mass spectrometry via an electrospray interface provides a powerful system for separation and characterization of a high number of biomolecules. The present paper describes a home-made sheathless interface and compares it with a commercial sheath-flow interface, using a separation method based on a peptide hormone mixture of therapeutic interest. In a previous work, we optimized the parameters involved in a sheath-flow interface and obtained good results in sensitivity and reproducibility. The sheathless interface is performed with a graphite-coated electrospray ionisation (ESI) tip attached to the separation capillary. We demonstrate that electrolyte composition is the main parameter affecting signal sensitivity and separation resolution. The effect of the nature and concentration of the organic solvent added to the separation electrolyte is carefully studied. Furthermore, a general comparison of both interfaces is made in terms of separation, reproducibility, and sensitivity obtained under the optimized conditions described. Advantages and disadvantages of both coupling setups have been evaluated.

Sheathless electrospray ionization interfaces for capillary electrophoresis–mass spectrometric detectionAdvantages and limitations

A review of sheathless interfaces for capillary electrophoresis (CE)-mass spectrometry (MS) is presented. The review discusses the on-line CE-MS system requirements, advantages and weaknesses of current sheathless interface designs for CE-electrospray ionization MS, and comparison between sheath flow and sheathless interfaces. The advantages and limitations of three sheathless designs are discussed and commented upon, these include single-capillary, two-capillary and three-piece designs.

A simple and robust conductive graphite coating for sheathless electrospray emitters used in capillary electrophoresis/mass spectrometry.

A graphite-polyimide mixture was used as a conductive coating for sheathless electrospray emitters. The coating procedure described is simple and inexpensive compared to previously described methods. An investigation of the stability of the conductive coating carried out by electrochemical methods revealed good performances during oxidative stress. In addition, no decrease in emitter performance was seen during continuous electrospray in the positive electrospray mode for two weeks. Fast capillary electrophoresis with attomole sensitivity demonstrated the excellent performance of the described sheathless interface when used in conjunction with an orthogonal time-of-flight mass spectrometer. The overall simplicity, stability and low cost of this type of sheathless emitter make the described approach highly suitable for any on-column coupling of low flow rate separation techniques to a mass spectrometer.

Capillary electrophoretic bioanalysis of therapeutically active peptides with UV and mass spectrometric detection after on-capillary preconcentration.

An earlier developed capillary electrophoresis (CE) system with an on-capillary adsorptive phase is investigated for its suitability to quantitate low concentrations of angiotensin II and gonadorelin in plasma. An off-line solid-phase extraction is used for sample preparation. The on-line preconcentration CE system allows multiple capillary volumes of sample solution to be injected, increasing the concentration sensitivity of CE with 3-4 orders of magnitude. Furthermore, possible influence of matrix salts can be ruled out by employing a rinsing step after sample application. Using short-wavelength UV detection, reproducibility and linearity in the low nanomolar range were satisfactory. The capillary could be efficiently regenerated using a programmed between-run rinsing procedure, allowing 20-30 large injections of sample extracts. Coating of the capillary improved the robustness of the method. Mass spectrometric detection via a previously reported sheathless interface increased the selectivity and sensitivity substantially. Recommendations are provided for the sample preparation process, the most critical part of the system. Further purification of the sample is required to allow the loading of larger sample volumes and to optimize the system's robustness.

Ion trap mass spectrometry as detector for capillary electrochromatography of peptides: Possibilities and limitations

Capillary electrochromatography has been coupled to an ion trap mass spectrometer via a sheath flow electrospray interface. Peptide analysis was performed on 25 cm columns packed with C18-bonded silica particles, and very fast separations were obtained. Full mass spectrometry and tandem mass spectrometry (MS/MS) detection were evaluated in terms of sensitivity and scanning speed. The separation of seven selected peptides and their detection by MS/MS in selected reaction monitoring mode was demonstrated. The performance of this sheath flow interface also was compared with the previously reported sheathless interface. Despite the loss in sensitivity (20–40-fold), the sheath flow interface was shown to be superior in terms of ruggedness, and allowed the use of higher electric fields to achieve faster analysis times. © 2001 John Wiley & Sons, Inc. J Micro Sep 13: 243–249, 2001

Ion trap mass spectrometry as detector for capillary electrochromatography of peptides: Possibilities and limitations

AbstractCapillary electrochromatography has been coupled to an ion trap mass spectrometer via a sheath flow electrospray interface. Peptide analysis was performed on 25 cm columns packed with C18‐bonded silica particles, and very fast separations were obtained. Full mass spectrometry and tandem mass spectrometry (MS/MS) detection were evaluated in terms of sensitivity and scanning speed. The separation of seven selected peptides and their detection by MS/MS in selected reaction monitoring mode was demonstrated. The performance of this sheath flow interface also was compared with the previously reported sheathless interface. Despite the loss in sensitivity (20–40‐fold), the sheath flow interface was shown to be superior in terms of ruggedness, and allowed the use of higher electric fields to achieve faster analysis times. © 2001 John Wiley & Sons, Inc. J Micro Sep 13: 243–249, 2001

Design of a sheathless capillary eletrophoresis-mass spectrometry probe for operation with a Z-spray ionization source.

The construction of a sheathless interface for capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI-MS), for operation with a Z-Spray source on a Micromass Quattro-LC triple quadrupole mass spectrometer is described. Designing the interface involved machining a probe compatible with the setup already in place on the mass spectrometer, i.e., MegaFlow-Z ESI. The probe was made of Lexan with the same dimensions as the ESI probe supplied with the instrument. The electrical connection at the electrospray end of the CE capillary was made possible by gold-coating (sheathless CE-ESI-MS). The probe design as well as the electrical and power supply requirements are described in detail. Experiments were performed using this interface, and CE separations of mixtures containing pmole and sub-pmole amounts of peptides were monitored by on-line MS. For a standard peptide mixture (10(-4) M), separation efficiency was typically characterized by N > 10(4) theoretical plates with S/N > 400. Using the same experimental setup, it was also possible to conduct on-line CE-ESI-tandem MS (MS/MS) experiments on the same peptide mixture, and to determine the sequence of the peptides. MS/MS scan functions for different precursor ions were used either alternately or sequentially and the results from both methods were compared. The possibility of peptide mass mapping was explored, and CE-ESI-MS results were obtained for the digestion products of equine myoglobin. Separation efficiencies and S/N values were similar to those obtained for standard peptides. A complete map of the digestion products was obtained.

Optimization of capillary electrophoresis conditions for coupling to a mass spectrometer via a sheathless interface.

When optimizing a capillary electrophoresis/electrospray ionization mass spectrometry (CE/ESI-MS) system, consideration has to be given not only to the separation but also to the electrospray stability. Methods developed for CE/UV analysis of drugs and peptides were considered and modified to be suitable for a CE/MS system with a robust sheathless interface. Different concentrations of the organic modifiers acetonitrile, methanol and 2-propanol were used in the separation buffer. The type and concentrations of these modifiers were also compared with reference to electrospray stability, sensitivity and time of analysis. In addition, different ionic strengths in the buffers were evaluated with reference to electrospray stability. The repeatability was used for the estimation of electrospray stability. The degree to which these parameters influenced the separation and the ESI stability was studied using a nine-peptide standard mixture and the antibiotic drugs bacampicillin and ampicillin as test substances. The analysis time and resolution were used as measures of the efficiency of the separation. A time-of-flight MS analyzer was used since it has the potential advantages of becoming a better fit for integration of CE with MS owing to the speed and sensitivity of this mass analyzer. The detection limit, i.e. 1 microM, for bacampicillin was comparable to what could be achieved with CE/MS on a quadrupole instrument using selected ion monitoring and sheath flow ESI.

Electrospray ionization mass spectrometry and on-line capillary zone electrophoresis - mass spectrometry for the characterization of citrate synthase

The enzyme citrate synthase from E. coli is a protein with a molecular weight (Mr) of 47 885 Da (wild type). This enzyme has been studied extensively, and its amino acid sequence has been characterized. This model protein has been used in this work for development and validation of methods involving capillary electrophoresis (CE) and electrospray ionization mass spectrometry (ESI-MS). The Mr determinations were conducted using sample infusion ESI-MS, and the tryptic digestion products of wild-type citrate synthase were characterized by on-line CE-ESI-MS coupled with a sheathless interface. On-line experiments were conducted on two different mass spectrometers, a Quattro-LC triple quadrupole instrument equipped with a Z-SprayTM source (Micromass), and a reflecting time-of-flight (TOF) mass spectrometer built in-house in the Time-of-Flight Laboratory, Department of Physics, University of Manitoba. This is the first article to be written on the interfacing of a Z-SprayTM source with CE. Unmodified fused silica capillaries gold-coated sheathless interfaces were used. The on-line CE separations yielded theoretical plate numbers greater than 104 on average. Selected ion electrophorograms (SIE) of the tryptic peptides recorded on the Quattro-LC displayed S/N ratios ranging from ca. 14 to 120 on raw data. These SIE enabled identification of each peptide. The use of reflecting time-of-flight mass spectrometry (TOFMS) afforded mass resolution values of ca. 6000 (m/deltamFWHM), which enabled isotopic resolution of the peptide components. CE-ESI-MS and CE-ESI-TOFMS experiments enabled the generation of a complete tryptic map of citrate synthase.Key words: capillary electrophoresis, electrospray ionization, mass spectrometry, citrate synthase, tryptic digestion, triple quadrupole analyzer, time-of-flight analyzer.

New sheathless interface for coupling capillary electrophoresis to electrospray mass spectrometry evaluated by the analysis of fatty acids and prostaglandins

A new interface for capillary electrophoresis electrospray ionization (CE–ESI) is presented. High voltage is applied at the outlet of the separation capillary by a stainless steel tube, a so called liner, through which the capillary is led. A compensating current between the liquid and the liner is maintained by a natural liquid film, which is built up at the outer surface of the capillary end. Operable potential ranges for differently treated capillary ends have been examined. The liner has been evaluated for the analysis of fatty acids and prostaglandins, all run with the ESI in the negative ionization mode. This simple stainless steel liner should fill the gap, which has prevented CE–MS from being the successful tool, which it has the potential for, namely fast and unattended measurements of analytes in the nM range in complex mixtures.

Analysis of metallothioneins by means of capillary electrophoresis coupled to electrospray mass spectrometry with sheathless interfacing.

Capillary electrophoresis (CE) coupled to electrospray mass spectrometry via sheathless interfacing has been applied to the analysis of mammalian metallothionein (MT) extracts. In a rabbit-liver extract, four (MT-2C, MT-2A, MT-2D and MT-2E) out of six known MT sub-isoforms were unambiguously identified under three CE-resolved peaks. A fourth peak was found to contain MT-1A and/or MT-2B, whose molecular masses differ by only 1 Da. Traces of non-N-acetylated MT-2D and MT-2E were observed in a fifth, minor peak. In a rat-liver extract, both MT-1 and MT-2 were resolved and identified. Non-N-acetylated MT-2 was also identified in a resolved, minor peak. Minimum detectable amounts of MTs have been estimated to be approximately 0.6 fmol per sub-isoform.

Hydroquinone as a buffer additive for suppression of bubbles formed by electrochemical oxidation of the CE buffer at the outlet electrode in capillary electrophoresis/electrospray ionization-mass spectrometry.

Hydroquinone was found to suppress bubble formation at the outlet electrode of a sheathless capillary electrophoresis/electrospray ionization-mass spectrometer by replacing the oxidation of water (2H2O(1)<-->O2(g) + 4H+ + 4e) with that of more easily oxidized hydroquinone (hydroquinone<-->p-benzoquinone + 2H+ + 2e). Formation of p-benzoquinone replaces the formation of oxygen gas, effectively suppressing gas bubble formation. Several electrode materials, including platinum, gold-coated stainless steel, and stainless steel wires, were tested. However, hydroquinone suppressed bubbles only at the platinum electrode. Combination of the in-capillary electrode sheathless interface using platinum wire, hydroquinone as a buffer additive, and pressure programming at the inlet of the capillary electrophoresis provided a rugged high efficiency interface for analysis of protein digests using CE/ESI-MS.

Electrospray ionization mass spectrometry and on-line capillary zone electrophoresis - mass spectrometry for the characterization of citrate synthase

The enzyme citrate synthase from E. coli is a protein with a molecular weight (Mr) of 47 885 Da (wild type). This enzyme has been studied extensively, and its amino acid sequence has been characterized. This model protein has been used in this work for development and validation of methods involving capillary electrophoresis (CE) and electrospray ionization mass spectrometry (ESI-MS). The Mr determinations were conducted using sample infusion ESI-MS, and the tryptic digestion products of wild-type citrate synthase were characterized by on-line CE-ESI-MS coupled with a sheathless interface. On-line experiments were conducted on two different mass spectrometers, a Quattro-LC triple quadrupole instrument equipped with a Z-SprayTM source (Micromass), and a reflecting time-of-flight (TOF) mass spectrometer built in-house in the Time-of-Flight Laboratory, Department of Physics, University of Manitoba. This is the first article to be written on the interfacing of a Z-SprayTM source with CE. Unmodified fused silica capillaries gold-coated sheathless interfaces were used. The on-line CE separations yielded theoretical plate numbers greater than 104 on average. Selected ion electrophorograms (SIE) of the tryptic peptides recorded on the Quattro-LC displayed S/N ratios ranging from ca. 14 to 120 on raw data. These SIE enabled identification of each peptide. The use of reflecting time-of-flight mass spectrometry (TOFMS) afforded mass resolution values of ca. 6000 (m/deltamFWHM), which enabled isotopic resolution of the peptide components. CE-ESI-MS and CE-ESI-TOFMS experiments enabled the generation of a complete tryptic map of citrate synthase.Key words: capillary electrophoresis, electrospray ionization, mass spectrometry, citrate synthase, tryptic digestion, triple quadrupole analyzer, time-of-flight analyzer.

Analysis of peptides, proteins, protein digests, and whole human blood by capillary electrophoresis/electrospray ionization-mass spectrometry using an in-capillary electrode sheathless interface

An in-capillary electrode sheathless interface was applied to the capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) analysis of mixtures of small peptides, proteins, and tryptic digests of proteins. The effects of different experimental parameters on the performance of this CE/ESI-MS interface were studied. The distance of the in-capillary electrode from the CE outlet and the length of the electrode inside the capillary had no significant effects on the CE separation and ESI behavior under the experimental conditions used. However, significant enhancement of the sensitivity resulted from the use of narrower CE capillaries. Using a quadrupole mass spectrometer, an aminopropylsilane-coated capillary, and a wide scan mass-to-charge ratio range of 500–1400, detection limits of approximately 4, 1, and 0.6 fmol for cytochrome c and myoglobin were achieved for 75-, 50-, and 30-μm inner diameter capillaries, respectively. Approximately one order of magnitude lower detection limits were achieved under the multiple-ion monitoring mode. The application of the in-capillary electrode sheathless interface to real-world samples was demonstrated by CE/ESI-MS analysis of a human blood sample.

Sensitive high-resolution analysis of biological molecules by capillary zone electrophoresis coupled with reflecting time-of-flight mass spectrometry

Off-line and on-line capillary zone electrophoresis–electrospray ionization time-of-flight mass spectrometry (CZE–ESI-TOF-MS) experiments were conducted using uncoated fused-silica capillaries coupled to a reflecting TOF mass spectrometer via a gold-coated sheathless interface. Off-line and on-line experiments were performed on standard mixtures of proteins and peptides. Samples collected off-line electrokinetically in plastic vials were analyzed by standard ESI–TOF-MS at the pmol level. Sheathless CZE–ESI-TOF-MS was first simulated in an off-line experiment, using a test bench, in order to select a suitable running electrolyte, to find the optimal electrospray potential, and also to test the gold-coated capillary tips. This enabled an ease of transition to on-line measurements. On-line CZE–ESI-TOF-MS measurements of the total ion electropherogram (TIE) and of selected ion electropherograms (SIE) on peptide mixtures demonstrated fmol-level sensitivity, with S/ N values of 250–400 on raw data (TIE mode) and of 30–760 (SIE mode). The use of reflecting TOF-MS afforded mass resolution values R>6000 ( m/Δ m FWHM) and enabled isotopic resolution of peptide components as well as mass accuracy in the 10 ppm range. These results were comparable with values observed with the usual ESI source on the same mass spectrometer, and thus demonstrated no loss in spectral quality from using the sheathless CE interface. On-line CE separation efficiency was equivalent to that obtained off-line for the separation of a peptide mixture, with N=35 000–87 000 theoretical plates. Separations of standard proteins yielded equivalent mass spectral resolution and accuracy with separation efficiencies of N=2800–5500 and S/ N values of 110–225 on raw data. The gold-coated sheathless CE–ESI interface was found to be relatively easy to prepare with the use of gold vapour deposition. The interface produced a stable electrospray for extended periods of time and was found to be robust and reliable.

A novel sheathless interface for capillary electrophoresis/electrospray ionization mass spectrometry using an in-capillary electrode

A simple and rugged sheathless interface for capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was designed using common laboratory tools and chemicals. The interface uses a small platinum (Pt) wire which is inserted into the CE capillary through a small hole near the terminus. The position of the wire inside the CE capillary and within the buffer solution is analogous to standard CE separation operations where the terminus of the CE capillary is placed inside a buffer reservoir along with a grounded platinum electrode. By combining the use of the in-capillary electrode interface with sharpening of the fused silica tip of the CE capillary outlet, a stable electrospray current was maintained for an extended period of time. The design was successfully applied to CE/ESI-MS separations and analysis of mixtures of peptides and proteins. A detection limit of approximately 4 femtomole (S/N=3) was achieved for detection of myoglobin utilizing a 75 μm-i.d. aminopropylsilane treated CE column and using a wide scan range of 550-1350 Da. The advantages of this new design include: (1) a stable CE and ESI current, (2) durability, (3) a reduced risk of sparking between the capillary tip and the inlet of the mass spectrometer, (4) lack of any dead volume, and (5) facile fabrication with common tools and chemicals.