Development of a fritless packed column for capillary electrochromatography-mass spectrometry

A novel procedure was developed for the fabrication of a fritless packed column for the coupling of capillary electrochromatography (CEC) to mass spectrometry (MS). The process involved the formation of internal tapers on two separate columns. Once the internal tapers are formed and the columns are packed, the untapered ends of each column were joined together by a commercially available connector. Several advantages of the fritless columns are described. First, the design used here eventually eliminates the need for any frits thus reducing the possibility of bubble formation seen with fritted packed columns. In addition, this is the first report in which the internal tapers are formed at both the inlet and outlet column ends making the fritless CEC-MS column more robust compared to only one report with externally tapered counterparts. Second, a comparison of internally tapered single frit packed CEC-MS (previously developed in our laboratory) column versus fritless CEC-MS column reported here shows that the latter provides better efficiency, suggesting no dead volume with equally good sensitivity and chiral resolution of (±)-aminoglutethimide. The fritless column procedure is universal and was used to prepare a series of columns with a variety of commercially available packing material (mixed mode strong cation exchange, SCX; mixed mode strong anion exchange, SAX; C-18) for the separation and MS detection of short chain non-chromophoric polar amines, long chain nonchromophic anionic surfactant as well as oligomers of non-chromophoric non-ionic surfactants, respectively. The fritless columns showed good intra-day repeatability and inter-day reproducibility of retention times, chiral and achiral resolutions and peak areas. Very satisfactory column-to-column and operator-to-operator reproducibility was demonstrated.     

Fast CEC-MS using poly(dimethylsiloxane) microinjector, short packed column, and low-sheath-flow interface

A fast CEC-MS approach based on a microinjector and a short CEC column was developed. Poly(dimethylsiloxane) was used as the substrate for microinjector fabrication. A short capillary column (～5 cm) packed with 5 μm octadecyl silica particles was inserted into the microinjector. The microinjector CEC device was interfaced to ESI-MS using a low-flow sheath liquid interface. The device delivers the advantages of sample introduction, pre-concentration, elution, and fast analysis as in chip-CEC yet avoids the difficulty of packing stationary material into the chip. The online pre-concentration and CEC-MS analysis capabilities of this device were demonstrated by analysis of a six-triazine mixture. A signal enhancement of 20-99-fold was achieved with a sample loading time of 180 s.     

Analysis of triazines by capillary electrochromatography/electrospray ionization-mass spectrometry using a low-flow sheath liquid interface

CEC-MS has been used for the analysis of eight-triazine herbicides. It showed significantly better S/N ratio than reversed EOF CE-MS and MEKC-MS, due to the lack of a surfactant in the separation buffer. By optimizing the pH, the organic content of the running buffer, and the separation potential, optimal separation was achieved within 18 min using a running buffer of pH 7.0, containing 70% v/v ACN, and an applied voltage of 17 kV. Gradient CEC showed superior separation when compared with isocratic elution. The combination of a tapered CEC column and a low-flow interface confers several advantages including better sensitivity, low dead volume, and independent control of the conditions used for CEC separation and ESI analysis.     

Simultaneous enantioseparation and sensitive detection of eight beta-blockers using capillary electrochromatography-electrospray ionization-mass spectrometry

The feasibility of using vancomycin chiral stationary phase (CSP) and polar organic eluent is investigated for simultaneous enantioseparation of eight beta-blockers using CEC coupled to ESI mass spectrometric detection (ESI-MS). The internally tapered capillaries were utilized to pack CEC-MS columns. As compared to externally tapered columns, the use of internally tapered columns demonstrated enhanced stability, durability, and reproducibility. A mixture containing methanol/ACN/acetic acid/triethylamine at 70:30:1.6:0.2 v/v/v/v was considered as optimum mobile phase since it provided a good compromise between resolution and analysis time. As expected, sheath liquid and ESI-MS parameters mainly influenced the detection sensitivity. Interestingly, structural information of beta-blockers was available by varying the MS fragmentor voltage using in-house CID in the scan mode. In order to maximize the chiral/achiral resolution, various column-coupling approaches using teicoplanin as complementary CSP to vancomycin were tested. Several changes in the elution order of beta-blockers were observed using multimodal CSPs with some improvement in chiral or achiral resolution. The quantitative aspects of the CEC-MS method were demonstrated using R- and S-talinolol as internal standards. The calibration curves of beta-blockers showed good linearity in the range of 3-600 microM. The enantiomer of beta-blockers at a concentration of 30 nM was detectable. Furthermore, both 0.1 and 1% of the S-enantiomer could be precisely quantified in the presence of 99.9 and 99% of the R-isomer of beta-blocker.     

Development of a low-flow multiplexed interface for capillary electrophoresis/electrospray ion trap mass spectrometry using sequential spray

A multiplexed CE-MS interface using four low-flow sheath liquid ESI sprayers has been developed. Because of the limited space between the low-flow sprayers and the entrance aperture of the ESI source, multichannel analysis is difficult using conventional rotating plate approaches. Instead, a multiplexed low-flow system was achieved by applying an ESI potential sequentially to the four low-flow sprayers, resulting in only one sprayer being sprayed at any given time. The synchronization of the scan event and the voltage relays was accomplished by using the data acquisition signal from the IT mass spectrometer. This synchronization resulted in the ESI voltage being sequentially applied to each of the four sprayers according to the corresponding scan event. With this design, a four-fold increase in analytical throughput was achieved. Because of the use of low-flow interfaces, this multiplexed system has superior sensitivity than a rotating plate design using conventional sheath liquid interfaces. The multiplexed design presented has the potential to be applied to other low-flow multiplexed systems, such as multiplexed capillary LC and multiplexed CEC.     

A sheathless poly(methyl methacrylate) chip-CE/MS interface fabricated using a wire-assisted epoxy-fixing method

Using a wire-assisted epoxy-fixing method, a sheathless CE/MS interface on a poly-(methyl methacrylate) (PMMA) CE chip has been developed. The sheathless chip-CE/MS interface utilized a tapered fused-silica tip and the electrical connection was achieved through a layered coating of conductive rubber. The wire-assisted method provided facile alignment of channels between the PMMA CE chip and an external capillary sprayer without the need for micromachining. Because the wire was in the channel during fixing, the risk of channel blockage by the epoxy was avoided. This chip CE device has minimal dead volume because the interstitial spaces were filled by a fast-fixing epoxy resin. The performance of the chip-CE-ESI-MS device was demonstrated with the analysis of peptide mixtures.     

Design and performance of a low-flow capillary electrophoresis-electrospray-mass spectrometry interface using an emitter with dual beveled edge

A low-flow electrophoresis-mass spectrometry (CE-MS) interface has been developed for interfacing capillary zone electrophoresis (CZE) with electrospray- ionization-mass spectrometry (ESI-MS). The interface consists of two parallel capillary columns (a separation column and a makeup column), and an emitter with a dual beveled edge. While maintaining a relatively low optimum flow rate, the dual-beveled-edge ESI emitter allows the use of a tip with larger orifice. Therefore, this interface is less prone to column blocking in comparison with a flat tip. Primarily attributed to low sample dilution and smaller initial droplet, the interface showed better sensitivity than a conventional sheath liquid interface. Furthermore, the interface was found to be more resistant to the presence of nonvolatile salts. By using 40 mM borate and 20 mM alpha-cyclodextrin (alpha-CD) as the running buffer, four major forms of gangliosides were detected by CE-MS.     

Fritless capillary electrochromatography

The preparation of packed capillaries with stable frits of good quality can be a hurdle to obtain efficient separations in capillary electrochromatography (CEC). Especially with particles smaller than 3 microm, frit preparation is cumbersome. Highly efficient separations using packed capillaries without frits are presented. Under appropriate CEC conditions the particles were retained by electrophoretic attraction towards the anode by a tapered capillary inlet, without the need of a frit at the outlet end. Such fritless capillaries, packed with 1.5 microm nonporous reversed-phase particles, allowed separations with efficiencies of more than 500,000 plates/m. Once the capillaries were conditioned properly, more than 100 separations could be performed with good repeatability. With respect to separation efficiency, fritless capillaries packed with 3 microm particles were comparable with standard CEC capillaries with frits. Examples of separations of steroids, a pesticide and its by-products, and cardiac glycosides under various CEC conditions are shown.     

Capillary electrochromatography/nanoelectrospray mass spectrometry for attomole characterization of peptides

The successful coupling of capillary electrochromatography (CEC) to an ion trap mass spectrometer via a nanoelectrospray interface (nESI) is described. Using a conductively coated tip butted to the end of a CEC column, it was possible to obtain a stable spray without any sheath liquid being employed. Selected small peptides were separated with CEC columns (100 microm i.d./25 cm long) packed with 3 microm Hypersil C8 or C18 bonded silica particles with an eluent composed of ammonium acetate/acetonitrile. Peptide mixtures of desmopressin, peptide A, oxytocin, carbetocin and [Met(5)]-enkephalin were detected in the mid-attomole range, which is the lowest amount analyzed using CEC combined with MS detection. It was also observed that sensitivity can be compromised at higher separation voltages. We demonstrate that CEC/nESI-MS, at the current stage of development, represents one of the most sensitive systems for peptide analysis.     

Capillary electrochromatography-mass spectrometry of cationic surfactants

The CEC-MS of alkyltrimethylammonium (ATMA+) ions with chain lengths ranging from C1-C18 is optimized using an internally tapered column packed with mixed mode reversed phase/strong cation exchange stationary phase. A systematic study of the CEC separation parameters is conducted followed by evaluation of the ESI-MS sheath liquid and spray chamber settings. First, the optimization of CEC separation parameters are performed including the ACN concentration, triethylamine (TEA) content, buffer pH and ammonium acetate concentration. Using 90% v/v ACN with 0.04% v/v TEA as mobile phase, the separation of longer chain C6-C18-TMA+ surfactants could be achieved in 15 min. Lowering the ACN concentration to 70% v/v provided resolution of shorter chain C1, C2-TMA+ from C6-TMA+ although the total analysis time increased to 40 min. Furthermore, variation of both the ACN and TEA content as well as ionic strength has found to significantly influence the retention of longer chain surfactants as compared to shorter chains. The optimum CEC conditions are 70% v/v ACN, 0.04% v/v TEA, pH 3.0 and 15 mM ammonium acetate. Next, the optimization of the ESI-MS sheath liquid composition is conducted comparing methanol to isopropanol followed by the use of experimental design for analysis of spray chamber parameters. Overall, the developed CEC-ESI-MS method allows quantitative and sensitive monitoring of ATMA+ from < or =10 microg/mL down to 10 ng/mL. Utilizing the optimized CEC-ESI-MS protocol, the challenging analysis of commercial sample Arquad S-50 ATMA+ containing cis-trans unsaturated and saturated soyabean fatty acid derivatives is demonstrated.     

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.     

Analysis of phospholipids using an open‐tubular capillary column with a monolithic layer of molecularly imprinted polymer in capillary electrochromatography‐electrospray ionization‐tandem mass spectrometry

In this study, an open‐tubular capillary electrochromatography (OT‐CEC) column with a monolithic layer of molecularly imprinted polymer (MIP) based on methacrylic acid, ethylene glycol dimethacrylate, and 4‐styrenesulfonic acid was utilized for the simultaneous separation and characterization of phospholipid (PL) molecular structures by interfacing with electrospray ionization‐tandem mass spectrometry (ESI‐MS‐MS). Introducing an MIP‐based monolith along with charged species at the OT column made it possible to separate PL molecules based on differences in head groups and acyl chain lengths in CEC. For the interface of OT‐CEC with ESI‐MS‐MS, a simple nanospray interface utilizing a sheath flow was developed and the resulting OT‐CEC‐ESI‐MS‐MS was able to separate PL standards (phosphatidylserines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acid, and lysophosphatidylglycerols). The developed method was applied to human urinary lipid extracts, and resulted in the separation and structural identification of 18 molecules by data‐dependent collision‐induced dissociation.     

Novel approach for fritless capillary electrochromatography

At present, the main limitation for the further adoption of capillary electrochromatography (CEC) in the (routine) laboratory is caused by the lack of reproducible and stable columns. The main source of column instability is concentrated in the frits needed to retain the packed bed inside the CEC capillary. The sintering process used to prepare the frits can be rather problematic and irreproducible, particularly for small stationary phase particles and wide column diameters. Since the (surface) composition of the frits is different from the bulk stationary phase packing, different electroosmotic flow (EOF) velocities are generated. This effect is assumed to be primarily responsible for rapid column destruction. In this contribution, a novel approach for the preparation of fritless CEC capillaries is presented and evaluated. Using 5 microm Hypersil ODS particles, separation efficiencies in the range of 130,000-200,000 plates/m were obtained. In a 100 microm inner diameter packed column, electrical currents up to 50 microA could be tolerated without negative effects such as bubble formation. The prepared CEC columns were found to be stable and could easily be operated continuously for several days without column damage. An additional advantage of the proposed tapering approach is that application of pressure on the in- and outlet vial during separation was not required to prevent bubble formation.     

Performance of a sheathless porous tip sprayer for capillary electrophoresis-electrospray ionization-mass spectrometry of intact proteins

The performance of a prototype porous tip sprayer for sheathless capillary electrophoresis-mass spectrometry (CE-MS) of intact proteins was studied. Capillaries with a porous tip were inserted in a stainless steel needle filled with static conductive liquid and installed in a conventional electrospray ionization (ESI) source. Using a BGE of 100 mM acetic acid (pH 3.1) and a positively charged capillary coating, a highly reproducible and efficient separation of four model proteins (insulin, carbonic anhydrase II, ribonuclease A and lysozyme) was obtained. The protein mass spectra were of good quality allowing reliable mass determination of the proteins and some of their impurities. Sheath-liquid CE-MS using the same porous tip capillary and an isopropanol-water-acetic acid sheath liquid showed slightly lower to similar analyte responses. However, as noise levels increased with sheath-liquid CE-MS, detection limits were improved by a factor 6.5-20 with sheathless CE-MS. The analyte response in sheathless CE-MS could be enhanced using a nanoESI source and adding 5% isopropanol to the BGE, leading to improved detection limits by 50-fold to 140-fold as compared to sheath liquid interfacing using the same capillary - equivalent to sub-nM detection limits for three out of four proteins. Clearly, the sheathless porous tip sprayer provides high sensitivity CE-MS of intact proteins.     

A beveled tip sheath liquid interface for capillary electrophoresis-electrospray ionization-mass spectrometry

A simple and durable sheath liquid interface for capillary zone electrophoresis-electrospray ionization-mass spectrometry (CZE-ESI-MS) has been developed. This interface utilized a beveled tip emitter and was found to be more sensitive than the conventional sheath liquid interface. The use of a beveled tip reduces the optimal flow rate and therefore decreases sample dilution. The interface utilized a 380 microm inner diameter and 400 microm outer diameter beveled tapered tip. Because of the large inner diameter and outer diameter of the tip, the interface is robust and can be easily implemented. The performance of this interface for CZE-ESI-MS and micelle electrokinetic capillary electrophoresis-electrospray-mass spectrometry, as demonstrated by the analysis of synthetic drugs and triazine mixtures, was significantly better than results obtained using a conventional sheath liquid interface.     

Improved column preparation and performance in capillary electrochromatography

Problems encountered in capillary electrochromatography, i.e. non-reproducible column manufacture, bubble formation during usage, short column lifetimes and limited choice of packing particles are addressed by the development of fritless or single-frit, internally tapered, segmented and dead-volume free coupled capillary columns. The Van Deemter plots measured demonstrate the performance of these high-quality capillaries which are suitable for capillary electrochromatography as well as for capillary high-performance liquid chromatographic applications.     

Analyses of sulfonamide antibiotics in meat samples by on-line concentration capillary electrochromatography-mass spectrometry

In this work, a series of poly(divinylbenzene-alkyl methacrylate) monolithic stationary phases, which were prepared by single step in situ polymerization of divinylbenzene and various alkyl methacrylates (butyl-, octyl-, lauryl- or stearyl methacrylate), were developed as separation columns of nine common sulfonamide antibiotics for capillary electrochromatography (CEC) coupled to mass spectrometry (MS). Results indicated that the sulfonamide's retention became weak with increased carbon chain length of alkyl methacrylate monomer (for example, t(R)=68 min and 21 min for butyl- and lauryl methacrylate, respectively). Among them, the poly(divinylbenzene-octyl methacrylate) (poly(DVB-OMA)) monolith was regarded as the optimal separation column as this provided better resolution within the shortest retention time. Moreover, the cross-sectional roughness of the monolithic column-end, that was used to couple to the ESI interface, strongly influenced the electrospray stability in the CEC-MS. Before the column was connected to the ESI-MS, a simple polishing was done to reduce the roughness of the column end that resulted to a great improvement in the signal stability. The relative standard deviations (RSDs) of the peak areas for the unpolished and polished ends of the poly(DVB-OMA) columns (n=5) were in the range of 46.1-60.2% and 8.9-16.4%, respectively. Furthermore, optimization of the mobile phase composition and the gradient elution strategy successfully determined the sulfonamide antibiotics in meat samples with as low as 10 μg/L level.     

Advances in column technology and instrumentation in capillary electrochromatography

Capillary electrochromatography (CEC) is an emerging technique gaining increased interest. Improvement of instrumentation and column technology will be of prime importance for the further development of this technique and its use in validated methods. In this paper, developments in column technology and instrumentation for CEC are reviewed with emphasis on developments within the last 3 years. Attention is directed to the employment of stationary phases specifically designed for CEC, the use of soft and rigid gels in place of packings, fritless packed capillaries, column dimensions, the optimization of injection and detection parameters, and gradient elution CEC.     

Analysis of non-covalent protein complexes by capillary electrophoresis--time-of-flight mass spectrometry

A capillary electrophoresis-electrospray ionisation time-of-flight mass spectrometry (CE-ESI-TOF-MS) method for characterisation of non-covalent protein complexes is described using a coaxial liquid sheath-flow sprayer. The CE capillary was connected to the mass spectrometer using a commercial CE-MS sprayer mounted on a ceramic holder of the ESI interface of the mass spectrometer. Using myoglobin (Mb) as an example of non-covalent protein complex, the effect on complex stability caused by organic modifiers added to the sheath liquid was analysed. Depending on the amount of methanol, either intact Mb or the apoprotein and the prosthetic heme group were detected.     

In-line coupling of low-pressure short capillary electrochromatography columns to electrospray ionisation mass spectrometry

A new in-house designed and constructed injection valve for capillary electrochromatography (CEC) based on a rotating injection part with compartments for the eluent as well as for the sample has been coupled to a mass spectrometer via a sheath flow electrospray ionisation (ESI) interface, using short capillary columns of 15 cm length. The CEC columns were packed with 3 microm C(18) bonded silica particles, and a mixture of peptides was analysed using an ammonium acetate/acetonitrile eluent. A significant increase in the signal-to-noise ratio was obtained when the peptides were dissolved in water with the same content of organic modifier as in the eluent with an addition of 0.5% (v/v) acetic acid. When the CEC analysis was performed without any additional pressure, the separation current sometimes dropped tremendously due to bubble formation, caused by different permeability in the first and packed part of the column causing an extremely low electroosmotic flow. The separation current was restored to its original value by applying only 7 bar at the inlet of the CEC column, and the separation performance for the test peptides was recovered. A comparison of the CEC performance of peptides in pure CEC mode and in low-pressure CEC mode is reported.