Two-dimensional electrophoretic separation of proteins using poly(methyl methacrylate) microchips

The work presented herein describes highly efficient, two-dimensional (2D) electrophoretic separations of proteins in a PMMA-based microchip. Sodium dodecyl sulfate microcapillary gel electrophoresis (SDS micro-CGE) and micellar electrokinetic chromatography (MEKC) were used as the separation modes for the first and second dimension of the electrophoresis, respectively. The microchip was prepared by hot embossing into PMMA from a brass mold master fabricated via high-precision micromilling. The microchip incorporated a 30-mm SDS micro-CGE and a 10-mm MEKC dimension length. Electrokinetic injection and separation were used with field strengths of up to 400 V/cm. Alexa Fluor 633 conjugated proteins, ranging in size from 38 to 110 kDa, were detected using laser-induced fluorescence with excitation/emission at 633/652 nm. Average plate numbers (N) of 4.8 x 10(4) and 1.2 x 10(4) were obtained in the SDS micro-CGE and MEKC separation dimensions, respectively, for the investigated proteins corresponding to plate heights (H) of 0.62 and 0.87 microm. Effluents from the first dimension (SDS micro-CGE) were repetitively transferred into the second dimension every 0.5 s of run time in the first dimension with the electrophoresis run time in the MEKC dimension being 10 s. The 2D separation was performed on the investigated proteins in approximately 12 min and provided a peak capacity of approximately 1000.     

Systematic Approach to Links between Separations in MEKC and Reversed-Phase HPLC

Retention factors and partition coefficients in micellar electrokinetic chromatography (MEKC) and reversed-phase high-performance liquid chromatography (RP-HPLC) are compared for a series of alkylbenzenes and substituted phenols. In both techniques, separations are based on partitioning between an aqueous phase and an alkyl phase. In MEKC, this was an SDS (C12) micellar pseudostationary phase, and in RP-HPLC an ODS 2 (C18) stationary phase. A nonporous silica (Micra 1.5-μm NPS), which has a low carbon loading, was used rather than a standard porous silica to avoid excessive retention in HPLC and to allow identical mobile phase conditions to be used in both separation modes. The average ratio of analyte retention factors, k(MEKC):k(HPLC), was found to be equal to the ratio β(MEKC):β(HPLC), where β is the phase ratio. This implies that partition coefficients, P, are similar in both MEKC and HPLC, since P = k/β, and that the dominant contribution to stability within each alkyl phase arises from hydrophobic interactions which are common to both separation media. Since partition coefficients are similar in MEKC and HPLC under aqueous buffer conditions, information on retention in one technique may be transferred to the other, provided that the phase ratios are known. In MEKC and HPLC, linear correlations of log octanol-water partition coefficients, K(ow), vs log k for the test compounds were transformed, knowing the phase ratio, to give log P values as a function of log K(ow). This allows quantitative links between MEKC and HPLC to be extended to include octanol-water partitioning. The addition of acetonitrile as an organic modifier over the concentration range 0-20% (v/v) was found to have a greater effect on k in HPLC than in MEKC. This could be a result of a decrease in the MEKC phase ratio due to an increase in the critical micelle concentration.     

Phospholipid bilayer coatings for the separation of proteins in capillary electrophoresis

The double-chained, zwitterionic phospholipid 1,2-dilauroyl-sn-phosphatidylcholine (DLPC, C12) was investigated for its use as a wall coating for the prevention of protein adsorption in capillary electrophoresis. DLPC forms a semipermanent coating at the capillary wall, which allows excess phospholipid to be removed from the capillary prior to electrophoretic separation. A DLPC-coated capillary allowed for the separation of both cationic and anionic proteins with efficiencies as high as 1.4 million plates/m. Migration time reproducibility was less than 1.3% RSD from run to run and less than 4.0% RSD from day to day. Protein recovery was as high as 93%. Cationic and anionic proteins could be separated over a pH range of 3-10, all yielding good efficiencies (N up to 1 million plates/m). The chain length of the phospholipid affected the performance of the wall coating. The C10 analogue of DLPC (DDPC) did not form a coating on the capillary wall while the C14 analogue of DLPC (DMPC) formed a stable coating that prevented protein adsorption to the same extent as its C12 counterpart.     

High-efficiency, two-dimensional separations of protein digests on microfluidic devices

High-efficiency, two-dimensional separations of tryptic digests were achieved using glass microfluidic devices. Following micellar electrokinetic chromatography (MEKC) separations in a 19.6-cm-long serpentine channel, the peptides were rapidly sampled into a 1.3-cm-long second-dimension channel, where they were separated by capillary electrophoresis (CE). The turns in the serpentine channel were asymmetrically tapered to minimize geometrical contributions to band broadening and to provide ample channel length for high-efficiency chromatographic separations. Analysis of rhodamine B injections routinely produced plate numbers of 230000 and 40000 in the first (MEKC) and second (CE) dimensions, respectively, corresponding to plate heights of 0.9 and 0.3 microm. The electric field strengths were 200 V/cm for MEKC and 2400 V/cm for CE. In analysis times less than 15 min, two-dimensional separation of bovine serum albumin tryptic digest produced a peak capacity of 4200 (110 in the first dimension and 38 in the second dimension). The system was used to identify a peptide from a tryptic digest of ovalbumin using standard addition and to distinguish between tryptic digests of human and bovine hemoglobin.     

Double-chained surfactants for semipermanent wall coatings in capillary electrophoresis

The double-chained cationic surfactant didodecyldimethylammonium bromide (DDAB) was found to form more stable coatings onto the walls of CE capillaries than similar single-chained surfactants such as cetyltrimethylammonium bromide (C16TAB). After removing DDAB from the buffer, the reversed EOF decreased only 3% over 75 min under continuous electrophoretic conditions. Also, the reversed EOF is 60% greater for DDAB than for C16TAB at pH 2. This greater coating stability is associated with a different aggregate structure for the surfactant at the capillary surface. The more homogeneous coating and greater surface coverage provided by DDAB allows the excess surfactant to be flushed from the capillary prior to performing electrophoretic separations. Separations of a basic protein mixture yielded quantitative recoveries, efficiencies ranging from 560,000 to 750,000 plates/m, and migration time reproducibility of 0.8-1.0% RSD (n = 10). This performance is similar to that of adsorbed cationic polymers (Polybrene, polyethyleneimine) but is achieved using a coating procedure that is over 10 times faster.     

Polymer-coated fibrous materials as the stationary phase in packed capillary gas chromatography

Synthetic polymer filaments have been introduced as the support material in packed capillary gas chromatography (GC). The filaments of the heat-resistant polymers, Zylon, Kevlar, Nomex, and Technora, were longitudinally packed into a short fused-silica capillary, followed by the conventional coating process for open-tubular GC columns. The separation of several test mixtures such as n-alkylbenzenes and n-alkanes was carried out with these polymer-coated fiber-packed capillary columns. With the coating by various polymeric materials on the surface of these filaments, the retentivity was significantly improved over the parent fiber-packed column (without polymer coating) as well as a conventional open-tubular capillary of the same length. The results demonstrated a good combination of Zylon as the support and poly(dimethylsiloxane)-based materials as the coating liquid-phase for the successful GC separation of n-alkanes and polycyclic aromatic hydrocarbons (PAHs), while successful applications for other separations such as poly(ethylene glycol) coating for the separation of alcohols were also obtained. From the results it has been suggested that the selectivity of the fiber-packed column could be tuned by selecting different coating materials, indicating the promising possibility for a novel usage of fine fibrous polymers as the support material that can be combined with newly synthesized coating materials specially designed for particular separations. Taking advantage of good thermal stability of the fibers, the column temperature could be elevated to higher than 350 degrees C with the combination of a short metallic capillary.     

Robust polymeric microchannel coatings for microchip-based analysis of neat PCR products

Several silica coatings have been evaluated for replicate PCR product analysis in capillaries and electrophoretic microchips. Silica coatings are an essential component to many electrophoretic separations, and this importance is magnified in microchips, where separation distances are minimized. Increasing the resistance of coatings to separation conditions improves the reproducibility and longevity of the coated microchip, which allows for the full potential of these devices (rapid separations, high through-put, and longevity) to be realized. In this study, several coating parameters have been evaluated experimentally and through the literature to produce a coating with high resistance to the separation conditions of interest, neat PCR product separations. Coating degradation induced under these conditions was tested for several coatings, and the influence of surface hydroxylation, surface hydration, silanization solvent, silanizing reagent, catalysis, endcapping, and polymerization procedure are discussed. Under the testing conditions, a coating (coating E) prepared by silanization with chlorodimethyloctylsilane in toluene with a polymer layer of poly(vinylpyrrolidone) attached by a hydrogen abstraction method [Srinivasan, K.; Pohl, C.; Avdalovic, N. Anal. Chem. 1997, 69, 2798-2805] was most resistant. This coating was tested for longevity on electrophoretic microchips and was compared to the traditional coating of polyacrylamide. The coatings produced similar resolution and efficiencies; however, coating E provided more reproducible migration times and had performed for 635 analyses when testing was terminated. This procedure provides a reproducible, resistant surface coating, thus allowing for replicate analysis of neat PCR product on microchips.     

Solute-solvent interactions in micellar electrokinetic chromatography. 6. Optimization of the selectivity of lithium dodecyl sulfate-lithium perfluorooctanesulfonate mixed micellar buffers

The optimization of the composition of mixed surfactants used as micellar electrokinetic chromatography (MEKC) pseudostationary phases is proposed as an effective method for the separation of complex mixtures of analytes. The solvation parameter model is used to select two surfactants (lithium dodecyl sulfate, LDS, and lithium perfluorooctanesulfonate, LPFOS) with contrasting solvation properties. Combination of these two surfactants allows variations of the solvation properties of MEKC pseudostationary phase along a wide range. Thus, the convenient variation of the proportion of both surfactants allows an effective control of the selectivity in such systems. An algorithm that predicts the overall resolution of a given mixture of compounds is described and applied to optimize the composition of the mixed surfactant for the separation of the mixture. The algorithm is based on the calculation of peak purities on simulated chromatograms as a function of the composition of the mixed LDS/LPFOS micellar buffer from data at several micellar buffer compositions. Successful separations were achieved for mixtures containing up to 20 compounds, in less than 12 min.     

Preparation and characterization of cross-linked phospholipid bilayer capillary coatings for protein separations

Analysis of protein and peptide mixtures via capillary electrophoresis is hindered by nonspecific adsorption of analytes to the capillary walls, resulting in poor separations and quantitative reproducibility. Phospholipid bilayer (PLB) coatings are very promising for improving protein and peptide separations due to the native resistance to nonspecific protein adsorption offered by PLBs; however, these coatings display limited chemical and temporal stability. Here, we show the preparation and characterization of a highly cross-linked, polymerized phospholipid capillary coating prepared using bis-SorbPC. Poly(bis-SorbPC) PLB coatings are prepared in situ within fully enclosed fused silica capillaries via self-assembly and radical polymerization. Polymerization of the PLB coating stabilizes the membrane against desorption from the surface and migration in an electric field, improves the temporal and chemical stability, and allows for the separation of both cationic and anionic proteins, while preserving the native resistance to nonspecific protein adsorption of natural PLBs.     

Incorporation of single-wall carbon nanotubes into an organic polymer monolithic stationary phase for mu-HPLC and capillary electrochromatography

Single-wall carbon nanotubes (SWNT) were incorporated into an organic polymer monolith containing vinylbenzyl chloride (VBC) and ethylene dimethacrylate (EDMA) to form a novel monolithic stationary phase for high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). The retention behavior of neutral compounds on this poly(VBC-EDMA-SWNT) monolith was examined by separating a mixture of small organic molecules using micro-HPLC. The result indicated that incorporation of SWNT enhanced chromatographic retention of small neutral molecules in reversed-phase HPLC presumably because of their strongly hydrophobic characteristics. The stationary phase was formed inside a fused-silica capillary whose lumen was coated with covalently bound polyethyleneimine (PEI). The annular electroosmotic flow (EOF) generated by the PEI coating allowed peptide separation by CEC in the counterdirectional mode. Comparison of peptide separations on poly(VBC-EDMA-SWNT) and on poly(VBC-EDMA) with annular EOF generation revealed that the incorporation of SWNT into the monolithic stationary phase improved peak efficiency and influenced chromatographic retention. The structures of pretreated SWNT and poly(VBC-EDMA-SWNT) monolith were examined by high-resolution transmission electron microscopy, Raman spectroscopy, scanning electron microscopy, and multipoint BET nitrogen adsorption/desorption.     

Separation of acidic and basic proteins by nanoparticle-filled capillary electrophoresis

We present the first example of the analysis of acidic and basic proteins by nanoparticle-filled capillary electrophoresis. Compared to the didodecyldimethylammonium bromide (DDAB)-coated capillary, the DDAB-capped gold nanoparticles (AuNPs) as pseudostationary phase were found to form more stable coating on the capillary wall, thus leading to greater separation efficiency and high reproducibility. In addition to their advantages for protein separation, DDAB-capped AuNPs can generate high reversed electroosmotic flow, which is 75% greater than DDAB at pH 3.5. To allow strong interactions with proteins, the AuNPs were modified with poly(ethylene oxide) via noncovalent bonding to form gold nanoparticles/polymer composites (AuNPPs). Using a capillary dynamically coated with DDAB-capped AuNPs and filled with AuNPPs under acidic conditions (10 mM phosphate, pH 3.5), we have demonstrated the separation of acidic and basic proteins with peak efficiencies ranging from 71 000 to 1 007 000 plates/m and relative standard deviations of migration time less than 0.6%. Additionally, the proposed method has been applied to the analyses of biological samples, including saliva, red blood cells, and plasma. With simplicity, high resolving power, and high reproducibility, the proposed method has shown great potential for proteomics applications and clinical diagnosis.     

A fluorescence detection scheme for capillary electrophoresis of N-methylcarbamates with on-column thermal decomposition and derivatization

This paper describes a fluorescence detection method for N-methylcarbamate (NMC) pesticides in micellar electrokinetic chromatography (MEKC) separation. Fulfillment of the fluorescence detection hinged on the discovery that quaternary ammonium surfactants (particularly cetyltrimethylammonium bromide, CTAB), besides serving as hydrophobic pseudophases in MEKC, are also capable of catalyzing the thermal decomposition of NMCs to liberate methylamine. Thus, a multifunctional MEKC medium consisting of borate buffer, CTAB, and derivatizing components (o-phthaldialdehyde/2-mercaptoethanol) was formulated, which allowed first normal MEKC separation, subsequent thermal decomposition, and finally in situ derivatization of NMCs. With careful optimization of the operation conditions, fluorescence detection of 10 NMC compounds was achieved, with column efficiencies typically higher than 50,000 and detection limits better than 0.5 ppm. The present work represents an unprecedented effort in capillary electrophoresis (CE), in which an intact capillary was consecutively utilized as chambers for separation, decomposition, derivatization, and detection, without involving any interfacing features. The success in the implementation of such a detection system resulted in strikingly simple instrumentation as compared with the traditional postcolumn fluorescence determination of NMCs by reversed-phase HPLC. Similar protocols should be workable in the determination of a wide range of pesticides and pharmaceuticals in CE formats.     

Chiral separations using polymeric surfactants and polyelectrolyte multilayers in open-tubular capillary electrochromatography

In this study, fused-silica capillaries are modified using a polyelectrolyte multilayer (PEM) coating procedure in open-tubular capillary electrochromatography. The PEM coating was constructed in situ with alternating rinses of positively and negatively charged polymers. The quaternary ammonium salt poly (diallyldimethylammonium chloride) was used as the cationic polymer, and the polymeric surfactant poly (sodium N-undecanoyl-l-leucylvalinate) was used as the anionic polymer. Previous studies have shown that the PEM-coated capillaries used for achiral separations have excellent reproducibilities and high stabilities against extreme pH values. In the current study, this PEM coating approach was applied to chiral separations of 1,1'-binaphthyl-2,2'-dihydrogenphosphate (BNP), 1,1'-bi-2-naphthol, secobarbital, pentobarbital, and temazepam. However, the PEM coating procedure used in the achiral studies needed to be significantly modified in order to achieve chiral separations. Optimal conditions were established by varying the additives (sodium chloride, 1-ethyl-3-methyl-1H-imidazolium hexafluorophosphate, 1-butyl-3-methylimidazolium tetrafluoroborate) in the polymer deposition solutions, the salt concentration, the column temperature, and the bilayer number. Reproducibilities were evaluated by use of the relative standard deviation (RSD) values of the electroosmotic flow (EOF) and the first peak ((R)-(+)-BNP). In all cases, the run-to-run and capillary-to-capillary RSD values of EOF were less than 0.5%, and the run-to-run RSD values of the (R)-(+)-BNP peak were less than 1%. In addition, more than 230 runs were performed on a single PEM-coated capillary.     

Control of electroosmotic flow and wall interactions in capillary electrophoresis capillaries by photografted zwitterionic polymer surface layers

A novel capillary with covalently bonded zwitterionic surface modification was prepared by photograft polymerization of the zwitterionic monomer N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betaine, onto the inner surface of a UV-transparent fused-silica capillary. Although the zwitterionic moieties in the resulting polymeric "tentacles" comprise both a positive quaternary ammonium group and a negative sulfonate group, the coating has a net zero charge. The electroosmotic flow (EOF) was therefore extensively suppressed on the grafted capillary compared to the native silica capillary and to the silica capillary that had been activated for graft polymerization by reaction with 3-(methacryloyl)oxypropyltrimethoxysilane. It was also found that the EOF can be varied by adding chaotropic anions or divalent cations such as perchlorate ion and magnesium ion to the running buffer, due to the interaction between these ions and zwitterionic functional group. This provides a new way of altering the EOF and the wall interaction without changing the pH or the overall ionic strength of the separation buffer. The influence of pH and ionic strength of separation buffer on the EOF were also investigated to optimize the separation conditions. Good separations of a mixture containing eight inorganic anions were achieved within 5 min under optimal conditions by capillary zone electrophoresis. The newly prepared capillary was also well suited for the separation of peptides or proteins.     

Factors affecting the behavior and effectiveness of phospholipid bilayer coatings for capillary electrophoretic separations of basic proteins

Phospholipid bilayer coatings can prevent adsorption of cationic proteins on the surface of fused silica capillaries used in capillary electrophoresis. However, the performance of such bilayer coatings is strongly dependent on solution conditions. The factors affecting the rate of formation of phospholipid bilayer coatings were investigated using the double-chained zwitterionic 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC, C(14)) as a model phospholipid. The effectiveness of these coatings for CE separations of model cationic lysozyme, cytochrome c, ribonuclease A, and alpha-chymotrypsinogen A was also assessed. Increasing the ionic strength of a 0.1 mM DMPC solution reduced capillary coat times from >2 hours in 2.5 mM Tris (pH 7.4) buffer to 3.4 min in 40 mM Tris and dramatically improved separation performance such that > or =1.4 x 10(5) plates/m were observed in capillaries coated for 5 min with 0.1 mM DMPC in 20 mM Tris-HCl (pH 7.4). The presence of Ca(2+) in the coating solution also increases the rate of formation of the phospholipid bilayer coating. The type of vesicle strongly affects its adsorption rate onto the silica surface. The time required to coat the capillary was 7.2 min for small unilamellar vesicles (SUVs) and 22.5 min for large unilamellar vesicles and excessively long for multilamellar vesicles. Highest efficiency protein separations were achieved with bilayer coatings prepared from SUVs. The coating rate was enhanced by using greater DMPC concentrations and unaffected by pH. The type of buffer present in the DMPC coating solution affects the coating behavior, with HEPES buffer yielding a faster coat time than either Tris or phosphate buffers. Histone H1 was separated on a 0.1 mM DMPC-coated capillary.     

Graphite-coated nanoelectrospray emitter for mass spectrometry

A new, more rapid method for coating nanoelectrospray emitters with graphite is to use a vacuum deposition chamber and a graphite carbon electrode. This method allows for mass production of nanoelectrospray emitters in a short period of time. The emitters are laser-pulled borosilicate glass micropipets and have tapers of around 4 microm i.d. The conductive coating applied to the emitter is only 20-30 nm thick, allowing for optical transparency with the borosilicate emitters. The conductive coating is stable for a number of hours at the high voltages used for nanoelectrospray ionization and is durable in both positive and negative ion modes-even during electrical discharge. This stability will make it possible to couple these emitters with online separations such as capillary liquid chromatography or capillary electrophoresis.     

Two-dimensional fluorescence correlation in capillary electrophoresis for peak resolution and species identification

A new spectroscopic dimension-fluorescence intensity correlation--is introduced to enhance peak resolution and species identification in capillary electrophoresis. In two-dimensional correlation CE, a conventional electropherogram is spread into two dimensions through cross-correlation analysis of fluorescence time response. A laser that is sinusoidally modulated in intensity is used as the excitation source. Three channels of information are collected during a CE run: the steady-state intensity, the ac amplitude, and the phase-resolved fluorescence intensity. The correlation between two chosen channels is then evaluated. A two-dimensional correlation electropherogram consists of a plot of the correlation intensity versus two axes of migration time. Through correlation analysis, species discrimination and peak resolution are significantly enhanced without having to physically separate the solutes. Two-dimensional correlation CE showed complete resolution between two overlapping sample peaks with a resolution of 0.28 in the conventional one-dimensional electropherogram. In separations of polycyclic aromatic hydrocarbons by micellar electrokinetic chromatography (MEKC), two-dimensional correlation analysis resolved all overlapping elution peaks unseparable by one-dimensional MEKC, demonstrating the utility of 2D correlation in separation method development. The capability of 2D correlation CE in species identification is demonstrated with a sequence of 39 consecutively injected peaks containing four fluorescent dyes. Species identification in sequencing is achieved without complex data treatment in two-dimensional correlation CE.     

Fabrication of isoreticular metal-organic framework coated capillary columns for high-resolution gas chromatographic separation of persistent organic pollutants

The unusual properties of metal-organic frameworks (MOFs), such as permanent nanoscale porosity, high surface area, uniformly structured cavities, and the availability of in-pore functionality and outer-surface modification, are advantageous for diverse applications. However, most existing methods for the synthesis of nanosized MOFs require an activation procedure or auxiliary stabilizing agents. Here we report a 1-min, room-temperature approach for the synthesis of nanosized isoreticular MOFs (IRMOFs) to fabricate IRMOF coated capillary columns for the high-resolution gas chromatographic separation of persistent organic pollutants (POPs), including polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs), polybrominated diphenylethers (PBDEs), and hexachlorocyclohexanes (HCHs). The developed method allows the synthesis of well-shaped nanosized IRMOFs within 1 min at room temperature without the need for any activation procedure or auxiliary stabilizing agents. The IRMOF coated capillary columns offer good separation efficiency that is generally comparable to that of a commercial HP-5MS column for POPs. The IRMOF-1 and IRMOF-3 coated capillary columns gave the theoretical plate values of 2293 and 2063 plates m(-1) for naphthalene, respectively, which are slightly smaller than those with a HP-5MS column (2845 plates m(-1)). The IRMOF-1 coated capillary column offered good resolution for the separation of several intractable PAH isomer pairs, such as anthracene/phenanthrene, benzo[a]anthracene/chrysene, and benzo[b]fluoranthene/benzo[k]fluoranthene, with resolutions of 3.0, 1.1, and 4.1, respectively, which were difficult to be baseline separated on a HP-5MS column with a resolution of 1.0. In addition, the IRMOF-1 and IRMOF-3 coated capillary columns offered a clear group separation of the PCB isomers and a linear range covering three orders of magnitude. The relative standard deviations for the five replicate separations of PAHs were 0.23-0.26% and 2.1-4.5% for retention time and peak area, respectively. The fabricated IRMOF coated capillary columns have been shown to be very promising for the separation of POPs with good reproducibility, high resolution, great selectivity, and a wide linear range.     

Condensation nucleation light scattering detection for capillary electrophoresis

We describe two means for interfacing condensation nucleation light scattering detection to capillary electrophoresis (CE). With the first method, a fused-silica capillary was used for the separation and the CE was grounded through a Nafion membrane that also connected the system to a microconcentric pneumatic nebulizer. Limits of detection (LODs) for underivatized amino acids were at the low microgram per milliliter level, and separation efficiencies were ～9 times lower than the optimum predicted for these species based on the injection plug width and axial dispersion by diffusion. LODs were limited by background nonvolatiles resulting from dissolution of fused silica at the high pHs used for the separations. An alternate system employed PEEK capillaries which acted as the separation capillary and also as the inner nebulizer capillary. In this case, the exit end of the capillary was coated with conductive paint which extended to the tip of the nebulizer, was in contact with the CE buffer, and was grounded to complete the CE circuit. Response was nonlinear and the separation efficiency of this system was somewhat lower than that for the Nafion membrane system. Response as peak heights for all of the amino acids and peptides studied was nearly identical on a mass basis. With this system, much lower background signals were obtained, and as a result, LODs for underivatized amino acids and peptides were below the 1 μg/mL level, corresponding to less than 10 pg or less than 100 fmol injected. Both systems were fairly simple, effective means to generate aerosols with the low flows of CE and should be applicable to interfacing of other aerosol-based detectors with CE.     

A low-makeup beveled tip capillary electrophoresis /electrospray ionization mass spectrometry interface for micellar electrokinetic chromatography and nonvolatile buffer capillary electrophoresis

A robust interface has been developed for interfacing micellar electrokinetic chromatography (MEKC) and nonvolatile buffer capillary electrophoresis (CE) to electrospray ionization mass spectrometry (ESI-MS). The interface consists of two parallel capillaries for separation (50 microm i.d. x 155 microm o.d.) and makeup (50 microm i.d. x 155 microm o.d.) housed within a larger capillary (530 microm i.d. x 690 microm o.d.). The capillaries terminate in a single tapered tip having a beveled edge. The use of a tapered beveled edge results in a greater tip orifice diameter (75 microm) than in a previous design from our laboratory (25 microm) that used a flat tip. While maintaining a similar optimum flow rate and consequently similar sample dilution, a 75-microm beveled emitter is more rugged than a 25-microm flat tip. Furthermore, the incorporation of a sheath liquid capillary allows the compositions of the final spray solution to be controlled. The application of this novel CE/ESI-MS interface was demonstrated for MEKC using mixtures of triazines (positive ion mode) and phenols (negative ion mode). The ability to perform CE/ESI-MS using a nonvolatile buffer was demonstrated by the analysis of gangliosides with a buffer consisting of 40 mM borate and 20 mM alpha-cyclodextrin.