Nanoparticle-based continuous full filling capillary electrochromatography/electrospray ionization-mass spectrometry for separation of neutral compounds

Highly efficient reversed-phase capillary electrochromatography (CEC) separations (plate numbers up to 700 000/m), with electrospray ionization mass spectrometry detection were achieved utilizing novel dextran-coated polymer nanoparticles as a pseudostationary phase. A continuous full filling (CFF) technique in which nanoparticles are continuously introduced into the capillary was employed for separation of neutral analytes (dialkyl phthalates), utilizing an orthogonal electrospray interface to prevent nanoparticles from entering the mass spectrometer. CFF-CEC benefits from that an entirely fresh column is employed for every analysis, avoiding carryover effects associated with stationary-phase contamination. The highly efficient separations obtained were accomplished by optimizing the organic modifier concentration in the electrolyte and by using a high nanoparticle concentration (5 mg/mL), to improve interparticle mass transfer and gain sufficient retention. Nanoparticles, with an average diameter of 600 nm, were prepared by polymerization of methacrylic acid and trimethylolpropane trimethacrylate, which in turn were coated with dextran. These nanoparticles formed stable suspensions in electrolytes having broad ranges of polarities, enabling straightforward optimization of the reversed-phase conditions.     

Anionic functionalized gold nanoparticle continuous full filling separations: importance of sample concentration

Electrically driven separations which contain nanoparticles offer detection and separation advantages but are often difficult to reproduce. To address possible sources of separation inconsistencies, anionic functionalized gold nanoparticles are thoroughly characterized and subsequently included in continuous full filling capillary electrophoresis separations of varying concentrations of three small molecules. Citrate stabilized gold nanospheres are functionalized with 11-mercaptoundecanoic acid, 6-mercaptohexanoic acid, or thioctic acid self-assembled monolayers (SAMs) and characterized using dynamic light scattering, extinction spectroscopy, zeta potential, and X-ray photoelectron spectroscopy prior to use in capillary electrophoresis. Several important trends are noted. First, the stability of these anionic nanoparticles in the capillary improves with increased ligand packing density as indicated by a ratio of absorbance collected at 520 to 600 nm. Second, increasing nanoparticle concentration from 0 to 2 nM (0-0.002(5)%, w/w) minimally impacts analyte migration times; however, when higher nanoparticle concentrations are included within the capillary, nanoparticle aggregation occurs which induces separation inconsistencies. Third, analyte peak areas are most significantly impacted as their concentration decreases. These trends are attributed to both sample enrichment and electrostatic interactions between the anionic carboxylic acid functionalized gold nanoparticles and sample. These important findings suggest that sample concentration-induced conductivity differences between the sample matrix and separation buffer as well as SAM packing density are important parameters to both characterize and consider when nanoparticles are used during continuous full filling separations and their subsequent use to enhance spectroscopic signals to improve in-capillary analyte detection limits.     

Reversed-phase electrochromatography with a monolithic microcolumn prepared in a 2.2-mm-inner diameter fused-silica tube

Capillary electrochromatography possesses advantages of high separation efficiency and velocity, but it also has its disadvantages due to its small inner diameter, such as poor detection sensitivity, low sample capacity, and some trouble in its column preparation. To overcome these shortcomings, a monolithic microcolumn with a surface area larger than 200 m2/g was prepared by sol-gel polycondensation of tetraethoxysilane-hydrochloric acid-poly(ethylene glycol) and filling with fine quartz sand in a 2.2-mm-i.d. fused-silica tube. The prepared microcolumn was used in the separation of aromatic compounds by reversed-phase electrochromatography. Some factors that affected electroosmotic flow were explored, such as electric field strength, buffer concentration, and buffer pH. Acetonitrile concentration in the mobile phase was investigated for phenol, benzene, and naphthalene separation. The separation results were satisfying with the electrochromatographic microcolumns. The detection limits of phenol, benzene, and naphthalene were 0.07, 0.26, and 0.04 mg/L, respectively.     

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.     

Open-tubular capillary electrochromatography coupled with electrospray ionization mass spectrometry for peptide analysis

In this study, the open-tubular electrochromatographic (OT-CEC) migration behavior of various peptides has been characterized using etched and chemically (n-octadecyl- and cholesterol-) modified capillaries, interfaced to an electrospray ionization mass spectrometer through a sheath liquid configuration. The stationary phases were fabricated by etching the inner wall of the fused-silica capillary and then chemically modifying the new surface through a silanization/hydrosilation reaction. Unlike some other OT-CEC stationary-phase preparation methods, leaching of the immobilized stationary phase and subsequent contamination of the electrospray ion source was largely avoided with this novel surface modification technology. The influence of the immobilized organic phases and those of the buffer electrolytes (pH, the type and content of organic solvent) on the retention and separation of the selected peptides was investigated. Significant peptide retention was found even at very low pH with both types of stationary phases, under conditions whereby the electrophoretic migration dominated the separation process. Due to the effective coverage of the etched surface by a silanization/hydrosilation reaction, adverse adsorption of charged analytes onto the capillary wall was minimized. As a result, very efficient and highly reproducible peptide separations were achieved over a broad pH range. Moreover, peptide-specific multizoning effects were observed. The origin of this novel phenomenon was explored. Compared to capillary electrophoresis electrospray ionization mass spectrometry system, much higher detection sensitivity could be obtained, since a larger amount of sample could be injected and stacked at the head of the open-tubular capillary column without deteriorating the separation performance. On the basis of these observations, these procedures have been adapted to allow the analysis of tryptic peptides generated from proteins.     

Electrochromatographic retention studies on a flavin-binding RNA aptamer sorbent

Aptamers are oligonucleotides that are isolated and amplified on the basis of their recognition of a target molecule. In this study, an RNA aptamer isolated and amplified on the basis of its affinity for flavin mononucleotide (FMN) was covalently bound to the inner walls of fused-silica capillaries. This aptamer recognizes the flavin moiety of both FMN and flavin adenine dinucleotide (FAD). When an attempt was made to evaluate these capillaries according to existing theory, the theory proved to be insufficient. We describe a new method to evaluate capillaries for use in open-tubular capillary electrochromatography (OTCEC) of charged analytes, which combines OTCEC and flow-counterbalanced capillary electrophoresis. This method enabled us to extract k' and evaluate k(CEC) values for these capillaries, and the dependence of these values on Mg(2+) concentration was explored. The k' values for these capillaries ranged from 0.0951 to 0.2530 and from 0.0255 to 0.1118 for FMN and FAD, respectively.     

Open tubular capillary electrochromatography of synthetic peptides on etched chemically modified columns

Two sets of peptides, each having structurally similar amino acid sequences, have been investigated by capillary electrochromatography (CEC) using etched chemically modified capillaries as the separation medium. In comparison to gradient RP-HPLC, the resolving power of the described CEC methods has been found to be superior. A number of variables have been examined with respect to optimization of the separation of these closely related peptides with several different etched chemically modified capillaries. These experimental variables included the nature of the bonded moiety, the pH, the organic modifier type, and the amount of organic modifier in the buffer electrolyte. Systematic variation of these parameters results in significant changes in the migrational behavior of the investigated peptides and provides important insight into the underlying molecular separation processes that prevail in open tubular CEC. Moreover, under optimized conditions, efficient separations characterized by highly symmetrical peaks were achieved. In addition, this study has permitted the long-term stability as well as the short-term and long-term reproducibility of the etched chemically modified capillaries to be documented.     

Characterization of open tubular capillary electrochromatography columns for the analysis of synthetic peptides using isocratic conditions.

In this paper, we report on investigations related to the performance characteristics of two different types of etched chemically (n-octadecyl- and cholesterol-) modified capillaries in the open tubular format of capillary electrochromatography (CEC) for the analysis of synthetic peptides. The results confirm that the nature of the surface chemistry used to modify the capillary wall and type of chemically bonded group employed can affect the selectivity as well as the resolution of peptide samples. The results are consistent with the participation of selective peptide interactions with the bonded phase, although other factors, such as the morphology of the capillary wall surfaces, appear to be also involved. Moreover, several surprising observations related to peptide-specific multi-zoning effects have been observed. Additional experimental variables that can also be utilized to affect the retention of peptides in this approach to OTCEC include the type and percentage of organic solvent modifier employed in the eluent and the pH of the buffer system. To evaluate the reproducibility of different batches of the n-octadecyl- and cholesterol-modified capillaries and the stability of the chemically modified surface, the OTCEC selectivity and peak shape behavior of two small basic molecules (serotonin and tryptamine) and two proteins (turkey and chicken lysozyme) were also investigated. Finally, the use of the "bubble" cell technology for creating the detector window has been shown to provide significantly higher detection sensitivity with peptides, as compared with the conventional capillary format.     

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.     

Capillary electrochromatography with fused-silica capillaries packed with copolymeric reversed-phase adsorbent and ion exchangers

Macroporous poly(styrene-divinylbenzene) (PSDVB), PRP-1, a reversed-phase adsorbent, and PSDVB-based strong acid cation exchangers and strong base and weak base anion exchangers were evaluated as stationary phases for capillary electrochromatography (CEC). Electroosmotic flow (EOF) for adsorbent and exchanger packed fused-silica capillaries for acetone as the marker increases with increasing ion exchange capacity, buffer organic solvent concentration, and applied voltage, is nearly independent of pH, and decreases with increased buffer ionic strength. For anion exchangers, EOF is reversed. Thiourea, acetone, acrylamide, nitromethane, propanal, and acetic acid were evaluated as EOF markers and undergo weak interaction with the PSDVB-based stationary phases. EOF in a basic buffer is greater than or equal to silica-based C-18 and cation exchanger packed capillaries. For an acidic buffer, EOF for a PRP-1 capillary is almost twice the C-18 packed capillary. As analyte hydrophobicity increases, retention and migration time increases for the PSDVB-based stationary phases. As exchange capacity increases, availability of the polymeric matrix for analyte partitioning decreases, causing analyte migration time to decrease. Increasing buffer organic solvent concentration decreases analyte retention. The PSDVB-based stationary phases provide good resolving power and reproducibility and are applicable to the CEC separation of neutral, weakly acidic, and basic analytes. Efficiency, however, is less than obtained with silica-based stationary phases. Because of stability in a strong acid buffer, the CEC separation of weak acids, where dissociation is suppressed, and weak bases as cations is possible. Separations of short-chain alkyl aldehydes, methyl ketones, aromatic hydrocarbons, substituted benzene derivatives, and short-chain carboxylic acids are described.     

An etched porous interface for on-line capillary electrophoresis-based two-dimensional separation system

The construction and evaluation of an on-column etched fused-silica porous junction for on-line coupling of capillary isoelectric focusing (CIEF) with capillary zone electrophoresis (CZE) are described. Where two separation columns were integrated on a single piece of fused-silica capillary through the etched approximately 4 to 5-mm length porous junction along the capillary. The junction is easily prepared by etching a short section of the capillary wall with HF after removing the polyimide coating. The etched section becomes a porous glass membrane that allows only small ions related to the background electrolyte to pass through when high voltage is applied across the separation capillary. The primary advantages of this novel porous junction interface over previous designs (in which the interface is usually formed by fracturing the capillary followed by connecting the two capillaries with a section of microdialysis hollow fiber membrane) are no dead volume, simplicity, and ruggedness, which is particularly well suited for an on-line coupling capillary electrophoresis-based multiple dimensional separation system. The performance of the 2D CIEF-CZE system constructed by such an etched porous junction was evaluated by the analyses of protein mixtures.     

Ion-pair chromatographic separation of water-soluble gold monolayer-protected clusters

We demonstrate the efficacy of ion-pair chromatography for separations of samples of charged, polydisperse, water-soluble gold nanoparticles protected by monolayers of N-acetyl-l-cysteine and of tiopronin ligands. These nanoparticle mixtures have 1-2-nm-diameter Au core sizes as estimated from UV-visible spectra of the separated components. This size range encompasses the transition from bulk metal to molecular properties. The nanoparticle mixtures were resolved, the smallest nanoparticles eluting first, on an octadecylsilyl (C18) column using isocratic elution with a methanol/water mobile phase containing tetrabutylammonium fluoride (Bu4N+F-) and phosphate buffer. The column retention increases with Bu4N+F- concentration, lowered pH, and decreasing methanol volume fraction. The retention mechanism is dominated by ion-pairing in either the mobile phase or at the stationary/mobile-phase interface. Size exclusion effects, used in many previous nanoparticle separations, are insignificant.     

High-performance capillary gel electrochromatography with replaceable media

Capillary gel electrochromatography is evaluated with an entangled polymer solution which is pumped into the capillary and run under fritless conditions. The polymer used has an acid backbone with grafted hydrophobic segments, the polyacid giving the electroosmotic flow and the hydrophobe segments providing the retentive component. Experimental evaluation of this type of system reveals performance similar to capillary electrophoresis and other forms of electrochromatography. The analysis of plate height data demonstrates that zone broadening is primarily due to diffusion with little contribution from nonequilibrium zone broadening. Hence, operation at high velocities (high voltages) is most desirable as opposed to most chromatographic methods. Some of the advantages of this type of experiment include being able to replace the retentive media in a few minutes, fast and reproducible high-performance separation, and having a retention mechanism similar to reversed-phase liquid chromatography. Disadvantages include a low retentive phase concentration and hence low sample loadability and limited solvent compatibility of the polymer. A number of different separations are demonstrated including separation of alkyl benzoates, alkylphenones, alkylbenzenes, oxidation inhibitors, and PAHs.     

Use of gold nanoparticles to enhance capillary electrophoresis.

We describe here the use of gold nanoparticles to manipulate the selectivity between solutes in capillary electrophoresis. Two different gold-based nanoparticles were added to the run buffer. In one case, the nanoparticles were stabilized with citrate ions, but in another study, the gold nanoparticles were capped with mercaptopropionate ions (thiol-stablized). Citrate-stabilized gold nanoparticles were used in conjunction with capillaries treated with poly(diallyldimethylammonium chloride) (PDADMAC). The positively charged PDADMAC layer on the capillary walls adsorbs the negatively charged gold nanoparticles. The model solutes that were used to study the effect of the presence of the citrate-stabilized gold nanoparticles are structural isomers of aromatic acids and bases. The presence of the PDADMAC layer and the PDADMAC plus the gold nanoparticles changes both the electroosmotic mobility and the observed mobility of the solutes. These changes in the mobilities influence the observed selectivities and the separations of the system. Thiol-stabilized gold nanoparticles were used without PDADMAC in the capillary. The model solutes studied in this part are various aromatic amines. In this case as well, the presence of the gold nanoparticles modifies the electroosmotic mobility and the observed mobility of the solutes. These changes in the mobilities are manifested in selectivity alterations. The largest change in the selectivities occurs at low concentrations of the gold nanoparticles in the run buffer. The presence of nanoparticles improves the precision of the analysis and increases the separation efficiency.     

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.     

Monolayer-protected gold nanoparticles as a stationary phase for open tubular gas chromatography

The use of a thin film of monolayer-protected gold nanoparticles (MPNs) as a stationary phase for gas chromatography (GC) is reported. Deposition of a MPN film was obtained in a 2-m, 530-microm-i.d. deactivated silica capillary using gravity to force the solution containing the MPN material through the capillary. By SEM analysis, the average film thickness was determined to be 60.7 nm. The retention behavior for the dodecanethiol MPN column was studied using four compound classes (alkanes, alcohols, aromatics, ketones), and retention orders were objectively compared to a commercially available column (AT-1, 100-nm film thickness). Separation of an eight-component mixture was performed using both isothermal and temperature-programming methods with the dodecanethiol MPN phase and compared to an isothermal separation with the AT-1 phase. The AT-1 phase separation had an efficiency, N, of 6200 (k' = 0.33) while the dodecanethiol MPN phase separation had an efficiency, N, of 5700 (k' = 0.21) for the same analyte, octane. The reduced plate height, h, for octane was found to be less than 1 at the optimum linear flow velocity, indicating the MPN column operated near the optimum possible performance level. Robustness of the MPN phase is also discussed with consistent performance observed over several months. Overall, MPNs appear promising as a stationary-phase material for GC and as an experimental platform to study their thermodynamic and mass-transfer properties.     

Chiral monolithic columns for enantioselective capillary electrochromatography prepared by copolymerization of a monomer with quinidine functionality. 1. Optimization of polymerization conditions, porous properties, and chemistry of the stationary phase

Monolithic columns for chiral capillary electrochromatography have been prepared within the confines of untreated fused-silica capillaries in a single step by a simple copolymerization of mixtures of O-[2-(methacryloyloxy)ethylcarbamoyl]-10,11-dihydroquinidine , ethylene dimethacrylate, and glycidyl methacrylate or 2-hydroxyethyl methacrylate in the presence of mixture of cyclohexanol and 1-dodecanol as a porogenic solvent. The porous properties of the monolithic columns can easily be controlled through changes in the composition of the binary porogenic solvent. Although both thermal- and UV light-initiated polymerizations afford useful capillary columns, monoliths prepared using the former approach exhibit better chromatographic properties. The ability to control pore size independently of the polymerization mixture composition enables the preparation of monoliths with varying percentages of the chiral monomer and cross-linker, as well as the optimization of their separation properties. Very good separations of model racemate (R,S)-N-3,5-dinitrobenzoylleucine were achieved using an optimized monolithic CEC column, with high efficiencies of up to 74000 plates/m for the retained peaks.     

Gradient elution in capillary electrochromatography

In analogy to pressure-driven gradient techniques in high-performance liquid chromatography, a system has been developed for delivering electroosmotically driven solvent gradients for capillary electrochromatography (CEC). Dynamic gradients with submicroliter per minute flow rates are generated by merging two electroosmotic flows that are regulated by computer-controlled voltages. These flows are delivered by two fused-silica capillary arms attached to a T-connector, where they mix and then flow into a capillary column that has been electrokinetically packed with 3-μm reversed-phase particles. The inlet of one capillary arm is placed in a solution reservoir containing one mobile phase, and the inlet of the other is placed in a second reservoir containing a second mobile phase. Two independent computer-controlled, programmable, high-voltage power supplies (0-50 kV) [Formula: see text] one providing an increasing ramp and the other providing a decreasing ramp [Formula: see text] are used to apply variable high-voltage potentials to the mobile phase reservoirs to regulate the electroosmotic flow in each arm. The ratio of the electroosmotic flow rates between the two arms is changed with time according to the computer-controlled voltages to deliver the required gradient profile to the separation column. Experiments were performed to confirm the composition of the mobile phase during a gradient run and to determine the change of the composition in response to the programmed voltage profile. To demonstrate the performance of electroosmotically driven gradient elution in CEC, a mixture of 16 polycyclic aromatic hydrocarbons was separated in less than 90 min. This gradient technique is expected to be well-suited for generating not only solvent gradients in CEC but also other types of gradients, such as pH and ionic strength gradients, in capillary electrokinetic separations and analyses.     

On-line concentration of proteins and peptides in capillary zone electrophoresis with an etched porous joint

A novel approach for on-line concentration of proteins and peptides in capillary electrophoresis (CE) is presented. A short section (approximately 0.5-1 cm) along the capillary was etched with HF. The etched section became a porous membrane that allowed electrical conductivity but prevented passage of the analyte ions. The capillary was isolated into two parts by the etched section. Thus, we were able to use three buffer vials to perform CE experiments in the capillary by applying high voltages independently. Concentration and separation were performed at the two respective regions. When high voltage was applied to the concentration capillary (between the inlet end and the etched section), proteins and peptides were concentrated at the etched portion, because the porous capillary wall allowed only small buffer ions to pass through and there was no electric field gradient beyond that point. After focusing, the narrow sample zone was introduced into the separation capillary (between the etched section and the outlet end) by hydrodynamic flow or by electroosmotic flow. Finally, conventional CE was carried out for separation of the analytes. Several different concentration schemes for proteins and peptides were successfully demonstrated by using this new approach.     

Nanoinjector for capillary electrophoresis and capillary electrochromatography

A rotary valve nanoinjector was devised for use in capillary electrophoresis (CE) and capillary electrochromatography (CEC). A fused-silica capillary tip was inserted in a small through-hole in the rotor. The narrow and short capillary tip, with an inner volume of 6-24 nL, was embedded in the hole using epoxy resin. The injection volume was confirmed chromatographically by comparing the peak areas obtained with the nanoinjector to those of a conventional injector. In addition, both the rotor and stator of the injector were made of a nonconducting material, polyimide resin, to be utilized for CE and CEC. The application of the nanoinjector for CE was demonstrated.