Physically adsorbed chiral stationary phase of avidin on monolithic silica column for capillary electrochromatography and capillary liquid chromatography

The physical adsorption method proposed previously has been successfully applied to a monolithic silica column. By virtue of the physical adsorption, a chiral stationary phase of avidin was prepared onto the silica monolith. The phase ratio of resulting stationary phase was evaluated with frontal analysis. The method proved to be comparable in phase ratio to the chemical bonding methods used in high-performance liquid chromatography (HPLC). Enantiomer separations were carried out in capillary electrochromatography (CEC) and capillary liquid chromatography (CLC) modes. Due to its larger phase ratio, the resulting column showed more powerful separation capability as compared to open-tubular CEC (OTCEC). Twelve chiral compounds were baseline-resolved. The resulting column showed high separation efficiency, with average theoretical plate numbers of 66 000/m for CLC and 122 000/m for CEC. Good reproducibility was observed, with RSD value less than 1.3% for retention time, retention factor and separation factor, and less than 6.6% for plate counts and resolution (n = 40). Fast separations were achieved with a short column. The test enantiomers were baseline-resolved within 4 min under CLC and CEC modes. In addition, field-enhanced sample injection (FESI) was coupled to CLC as well as CEC to improve the detection sensitivity.     

Alkylthiol gold nanoparticles in sol-gel-based open tubular capillary electrochromatography

A novel open-tubular capillary electrochromatography (OTCEC) column was prepared by immobilizing dodecanethiol gold nanoparticles on prederivatised fused-silica capillary columns with sol-gel technology. 3-Mercaptopropyl-trimethoxysilane (MPTMS) was selected as sol-gel precursor to develop a sol-gel layer on the inner wall of the capillary, prior to assembly of dodecanethiol gold nanoparticles onto the generated sol-gel layer through specific interaction between the gold nanoparticles and surface terminating thiol groups. The electrochromatographic behaviour of the sol-gel gold nanoparticle capillary was compared with a gold nanoparticle capillary prepared via MPTMS surface functionalisation, through variation of the percentage of the organic modifier, pH, and separation voltage. Efficient separation for a "reversed-phase" test mixture of thiourea, naphthalene, and biphenyl and for selected polycyclic aromatic hydrocarbons (PAHs) was obtained on the sol-gel based gold nanoparticle capillaries. OTCEC separations of three selected drug substances (propiophenone, benzoin, and warfarin) were also demonstrated. Scanning electron microscopy was used for the characterization of the sol-gel gold nanoparticle capillary surface. The results confirm that dodecanethiol gold nanoparticles, bound on the sol-gel-based inner layer of fused-silica capillary, can provide sufficient solute-bonded phase interactions for OTCEC with reproducible retention as well as characteristic reversed-phase behaviour.     

On-line sample enrichment for the determination of proteins by capillary zone electrophoresis with poly(vinyl alcohol)-coated bubble cell capillaries

Field-amplified sample stacking (FASS) is used to separate basic proteins in a poly-(vinyl alcohol)-coated bubble cell capillary. To our knowledge, this is the first paper describing the on-column stacking of proteins (as cations) using FASS in bubble cell capillary. The bubble cell capillary is fabricated using a one-step method. Cetyltrimethylammonium chloride is added into the running buffer to reverse the EOF and, thus, to pump the water plug out during the sample stacking step. The effect of the water plug lengths and sample injection durations were investigated and optimized. The results obtained were compared with those for the normal capillary without bubble cell in terms of resolution and sensitivity enhancement. Under the optimal condition, this method can improve the sensitivity of the peak areas ranging from 5000- to 26 000-fold. The RSDs (n = 5) of the migration time and peak area are satisfactory (less than 0.6 and 12%, respectively). Application of the capillary electrophoresis method with bubble cell, FASS, and UV detection thereby leads to the determination of these proteins at concentrations ranging from 3 to 10 ng/mL, based on a signal-to-noise ratio of 3:1.     

Simultaneous analysis of polyhydroxylated alkaloids by capillary electrophoresis using borate complexation and evaluation of sweeping technique for sensitivity improvement

Summary Capillary zone electrophoresis coupled to UV detection was used for the simultaneous analysis of naturally occurring polyhydroxylated alkaloids. This separation was based on anin-situ complexation with borate ions. The effect of parameters such as borate concentration, capillary temperature and analyte molecular structure on migration times and selectivity were discussed. The best separation was obtained with a fused silica capillary (48.5 cm total length ×50 μm I.D., with a bubble factor of 3), 80 mM sodium tetraborate aqueous solution at pH 9.2 and temperature of 20°C. The method was validated and showed good data in terms of migration time and peak area reproducibility, selectivity, linearity and accuracy. The validated method was applied to determine miglitol in commercially available pharmaceutical tablets. To further improve method sensitivity, a sweeping technique involving borate ions was evaluated. This technique was found very sensitive to the analyte complexation with borate, borate concentration, and temperature as well as sample matrix. In the case of miglitol, a 35-fold improvement in peak height was achieved.     

Protein separation by open tubular capillary electrochromatography employing a capillary coated with phenylalanine functionalized tentacle-type polymer under both cathodic and anodic electroosmotic flows

The use of a phenylalanine (Phe) functionalized tentacle-type polymer coated capillary column for protein separation by open tubular capillary electrochromatography (OTCEC) was demonstrated in this work. The tentacle-type stationary phase was prepared from silanized fused-silica capillaries of 50 microm I.D. by glycidyl methacrylate graft polymerization and subsequent Phe functionalization. Due to the amphoteric functional groups of the Phe bonded on the tentacle-type polymer stationary phase, protein separation in the prepared column can be performed under both cathodic and anodic electroosmotic flow (EOF) by varying the pH values of the mobile phase. Model proteins including ribonuclease A (RNase A), myoglobin, transferrin, insulin were baseline separated under cathodic EOF with a mobile phase of pH 8.8. Comparison between the separation result of the four proteins under conditions of OTCEC and capillary zone electrophoresis indicates that the migration behavior of the four proteins in the prepared column was the result of the interplay of chromatographic retention and electrophoretic migration. Besides, three basic proteins including RNase A, cytochrome c (Cyt-c) and lysozyme (Lys) were fully resolved under anodic EOF with an acidic running buffer (pH 2.5). The elution order was the same as the isoelectric point values of the proteins (RNase A相似文献   

Improving detection in capillary electrophoresis with laser induced fluorescence via a bubble cell capillary and laser power adjustment

Bubble cells have been frequently employed in capillary electrophoresis (CE) to increase the light path length with UV detection to provide an increase in the observed sensitivity of CE; however this approach has not been commonly used for laser-induced fluorescence detection (LIF) with CE. In this paper we study the influence of laser power on the sensitivity of detection in using conventional and enlarged fused silica capillaries for CE with LIF. When using the bubble cell capillary, the laser power must be decreased relative to use of the conventional capillary to reduce the effects of photodegradation of the species being illuminated by the laser. Even though the light intensity was decreased, an increase in sensitivity of detection was observed for most compounds when a bubble cell was used. This increase ranged from a factor of 8 for riboflavin (410 nm excitation) to 3.2 for most aromatic compounds (266 nm excitation), when using a 3x bubble cell compared with a conventional capillary. The bubble cell capillary was used for native detection of IgG by LIF at 266 nm. A limit of detection of 60 ng mL(-1) was obtained from a 20 pg injection, which was 40 times more sensitive than silver staining in conventional SDS/PAGE.     

Open tubular capillary electrokinetic chromatography in etched fused-silica tubes

This review describes an open tubular approach to capillary electrochromatography (OTCEC) that first etches the inner surface of the fused-silica tube using ammonium hydrogen diflouride. This process can increase the inner surface area significantly. The new surface is then chemically modified to attach a bonded stationary phase using a silanization/hydrosilation reaction process. The surfaces are characterized spectroscopically by diffuse reflectance infrared Fourier transform and by electroosmotic flow measurements. Applications of OTCEC columns with C18, diol and chiral stationary phases are described.     

Quantification of ganciclovir in human plasma using capillary electrophoresis

A fast, simple, specific capillary electrophoretic method in the MEKC mode for the quantification of the antiviral drug ganciclovir is described. The separation was obtained using a 50 microm id fused-silica capillary, 60 mM borax buffer (pH 9.25) containing 40 mM SDS using ethenoadenosine as the internal standard. Sample preparation was done by ultrafiltration with a Microcon 30 000 kDa filter. The analytes were detected with UV detector at 254 nm. A sufficient sensitivity was achieved by using a bubble cell capillary. The linear range was from 0.5 to 10 mg/L with a LOQ of 0.5 mg/L. Correlation coefficients were better than 0.999 whereas inter- and intraday precision and accuracy were less than 10.7%. The analysis of patients' samples after administration of ganciclovir indicates that the method is suitable for drug monitoring in the clinic.     

Fast determination of sugars in Coke and Diet Coke by miniaturized capillary electrophoresis with amperometric detection

The fast separation capability of a novel miniaturized capillary electrophoresis with amperometric detection (CE-AD) system was demonstrated by determining sugar contents in Coke and diet Coke with an estimated separation efficiency of 60,000 TP/m. Factors influencing the separation and detection processes were examined and optimized. The end-capillary 300 microm Cu wire amperometric detector offers favorable signal-to-noise characteristics at a relatively low potential (+0.50 V vs. Ag/AgCl) for detecting sugars. Three sugars (sucrose, glucose, and fructose) have been separated within 330 s in a 8.5 cm length capillary at a separation voltage of 1000 V using a 50 mM NaOH running buffer (pH 12.7). Highly linear response is obtained for the above compounds over the range of 5.0 to 2.0 x 10(2) microg/mL with low detection limit, down to 0.8 microg/mL for glucose (S/N = 3). The injection-to-injection repeatability for analytes in peak current (RSD < 3.6%) and for migration times (RSD < 1.4%) was excellent. The new miniaturized CE-AD system should find a wide range of analytical applications involving assays of carbohydrates as an alternative to conventional CE and micro-CE.     

Quantitation and on-line concentration of enantiomers in open-tubular capillary electrochromatography.

The feasibility of open-tubular capillary electrochromatography (OTCEC) with UV detector for quantitation of enantiomers is explored, and a simple on-line sample concentration method to improve detection sensitivity of negatively charged enantiomers more than 1000-fold is described. With a capillary of 25 microm ID, the limits of detection (LODs) for absolute concentration and for enantiomeric ratio are 10(-6) M and 0.6-0.8% (signal-to-noise ratio S/N = 10). Good linearity and reproducibility are observed. The detection sensitivity is enhanced by combination with field-enhanced sample injection (FESI). A water plug is introduced hydrodynamically into the capillary inlet end and then the sample solution prepared with water is introduced with electrokinetic injection. With this concentration technique, the LOD for absolute concentration is reduced to a 10(-9) M level. On the other hand, due to the peak-sharpening effect of FESI, the LOD for enantiomeric ratio for the first-eluted enantiomer is significantly improved, being 0.3%. Effects of the injection conditions, such as length of water plug, buffer concentration, injection voltage, and injection time on the enrichment efficiency are investigated. Online concentration of a racemic compound with two chiral centers is demonstrated.     

Optimal configuration of capillary electrophoresis microchip with expansion chamber in separation channel

This study develops a novel capillary electrophoresis (CE) microfluidic device featuring a conventional cross-form injection system and an expansion chamber located at the inlet of the separation channel. The combined injection system/expansion chamber arrangement is designed to deliver a high-quality sample band into the separation channel such that the detection performance of the device is enhanced. Numerical simulations are performed to investigate the electrokinetic transport processes in the microfluidic device and to establish the optimal configuration of the expansion chamber. The results indicate that an expansion chamber with an expansion ratio of 2.5 and an expansion length of 500 microm delivers a sample plug with the correct shape and orientation. With this particular configuration, the peak intensities of the sample are sharp and clearly distinguishable in the detection region of the separation channel. Therefore, this configuration is well suited for capillary electrophoresis applications which require a highly sensitive resolution of the sample plug. The novel CE microfluidic device developed in this study has an exciting potential for use in high-performance, high-throughput chemical analysis applications and in many other applications throughout the field of micro-total-analysis-systems.     

Multimodal open-tubular capillary electrochromatographic analysis of amines and peptides

This study describes a comparison of different modes of open-tubular electrochromatography (OTCEC) in bare and etched capillaries. To carry out the investigation, the separation of impurities of two synthetic peptides and the separation of a mixture of five heterocyclic aromatic amines were studied. Three different types of stationary phase were evaluated: (i) fluorosurfactants (anionic and zwitterionic) adsorbed in the inner wall of the capillary (electrochromatography with dynamically modified stationary phases (DMS)CEC); (ii) physically adsorbed polymers (DMA-SO(3-) and DMA-N(+)(CH(3))(3)) and (iii) chemically modified capillaries (C(18), cholesteryl 10-undecanoate and diol). The results confirm that electrochromatography can be a viable alternative to capillary electrophoresis (CE) and liquid chromatography, more established separation techniques. It is possible to differentiate some minor species for the synthetic peptides that cannot be resolved by CE or high-performance liquid chromatography (HPLC). Moreover the separation of the amine mixture depends strongly on the stationary phase used.     

Separation of Trp-Arg and Arg-Trp using G-quartet-forming DNA oligonucleotides in open-tubular capillary electrochromatography

DNA oligonucleotides that form intramolecular G-quartet structures were investigated as stationary phase reagents for separation of mixtures of the isomeric dipeptides Trp-Arg and Arg-Trp in open-tubular capillary electrochromatography (OTCEC). The oligonucleotides included a thrombin-binding aptamer that forms a biplanar G-quartet structure and an oligonucleotide that forms a 4-plane G-quartet structure. Fluorescence, circular dichroism and UV-visible absorbance spectroscopies were used in batch solution studies to indicate interactions between the dipeptides and the biplanar G-quartet structure. Results for OTCEC separations were compared with results obtained for capillary zone electrophoresis separations on a bare capillary. Temperature studies suggest that resolution is improved when the G-quartet structure is partially destabilized, but control experiments in which potassium chloride was not included in the mobile phase indicate that the G-quartet structure nevertheless plays a role in the separations.     

Single bubble in-tube microextraction coupled with capillary electrophoresis

Headspace (HS) extraction is a sample pretreatment technique for volatile and semivolatile organic compounds in a complex matrix. Recently, in-tube microextraction (ITME) coupled with CE using an acceptor plug placed in the capillary inlet was developed as a simple but powerful HS extraction method. Here, we present single bubble (SB) ITME using a bubble hanging to the capillary inlet immersed in a sample donor solution as a HS of submicroliter volume (∼200 nL). The analytes evaporated to the bubble were extracted into the acceptor phase through the capillary opening, then electrophoresis of the enriched extract was carried out. Since the bubble volume was much smaller than a conventional HS volume (∼1 mL), it was filled with the evaporated analytes rapidly and the analytes could be enriched much faster compared to conventional HS-ITME. Owing to the high surface-to-volume ratio of the SB, 5 min SB-ITME yielded the enrichment factor values similar to those of 10 min HS-ITME. When 5 min SB-ITME at room temperature was applied to a tap water sample, the enrichment factors of 2,4,6-trichlorophenol (TCP), 2,3,6-TCP, and 2,6-dichlorophenol were 53, 41, and 60, respectively, and the LOQs obtained by monitoring the absorbance at 214 nm were 5.6–8.3 ppb, much lower than 200 ppb, the World Health Organization guideline for the maximum permissible concentration of 2,4,6-TCP in drinking water.     

Toward million-fold sensitivity enhancement by sweeping in capillary electrophoresis combined with thermal lens microscopic detection using an interface chip

This paper reports a thermal lens microscope (TLM) detection coupled with capillary electrophoresis (CE) by using an interface chip (IFChip) to achieve highly sensitive detection with high reproducibility. Fused silica capillaries with an inner diameter of 50 microm were directly connected to a microchannel on the IFChip. In comparison with an on-capillary detection method in CE-TLM, ca. 10-fold improvements in the reproducibility for peak height were obtained by using IFChips. The detection limit of an azo dye was estimated to be 3.6 x 10(-7)M (100 ppb), which was above 100-times lower than that of conventional absorbance detection. Toward further improvement of the detectability for nonfluorescent compounds, on-line sample preconcentration by sweeping was applied to the CE-TLM using the IFChip. Due to the sweeping effect, 3900000-fold increase in the sensitivity was successfully achieved.     

New technique for capillary electrophoresis directly coupled with end-column electrochemiluminescence detection

A new end-column electrochemiluminescence (ECL) detection technique coupling to capillary electrophoresis (CE) is characterized. A 300 microm diameter Pt working electrode was used to directly couple with a 75 microm inner diameter separation capillary without an electric field decoupler. The hydrodynamic cyclic voltammogram (CV) of Ru(bpy) 3 2+ showed that electrophoretic current did not affect the ECL reaction. The presence of high-voltage (HV) field only resulted in the shift of the ECL detection potential. The distance of capillary to electrode was an important parameter for optimizing detection performance as it determined the characteristics of mass transport toward the electrode and the actual concentration of Ru(bpy) 3 2+ in the detection region. The optimum distance of capillary to electrode was decided by the inner diameter of the capillary, too. For a 75 microm capillary, the working electrode should be placed away from the capillary outlet at a distance within the range of 220-260 microm. The effects of pH value of ECL solution and molecular structure of analytes on peak height and theoretical plate numbers were discussed. Using the 75 microm capillary, under the optimum conditions, the method provided a linear range for tripropylamine (TPA) between 1 x 10(-10) and 1 x 10(-5) mol/L with correlation coefficient of 0.998. The detection limit (signal-to-noise ratio S/N = 3) was 5.0 x 10(-11) mol/L. The relative standard deviation in peak height for eight consecutive injections was 5.6%. By this new technique lidocaine spiked in a urine sample was determined. The method exhibited the linear range for lidocaine from 5.0 x 10(-8) to 1.0 x 10(-5) mol/L with correlation efficient of 0.998. The limit of detection (S/N = 3) was 2.0 x 10(-8) mol/L.     

Graphene oxide and reduced graphene oxide as novel stationary phases via electrostatic assembly for open‐tubular capillary electrochromatography

We present here the application of graphene oxide (GO) and reduced graphene oxide (GOOH) sheet as novel stationary phases for open‐tubular CEC (OTCEC) separation based on electrostatic assembly. The inner walls of a bare capillary column was first modified by ionic assembly of poly (diallyldimethylammonium chloride) (PDDA), and then negatively charged GO or GOOH was easily assembled on a positively charged interior walls of the capillary by electrostatic force. Scanning Electron Microscope images showed that GO and GOOH can still maintain sheet‐layer‐like structure when coated onto the capillary via electrostatic assembly. The chromatographic properties of the GO and GOOH coated columns were evaluated via OTCEC separations of various kinds of analytes, including three acid nitrophenol isomers, three basic nitroaniline isomers, and four neutral PAHs. Efficient separations of all the analytes were achieved with optimized buffer pH and organic additive. The reproducibility and stability of the GO or GOOH coated columns were investigated. Our results indicate the capability of application GO or GOOH sheet in OTCEC separation, which can be coated on the inner wall of fused‐silica capillary via electrostatic assembly.     

Quantification and evaluation of Joule heating in on-chip capillary electrophoresis

We present the use of a novel, picoliter volume interferometer to measure, for the first time, the extent of Joule heating in chip-scale capillary electrophoresis (CE). The simple optical configuration for the on-chip interferometric backscatter detector (OCIBD) consists of an unfocused laser, an unaltered silica chip with a half-cylinder channel and a photodetector. Using OCIBD for millidegree-level noninvasive thermometry, temperature changes associated with Joule heating (2.81 degrees C above ambient) in on-chip CE have been observed in 90 microm wide and 40 microm deep separation channels. The temporal response of Joule heating in isotropically etched channels was exponential, with it taking an excess of 2.7 s to reach equilibrium. Buffer viscosity changes have also been derived from empirical on-chip thermometry data, allowing for the determination of diffusion coefficients for solutes when separated in heated buffers. In addition, OCIBD has allowed the reduction in separation efficiency to be estimated in the absence of laminar flow and due to increased molecular diffusion and lower buffer viscosity. A 7% reduction in separation efficiency was determined for a high current drawing buffer such as Tris-boric acid under an applied field of just 400 V/cm. Results indicate that heating effects in on-chip CE have been underestimated and there is a need to readdress the theoretical model.     

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.     

Efficiency studies in nonaqueous capillary electrophoresis

Nonaqueous capillary electrophoresis (NACE) is a relatively new area with several advantages that include enhanced efficiency and improved detection sensitivity. The goal of this study was to investigate the influence of NACE compared to aqueous CE on the separation efficiency of oligosaccharides. The applied voltage and buffer concentration were optimized for the aqueous and nonaqueous buffer media to minimize the band broadening effects of Joule heating and electrophoretic dispersion. At the optimized conditions a 1.5-fold enhancement in efficiency was obtained with the nonaqueous buffer medium.