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.     

Capillary electrophoresis absorption detection using fiber-loop ring-down spectroscopy

The application of phase-shift, fiber-loop, ring-down spectroscopy (PS-FLRDS) as an on-line detector for capillary electrophoresis (CE) of biomolecules is demonstrated. CE was conducted using a custom-designed capillary/fiber interface coupled to an absorption detector, which is based on the ring-down of an optical signal in a closed fiber waveguide loop. The ring-down times were obtained by measuring the phase difference between intensity modulated light entering and exiting the fiber loop. The incorporation of a microlens to enhance transmission through the sample gap led to an improvement of the sensitivity by up to 80% compared to the square-cut fiber and a reduction in the detection limit. The performance of the PS-FLRDS absorption technique as an online detector was characterized by flow injection through a capillary. Good repeatability and linear response were obtained, and the detection limit using the lensed fiber/capillary interface system was determined to be alpha(min) = 1.6 cm(-1) for an absorption path of approximately 30 microm. PS-FLRDS coupled to CE was also applied to the analysis of human serum albumin (HSA) by using a NIR dye as a noncovalent label. The excess free dye and the dye/protein complex were resolved. The labeling coefficient was determined to be approximately 6, and good repeatability of peak areas (RSD = 8.7%) was obtained for the analysis of HSA. Furthermore, an excellent linear response (R2 > 0.99) was obtained between the peak areas and concentrations of HSA. The detection limit of labeled HSA was determined to be 1.67 microM.     

Capillary electrophoresis to mass spectrometry interface using a porous junction

A new capillary electrophoresis interface to electrospray ionization mass spectrometry (CE/ESI-MS) is introduced in which the electrical connection to the CE capillary outlet/ESI electrode is achieved by transfer of small ions related to the background electrolyte (BGE) through a porous section near the CE capillary outlet. In this design, only a small section of the capillary wall is made porous. The porous section is created by first thinning a small section of the capillary wall by drilling a well into it and then etching the remaining thin wall porous. This design has two advantages over previous designs (in which the whole circumference of the capillary was made porous): first, the capillary interface is more robust because only a small section of it is made porous, and therefore, no liquid junction is needed to secure the porous section. The electrical connection is achieved simply by inserting the capillary outlet containing the porous junction into the existing ESI needle and filling the needle with the BGE. Second, the time required to make the fused silica porous is reduced from approximately 1 h to a few minutes. In addition, there is no dead volume associated with the porous design, and because the actual metal/liquid contact occurs outside of the CE capillary, bubble formation due to redox reactions of water at the electrode does not affect CE/ESI-MS performance. The performance of this interface is demonstrated by the analyses of peptide and protein mixtures.     

Capillary electrophoresis coupled on-line with ultraviolet resonance Raman spectroscopy

Capillary electrophoresis (CE) and resonance Raman spectroscopy (RRS) with excitation in the deep ultraviolet (UV) region (lambda(ex): 244 or 257 nm) were coupled on-line. The potential of this hyphenated technique, denoted as CE-UV-RRS, for analyte confirmation/identification purposes was explored with aromatic sulfonic acids and nucleotides as test compounds. Good-quality UV-RRS spectra could be recorded on-the-fly. Identification limits for the nucleotides were in the 10-125 microg/mL range. The RRS spectra showed sufficient characteristic features to enable analyte confirmation. In addition, the identification power of UV-RRS was studied with substituted pyrenes as model compounds. The compounds were distinguishable on the basis of their RRS spectra at 244 nm.     

Capillary electrophoresis of amino sugars with laser-induced fluorescence detection

3-(4-Carboxybenzoyl)-2-quinolinecarboxaldehyde has been utilized as a precolumn derivatization agent for various amino sugars. Constituents of various biological mixtures can be converted to highly fluorescent isoindole derivatives, separated by high-performance capillary electrophoresis and determined at attomole (10(-18) mol) levels by a laser-induced fluorescence detector. This method has been applied to the analysis of monosaccharides and acid-hydrolyzed polysaccharides. Carbohydrate moieties derived from a glycoprotein were also tagged and determined.     

Capillary electrophoresis chips with a sheath-flow supported electrochemical detection system

Microfabricated capillary electrophoresis chips containing an integrated sheath-flow electrochemical detector are developed with the goal of minimizing the influence of separation voltages on end-column detection while maintaining optimum performance. The microdevice consists of an upper glass wafer carrying the etched separation, injection, and sheath-flow channels and a lower glass wafer on which gold- and silver-plated electrodes have been fabricated. The sheath-flow channels join the end of the separation channel from each side, and gravity-driven flow carries the analytes to the electrochemical detector placed at working distances of 100, 150, 200, and 250 microm from the separation channel exit. The performance of this detector is evaluated using catechol and a detection limit of 4.1 microM obtained at a working distance of 250 microm. Detection of DNA restriction fragments and PCR product sizing is demonstrated using the electroactive intercalating dye, iron phenanthroline. Additionally, an allele-specific, PCR-based single-nucleotide polymorphism typing assay for the C282Y substitution diagnostic for hereditary hemochromatosis is developed and evaluated using ferrocene-labeled primers. This study advances the feasibility of high-speed, high-throughput chemical and genetic analysis using microchip electrochemical detection.     

Microchip capillary electrophoresis with solid-state electrochemiluminescence detector

We report microchip capillary electrophoresis (CE) coupling to a solid-state electrochemiluminescence (ECL) detector. The solid-state ECL detector was fabricated by immobilizing tris(2,2'-bipyridyl)ruthenium(II) (TBR) into an Eastman AQ55D-silica-carbon nanotube composite thin film on an indium tin oxide (ITO) electrode. After being made by a photolithographic method, the surface of the ITO electrode was coated with a thin composite film through a micromolding in capillary (MIMIC) technique using a poly(dimethylsiloxane) (PDMS) microchannel with the same pattern as an ITO electrode. Then the TBR was immobilized via ion exchange by immersing the ITO electrode containing the thin film in TBR aqueous solution. The whole system was built by reversibly sealing the TBR-modified ITO electrode plate with a PDMS layer containing electrophoresis microchannels. The results indicated that the present solid-state ECL detector displayed good durability and stability in the microchip CE-ECL system. Proline was selected to perform the microchip device with a limit of detection of 2 microM (S/N=3) and a linear range from 25 to 1000 microM. Compared with the CE-ECL of TBR in aqueous solution, while the CE microchip with solid-state ECL detector system gave the same sensitivity of analysis, a much lower TBR consumption and a high integration of the whole system were obtained. The present system was also used for medicine analysis.     

Capillary electrophoresis with electrochemical detection for chiral separation of optical isomers

The enantiomers of two amine derivatives were directly separated by capillary electrophoresis (CE), employing β-cyclodxtrin (β-CD) as a chiral additive in strongly alkaline solutions. The analytes were detected by electrochemistry, using a copper disk electrode at +675 mV vs Ag/AgCl reference electrode. Both the free enantiomers and the enantiomer-cyclodxtrin inclusion complexes could be detected using this approach, although the complexed forms gave lower oxidation currents than the free forms. Factors affecting the chiral CE separation of the analytes, such as working potential, concentration of running buffer and β-CD, and applied voltage, were extensively investigated. Under the optimum conditions, baseline separation of the enantiomers could be accomplished in less than 18 min. In addition, a successful application of the method to the enantiomeric purity determination confirmed its validity and practicability.     

Chemical oxidation of p-hydroxyphenylpyruvic acid in aqueous solution by capillary electrophoresis with an electrochemiluminescence detection system

A system of capillary electrophoresis with electrochemiluminescence detection (CE-ECL) together with UV spectroscopic and electrochemical methods were used to study the chemical oxidation of p-hydroxyphenylpyruvic acid (pHPP) by dissolved oxygen in aqueous solution. The pHPP was observed to be readily oxidized by dissolved oxygen in alkaline solution and yielded a compound that strongly enhanced the electrochemiluminescence of Ru(bpy)23+. This compound was separated and detected by a new CE-ECL system and revealed to be oxalate by being compared with an authentic sample of oxalate. The chemical oxidation mechanism of pHPP by dissolved oxygen was discussed in this paper.     

Portable microcoil NMR detection coupled to capillary electrophoresis

High-efficiency separation techniques, such as capillary electrophoresis (CE), coupled to a nondestructive nuclear magnetic resonance (NMR) spectrometer offer the ability to separate, chemically identify, and provide structural information on analytes in small sample volumes. Previous CE-NMR coupled systems utilized laboratory-scale NMR magnets and spectrometers, which require very long separation capillaries. New technological developments in electronics have reduced the size of the NMR system, and small 1-2 T permanent magnets provide the possibilities of a truly portable NMR. The microcoils used in portable and laboratory-scale NMR may offer the advantage of improved mass sensitivity because the limit of detection (LOD) is proportional to the coil diameter. In this work, CE is coupled with a portable, briefcase-sized NMR system that incorporates a microcoil probe and a 1.8 T permanent magnet to measure (19)F NMR spectra. Separations of fluorinated molecules are demonstrated with stopped- and continuous-flow NMR detection. The results demonstrate that coupling CE to a portable NMR instrument is feasible and can provide a low-cost method to obtain structural information on microliter samples. An LOD of 31.8 nmol for perfluorotributylamine with a resolution of 4 ppm has been achieved with this system.     

Fabrication of a poly(dimethylsiloxane)-based electrochemiluminescence detection cell for capillary electrophoresis

An easy but effective technique is described here for quick fabrication of low-cost electrochemiluminescence detection cells for capillary electrophoresis. The technique is based on molding of poly(dimethylsiloxane) (PDMS) with a capillary column inserted into a pipet tip. Two access holes are left in the PDMS slab; they provide neat accommodations for the separation capillary and the working electrode made with the same type of tip. Since the access holes are well-aligned, the electrode and the capillary are automatically aligned; thus, end-column detection is easily obtained. Fabrication of the detection cell is straightforward; no micromechanical operation is included. Also the principle for the procedure makes it possible to efficiently batch production detection cells with good reproducibility. Because of the end-column scheme, the cell can be adopted for electrophoresis with electrochemical detection as well.     

Capillary electrophoresis with lamp-based wavelength-resolved fluorescence detection for the probing of protein conformational changes

Native protein fluorescence spectra encompass information on protein conformation. In this study, capillary electrophoresis (CE) combined with lamp-based wavelength-resolved fluorescence detection (wrFlu) is presented as a novel tool for the analysis of protein mixtures and the monitoring of protein unfolding. The CE-wrFlu system provides three-dimensional data (time, emission wavelength, intensity) from which electropherograms and accurate emission spectra of separated proteins can be extracted. For model proteins, linear detector responses (peak height vs concentration) were obtained (R(2) > 0.96) with detection limits (LODs) in the 6-32 nM range. The minimum protein concentration required for precise determination of the maximum emission wavelength by CE-wrFlu was about 15 times the LOD. Unfolding of various model proteins was induced by protein incubation and analysis in background electrolyte (BGE) containing 7.0 M urea. CE-wrFlu of the unfolded species revealed peaks with clear red-shifted spectra, which adequately corresponded to reference spectra obtained on a standard spectrophotometer. Moreover, unfolded proteins showed a significant decrease in effective electrophoretic mobility (after correction for BGE viscosity) due to the increase of their molecular hydrodynamic radii. It is concluded that the CE-wrFlu system provides two independent indicators for changes in protein folding and will allow the simultaneous assessment of protein purity and conformation.     

Capillary electrophoresis microchips for separation and detection of organophosphate nerve agents

A miniaturized analytical system for separating and detecting toxic organophosphate nerve agent compounds, based on the coupling of a micromachined capillary electrophoresis chip with a thick-film amperometric detector, is described. Factors influencing the on-chip separation and detection processes have been optimized. Using a MES buffer (20 mM, pH 5.0) running buffer, a 72-mm-long separation channel, and a separation voltage of 2000 V, baseline resolution is observed for paraoxon, methyl parathion, fenitrothion, and ethyl parathion in 140 s. Such miniaturization and speed advantages are coupled to submicromolar detection limits and good precision. Applicability to spiked river water samples is demonstrated, and the implications for on-site environmental monitoring and rapid security screening/warning are discussed.     

Chlorinated phenol analysis using off-line solid-phase extraction and capillary electrophoresis coupled with amperometric detection and a boron-doped diamond microelectrode

The analysis of chlorinated phenols (2-chlorophenol, 3-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol, 2,4,6-trichlorophenol, pentachlorophenol) in river water was accomplished using off-line solid-phase extraction (SPE) and capillary electrophoresis coupled with electrochemical detection. A key to the sensitive, reproducible, and stable detection of these pollutants was the use of a boron-doped diamond microelectrode in the amperometric detection mode. An off-line SPE procedure was utilized to extract and preconcentrate the pollutants prior to separation and detection, with ENVI-Chrom P, a highly cross-linked styrene-divinylbenzene copolymer, being employed as the sorbent. Pollutant recoveries in the 95-100% range with relative standard deviations of 1-4% were achieved. The diamond microelectrode provided a low and stable background current with low peak-to-peak noise. The oxidative detection of the pollutants was accomplished at +1.05 V vs Ag/AgCl without the need for electrode pretreatment. The method was evaluated in terms of the linear dynamic range, sensitivity, limit of quantitation, response precision, and response stability. A reproducible electrode response was observed during multiple injections of the chlorinated phenol solutions with a relative standard deviation of < or =5.4%. Good electrode response stability was observed over many days of continuous use with no significant electrode deactivation or fouling. The separation efficiencies for all six pollutants were greater than 170,000 plates/m. The minimum concentration detectable for all six ranged from 0.02 to 0.2 ppb (S/N > or = 3) using a 250:1 preconcentration factor.     

High-voltage capacitively coupled contactless conductivity detection for microchip capillary electrophoresis

Contactless conductivity detection was carried out on a planar electrophoresis device by capacitive coupling using an ac excitation voltage of 500 V(p-p) and a frequency of 100 kHz. It was possible to carry out detection in this way through a cover plate of 1 mm thickness. Better sensitivity is obtained, however, by placing the electrodes into troughs that allow tighter coupling to the separation channel. The 3 x S/N detection limits are 0.49, 0.41, and 0.35 microM for the small inorganic ions K+, Na+, and Mg2+. The detection of heavy metals is demonstrated with the example of Mn2+, Zn2+, and Cr3+ with detection limits of 2.1, 2.8, and 6.8 microM, respectively. The universal nature of the method is further illustrated by the detection of citric and lactic acids, which are of interest in food and beverage analysis, and detection of three antiinflammatory nonsteroid drugs, 4-acetamidophenol, ibuprofen, and salicylic acid, as examples of species of pharmaceutical interest.     

Capillary electrophoresis with in-capillary solid-phase extraction sample cleanup

An in-capillary, solid-phase extraction (SPE)-capillary electrophoresis (CE) method, with not only high preconcentration factor but also no adsorption on the inner capillary wall of absorbing species in real complex samples, has been developed with a hole-opened capillary. The SPE sorbents approximately 3 mm in length was packed in the inlet end of the capillary. A hole approximately 30 microm in diameter was opened after the sorbents on the capillary. Sample solutions were loaded from the inlet end, and the sample wastes flowed out from the hole. After a certain time of the sample loading, a 1.5-mm-long methanol plug was introduced from the inlet end and forced to pass by the sorbents and the hole. Then, a separation voltage was applied between the hole and the outlet end of the capillary to carry out normal CE. When the sample loading time was 120 min, CE peak heights of the 2,4-dichlorophenol and 2,4,5-trichlorophenol were proportional to their concentration in a range of 0.08-5 ng/mL, and their detection limits were 25 and 17 pg/mL, respectively. A 16,000-fold sensitivity enhancement was obtained for CE of the chlorophenols with only a little decrease in CE separation efficiency. It was also demonstrated with the mixture of the chlorophenols and a surfactant cetyltrimethylammonium bromide that the present method could eliminate the adsorption problem of absorbing species on the inner wall during sample loading. Furthermore, the SPE-CE was directly applied to determination of chlorophenols on the level of 0.02 ppb in downstream water of a river, and the results agreed well with those obtained with off-line SPE-HPLC experiments.     

Microchip capillary electrophoresis with an integrated indium tin oxide electrode-based electrochemiluminescence detector

This paper describes an indium tin oxide (ITO) electrode-based Ru(bpy)3(2+) electrochemiluminecence (ECL) detector for a microchip capillary electrophoresis (CE). The microchip CE-ECL system described in this article consists of a poly(dimethylsiloxane) (PDMS) layer containing separation and injection channels and an electrode plate with an ITO electrode fabricated by a photolithographic method. The PDMS layer was reversibly bound to the ITO electrode plate, which greatly simplified the alignment of the separation channel with the working electrode and enhanced the photon-capturing efficiency. In our study, the high separation electric field had no significant influence on the ECL detector, and decouplers for isolating the separation electric field were not needed in the microchip CE-ECL system. The ITO electrodes employed in the experiments displayed good durability and stability in the analytical procedures. Proline was selected to perform the microchip device with a limit of detection of 1.2 microM (S/N = 3) and a linear range from 5 to 600 microM.