Recent advances in enrichment techniques for trace analysis in capillary electrophoresis

CE is gaining great popularity as a well‐established separation technique for many fields such as pharmaceutical research, clinical application, environmental monitoring, and food analysis, owing to its high resolving power, rapidity, and small amount of samples and reagents required. However, the sensitivity in CE analysis is still considered as being inferior to that in HPLC analysis. Diverse enrichment methods and techniques have been increasingly developed for overcoming this issue. In this review, we summarize the recent advances in enrichment techniques containing off‐line preconcentration (sample preparation) and on‐line concentration (sample stacking) to enhancing sensitivity in CE for trace analysis over the last 5 years. Some relatively new cleanup and preconcentration methods involving the use of dispersive liquid–liquid microextraction, supercritical fluid extraction, matrix solid‐phase dispersion, etc., and the continued use and improvement of conventional SPE, have been comprehensively reviewed and proved effective preconcentration alternatives for liquid, semisolid, and solid samples. As for CE on‐line stacking, we give an overview of field amplication, sweeping, pH regulation, and transient isotachophoresis, and the coupling of multiple modes. Moreover, some limitations and comparisons related to such methods/techniques are also discussed. Finally, the combined use of various enrichment techniques and some significant attempts are proposed to further promote analytical merits in CE.     

On-line preconcentration strategies for trace analysis of metabolites by capillary electrophoresis

Analysis of low concentrations of metabolites is required for new fields of biological research, such as metabolomics. In this review, recent work in our laboratory aimed at developing improved strategies for on-line sample preconcentration of metabolites by capillary electrophoresis (CE) is presented. Dynamic pH junction, sweeping and dynamic pH junction-sweeping represent three complementary methods for electrokinetic focusing of large volumes of sample directly on-capillary. Focusing selectivity and focusing efficiency are two factors that can be used to assess the suitability of each method for different classes of metabolites. Buffer properties can be selected to enhance the focusing of specific types of metabolites based on knowledge of the analyte physicochemical properties. The application of on-line preconcentration CE for trace analysis of metabolites in real samples of interest, such as biological fluids and cellular extracts, is also demonstrated. Under optimum conditions, up to three orders of magnitude increase in concentration sensitivity can be realized for several classes of metabolites, including catecholamines, purines, nucleosides, nucleotides, amino acids, steroids and coenzymes. Recent work on hyphenating on-line preconcentration with multiplexed CE is highlighted as a promising platform for sensitive and high-throughput analyses of metabolites.     

Novel cationic polyelectrolyte coatings for capillary electrophoresis

The use of bare fused silica capillary in CE can sometimes be inconvenient due to undesirable effects including adsorption of sample or instability of the EOF. This can often be avoided by coating the inner surface of the capillary. In this work, we present and characterize two novel polyelectrolyte coatings (PECs) poly(2‐(methacryloyloxy)ethyl trimethylammonium iodide) (PMOTAI) and poly(3‐methyl‐1‐(4‐vinylbenzyl)‐imidazolium chloride) (PIL‐1) for CE. The coated capillaries were studied using a series of aqueous buffers of varying pH, ionic strength, and composition. Our results show that the investigated polyelectrolytes are usable as semi‐permanent (physically adsorbed) coatings with at least five runs stability before a short coating regeneration is necessary. Both PECs showed a considerably decreased stability at pH 11.0. The EOF was higher using Good's buffers than with sodium phosphate buffer at the same pH and ionic strength. The thickness of the PEC layers studied by quartz crystal microbalance was 0.83 and 0.52 nm for PMOTAI and PIL‐1, respectively. The hydrophobicity of the PEC layers was determined by analysis of a homologous series of alkyl benzoates and expressed as the distribution constants. Our result demonstrates that both PECs had comparable hydrophobicity, which enabled separation of compounds with log P_o/w > 2. The ability to separate cationic drugs was shown with β‐blockers, compounds often misused in doping. Both coatings were also able to separate hydrolysis products of the ionic liquid 1,5‐diazabicyclo[4.3.0]non‐5‐ene acetate at highly acidic conditions, where bare fused silica capillaries failed to accomplish the separation.     

Determination of trace metals by capillary electrophoresis

A new analytical procedure is developed using a strong complexing agent, 1,10-phenanthroline (Phen), for direct UV detection of Zn, Mn, Cu, Co, Cd, and Fe at microg/L concentrations in environmental water samples. The metal chelates formed showed different electrophoretic mobilities and solved the comigration problem for capillary electrophoresis (CE) separation of free metal ions. To obtain stable metal-Phen chelates during the capillary zone electrophoresis (CZE) run, both pre-column and on-column complexation are required and threefold excess of Phen over metal ions should be added to the sample. The optimized background electrolyte (BGE) consists of 30 mM hydroxylamine hydrochloride and 0.1% methanol at pH 3.6. Under hydrodynamic sampling, CE run at + 20 kV in 65 cm x 0.05 mm ID fused-silica column with detection at 265 nm, baseline separation, satisfactory working ranges (10 microg/L to 5.5 mg/L), sensitive detection limits (1-3 microg/L), good repeatability for migration times (relative standard deviation, RSD 0.36-0.81%, n = 5), peak area (RSD 3.2-4.2%, n = 5) and peak height (RSD 3.2-4.5%, n = 5) were obtained for the metal cations investigated. The reliability of the method was established by parallel determination using the inductively coupled plasma-atomic emission spectrometry (ICP-AES) method giving results within statistical variation. The procedure developed is shown to provide a quick, sensitive, precise, and economic method for simultaneous determination of metal cations that can form stable chelates with Phen.     

Combination of liquid-phase microextraction and on-column stacking for trace analysis of amino alcohols by capillary electrophoresis

We described a new method for the enrichment of basic drugs present in water samples via liquid-phase microextraction (LPME) combined with on-column stacking in capillary electrophoresis. Two steps were employed to enhance the detection sensitivity of four amino alcohols. The analytes were first extracted from aqueous sample (donor solution) that were adjusted to basic through a thin layer of 1-octanol entrapped within the pores of a polypropylene hollow fiber, and then into a 5-microl acidic acceptor solution inside the hollow fiber. The extract was then further enriched through on-column stacking in capillary electrophoresis. With this two-step enrichment procedure, the method provided 72-110-fold preconcentration of the target amino alcohols. The limits of detection were 0.08-0.5 microg/ml. Relative standard deviation (n=6) ranged between 4.3 and 6.9% for the studied drugs utilizing 2-amino-1-phenylethanol as internal standard. The extraction of amino alcohols in spiked urine samples was evaluated using the developed procedure.     

Novel approach to the analysis and use of fullerenes in capillary electrophoresis

The analysis and use of fullerenes in capillary electrophoresis (CE) was investigated. Sodium dodecyl sulfate (SDS) was used to solubilize fullerenes C60, C70, and a mixture of C60 and C70 in water. The behavior of the solutions of the C60- and C70-SDS complexes was examined by CE with on-line UV-Vis diode array detection. This study included the use of a C60-SDS complex as a new method of micellar electrokinetic chromatography (MEKC) for the separation of polycyclic aromatic hydrocarbons (PAHs) using CE with uniwavelength detection. Since SDS micelles act as a pseudostationary phase in which the PAH compounds partition with their hydrophobic interior, the addition of C60 within the micelles enhanced separation of the PAHs. The preliminary results using C60-MEKC with SDS were compared to those obtained with MEKC with SDS. The capillary electrophoretic separations were performed in 10 mM borate-phosphate buffer with 100 mM SDS at pH 9.5.     

Novel covalently coated diazoresin/polyvinyl alcohol capillary column for the analysis of proteins by capillary electrophoresis

A novel method for the preparation of covalently linked capillary coatings of PVA was demonstrated using photosensitive diazoresin (DR) as coupling agents. Layer‐by‐layer self‐assembly film of DR and PVA based on hydrogen bonding was first fabricated on the inner wall of capillary, then the hydrogen bonding was converted into covalent bonding after treatment with UV light through the unique photochemistry reaction of DR. The covalently bonded coatings suppressed basic protein adsorption on the inner surface of capillary, and thus a baseline separation of lysozyme, cytochrome c and BSA was achieved using CE. Compared with bare capillary or noncovalently bonded DR/PVA coatings, the covalently linked DR/PVA capillary coatings not only improved the CE separation performance for proteins, but also exhibited good stability and repeatability. Due to the replacement of highly toxic and moisture‐sensitive silane coupling agent by DR in the covalent coating preparation, this method may provide a green and easy way to make the covalently coated capillaries for CE.     

Novel wavelength-resolved fluorescence detection for a high-throughput capillary electrophoresis system under a diascopic configuration

This paper presents a novel method regarding a wavelength-resolved fluorescence detection scheme for high-throughput analysis of bio-samples in a micro-CE chip. Instead of using the conventional laser-induced fluorescence (LIF) microscope equipped with delicate spatial filters and complex control systems, this study adopts a hollow cone illumination generated using a dark-field condenser for exciting fluorescence in the microchannel and an ultraviolet-visible-near-infrared (UV-Vis-NIR) spectrometer for detecting the emission signals. Experimental results show that the proposed system is feasible for simultaneously detecting a mixed sample composed of Atto 610, Rhodamine B and fluorescein isothiocyanate (FITC) fluorescent dyes in a single test run. Furthermore, a mixed bio-sample composed of two mixed 16-mer single-stranded DNAs labeled with Cy3 and FITC fluorescent dyes is also successfully detected with the proposed system. The measured limit of detection (LOD) for detecting FITC of the proposed system can be as low as 5.4x10(-6)M (S/N=3). This proposed detection method has shown its potential on RNA identification and DNA sequencing applications.     

Neurotransmitter sampling and storage for capillary electrophoresis analysis

Quantitative analysis of signaling molecules from single cells and cellular materials requires careful validation of the analytical methods. Strategies have been investigated that enable single neurons and neuronal tissues to be stored before being assayed for many low-weight, biologically active molecules, such as serotonin, dopamine, and citrulline. Both metacerebral cell and pedal ganglia homogenates isolated from Pleiuohbrain-Chae californica have been studied by capillary electrophoresis with two complimentary laser-induced fluorescence detection methods. For homogenized ganglia samples, several cellular analytes (such as arginine and citrulline) are unaffected by standing at room temperature for days. Many other analytes in the biological matrix, including the catecholamines and indolamines, degrade by 20% within 10 h at room temperature. Rapidly freezing samples or preserving them with ascorbic acid preserves more than 80% of the dopamine and about 70% of the serotonin even after five days. In addition, serotonin and dopamine remain completely stable for at least five days by combining the ascorbic acid preservation and freezing at -20 degrees C. The timing of preservation is critical in maintaining the original composition of the biological samples. Using our optimum storage protocol of freezing the sample within 2 h after isolation, we can store frozen homogenate ganglia samples for more than four weeks before assay while still obtaining losses less than 10% of the original serotonin and dopamine. The nanoliter-volume single cell samples, however, must be analyzed within 4 h to obtain losses of less than 10% for serotonin related metabolites.     

Electrochemiluminescence detection system for microchip capillary electrophoresis and its application to pharmaceutical analysis

We describe an efficient and easily fabricated electrochemiluminescence detection system for microchip capillary electrophoresis. A 300-μm-diameter platinum disc working electrode was embedded in a titanium tube which provides an adequate holding for working electrode and acts as counter electrode. We also have designed a simplified detection cell with a guide channel for the electrode. The integrated working-counter electrode can be easily aligned to the outlet of the separation channel through the guide channel. The functionality of the system was demonstrated by separation and detection of proline and tripropylamine. The response to proline is linear in the range from 5 μM to 5,000 μM, and the detection limit is 1.0 μM (S/N?=?3). The system was further applied to the determination of chlorpromazine hydrochloride in pharmaceutical formulations. The system is believed to have potential applications in pharmaceutical analysis.

Neurotransmitter sampling and storage for capillary electrophoresis analysis

Quantitative analysis of signaling molecules from single cells and cellular materials requires careful validation of the analytical methods. Strategies have been investigated that enable single neurons and neuronal tissues to be stored before being assayed for many low-weight, biologically active molecules, such as serotonin, dopamine, and citrulline. Both metacerebral cell and pedal ganglia homogenates isolated from Pleurobranchaea californica have been studied by capillary electrophoresis with two complimentary laser-induced fluorescence detection methods. For homogenized ganglia samples, several cellular analytes (such as arginine and citrulline) are unaffected by standing at room temperature for days. Many other analytes in the biological matrix, including the catecholamines and indolamines, degrade by 20% within 10 h at room temperature. Rapidly freezing samples or preserving them with ascorbic acid preserves more than 80% of the dopamine and about 70% of the serotonin even after five days. In addition, serotonin and dopamine remain completely stable for at least five days by combining the ascorbic acid preservation and freezing at –20 °C. The timing of preservation is critical in maintaining the original composition of the biological samples. Using our optimum storage protocol of freezing the sample within 2 h after isolation, we can store frozen homogenate ganglia samples for more than four weeks before assay while still obtaining losses less than 10% of the original serotonin and dopamine. The nanoliter-volume single cell samples, however, must be analyzed within 4 h to obtain losses of less than 10% for serotonin related metabolites. Received: 23 August 2000 / Revised: 2 November 2000 / Accepted: 7 November 2000     

Automated two-dimensional separation flow system with electrochemical preconcentration, stripping, capillary electrophoresis and contactless conductivity detection for trace metal ion analysis

This paper describes the automation of a fully electrochemical system for preconcentration, cleanup, separation and detection, comprising the hyphenation of a thin layer electrochemical flow cell with CE coupled with contactless conductivity detection (CE-C?D). Traces of heavy metal ions were extracted from the pulsed-flowing sample and accumulated on a glassy carbon working electrode by electroreduction for some minutes. Anodic stripping of the accumulated metals was synchronized with hydrodynamic injection into the capillary. The effect of the angle of the slant polished tip of the CE capillary and its orientation against the working electrode in the electrochemical preconcentration (EPC) flow cell and of the accumulation time were studied, aiming at maximum CE-C?D signal enhancement. After 6 min of EPC, enhancement factors close to 50 times were obtained for thallium, lead, cadmium and copper ions, and about 16 for zinc ions. Limits of detection below 25 nmol/L were estimated for all target analytes but zinc. A second separation dimension was added to the CE separation capabilities by staircase scanning of the potentiostatic deposition and/or stripping potentials of metal ions, as implemented with the EPC-CE-C?D flow system. A matrix exchange between the deposition and stripping steps, highly valuable for sample cleanup, can be straightforwardly programmed with the multi-pumping flow management system. The automated simultaneous determination of the traces of five accumulable heavy metals together with four non-accumulated alkaline and alkaline earth metals in a single run was demonstrated, to highlight the potentiality of the system.     

Integrated circuit microchip system with multiplex capillary electrophoresis module for DNA analysis

In this paper, we describe the use of an integrated circuit (IC) microchip system as a detector in multiplex capillary electrophoresis (CE). This combination of multiplex capillary gel electrophoresis and the IC microchip technology represents a novel approach to DNA analysis on the microchip platform. Separation of DNA ladders using a multiplex CE microsystem of four capillaries was monitored simultaneously using the IC microchip system. The IC microchip-CE system has advantages such as low cost, rapid analysis, compactness, and multiplex capability, and has great potential as an alternative system to conventional capillary array gel electrophoresis systems based on charge-coupled device (CCD) detection.     

Applications of capillary electrophoresis for industrial analysis

Capillary electrophoresis techniques, particularly isotachophoresis and zone electrophoresis, are useful for the determination of various analytes in a wide range of sample matrices. This paper reports applications of capillary electrophoresis developed for detergent, food additive, herbicide, animal nutrition and biotechnology samples. Major advantages of capillary electrophoresis include versatility and speed of method development. Options for small ion analysis using modern, fused silica capillary instruments are discussed.     

DNA fragment analysis by an affordable multiple-channel capillary electrophoresis system

We are demonstrating a cost-effective multichannel capillary electrophoresis system for a high-efficiency double-stranded DNA (dsDNA) fragments analysis. This bench-type high-performance DNA analysis (HDA) system uses fluorescence-type detection with inexpensive solid-state light sources and nonmoving integrated emission collection micro-optics. DNA samples are analyzed simultaneously by using a multiple usage and disposable multicapillary cartridge, which contains integrated capillary channels, optical fibers and an integrated sieving gel reservoir. Using commercially available dsDNA size markers as indicators, the HDA system provides high resolving power in 7 min separations. The system can hold a total of 192 samples in two 96-well polymerase chain reaction (PCR) plates, which can be automatically analyzed within 2.5 h. This affordable system can be used in laboratories to replace slab gel electrophoresis for routine and high-throughput dsDNA analysis.     

Pesticide analysis by capillary electrophoresis

In this work, a critical and updated revision of the current situation of the analysis of pesticides by Capillary Electrophoresis (CE) is presented. The review has been written in two main sections. The first one presents a thorough revision of the various offline and on-line sample preconcentration procedures that have been used in conjunction with CE to analyze these compounds. The second part reviews the various detection strategies (i.e., UV, LIF, MS, and electrochemical) and CE modes that have been applied to the analysis of pesticides. Future trends that can be expected from this hot research area are also discussed.     

Performance of metal complex substituted polysiloxanes in capillary electrophoresis and capillary electrochromatography

Two novel polysiloxanes containing the metal complex, Co(TACN)(3+)2 (TACN= 1,4,7-triazacyclononane) were used as coatings for capillary electrophoresis (CE) and capillary electrochromatography (CEC). Through crosslinking and covalent bonding, the positively charged polymers were bonded to silica supports. In both CE and CEC, these coatings exhibited strong, pH-independent, and anodic electroosmotic flow (EOF), and had excellent long-term stability. Successful separations of aromatic acids were achieved in CE. In CEC, separation of alkylbenzenes (7 min) and basic compounds (20 min) was achieved with higher resolving power than conventional octadecyl silica packings. These polymers represent a new class of coatings for CE and CEC that generate pH-independent EOF.     

Investigation of preconcentration strategies for the trace analysis of multi-residue pesticides in real samples by capillary electrophoresis

In this work, on-line preconcentration strategies were investigated for the multi-residue analysis of pesticides in drinking water and vegetables using micellar electrokinetic chromatography. Among the on-line strategies, sweeping and stacking with reverse migration of micelles (SRMM), with and without the insertion of a plug of water before sample injection, were contrasted. A new version of SRMM was also introduced. The modification consisted of momentarily applying a positive voltage at the inlet vial right after sample has been injected, increasing the efficiency by which the analytes are captured. Nine pesticides from different classes, carbendazim (benzimidazole), simazine, atrazine, propazine and ametryn (triazine), diuron and linuron (urea), carbaryl and propoxur (carbamate), were baseline separated in less than 6 min with a electrolyte composed of 20 mmol l(-1) phosphate buffer at pH 2.5, containing 25 mmol l(-1) sodium dodecyl sulfate and 10% methanol. Limits of detection (LODs) in the order of 2-46 microg l(-1) for the pesticides under investigation were obtained solely using the on-line strategies. Enrichment factors of 3-18-fold were obtained. These factors were computed as the improvement of the concentration LODs with respect to the reference condition (injection of 10 s at 2.5 kPa pressure). The proposed methodologies were applied to the analysis of pesticides in complex matrices such as carrot extracts where the detection of 2.5 microg l(-1) was illustrated. By combining off-line solid-phase extraction and the proposed on-line strategies, the detection of pesticides in drinking water at the 0.1 microg l(-1) level was conceived.     

Automated sampling system for the analysis of amino acids using microfluidic capillary electrophoresis

An improved automated continuous sample introduction system for microfluidic capillary electrophoresis (CE) is described. A sample plate was designed into gear-shaped and was fixed onto the shaft of a step motor. Twenty slotted reservoirs for containing samples and working electrolytes were fabricated on the “gear tooth” of the plate. A single 7.5-cm long Teflon AF-coated silica capillary serves as separation channel, sampling probe, as well as liquid-core waveguide (LCW) for light transmission. Platinum layer deposited on the capillary tip serves as the electrode. Automated continuous sample introduction was achieved by scanning the capillary tip through the slots of reservoirs. The sample was introduced into capillary and separated immediately in the capillary with only about 2-nL gross sample consumption. The laser-induced fluorescence (LIF) method with LCW technique was used for detecting fluorescein isothiocyanate (FITC)-labeled amino acids. With electric-field strength of 320 V/cm for injection and separation, and 1.0-s sample injection time, a mixture of FITC-labeled arginine and leucine was separated with a throughput of 60/h and a carryover of 2.7%.