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A gradient system was developed for the separation of proteins on a femtoliter scale utilizing nanofluidic channels. In the history of chromatography, miniaturization of the separation column has been important for efficient separation and downsizing of instruments. Previously, our group developed a small and highly efficient chromatography system utilizing nanofluidic channels, although a flexible design of the gradient was difficult and separation of proteins was not achieved. Here, we propose a flexible gradient system using standard HPLC pumps and an auxiliary mixer with a simple sample injection system. In contrast to our previous sample injection system using pressure balance, the system enables a femtoliter-scale sample injection which is compatible with gradient elution using HPLC pumps. The system was carefully designed, verified for sample injection and gradient elution, and finally applied to the separation of proteins from model and real samples. This femtoliter-scale, efficient separation system will contribute to omics studies at the single-cell level.

Micellar electrokinetic chromatography (MEKC), a separation mode of capillary electrophoresis (CE), has enabled the separation of electrically neutral analytes. MEKC can be performed by adding an ionic micelle to the running solution of CE without modifying the instrument. Its separation principle is based on the differential migration of the ionic micelles and the bulk running buffer under electrophoresis conditions and on the interaction between the analyte and the micelle. Hence, MEKC's separation principle is similar to that of chromatography. MEKC is a useful technique particularly for the separation of small molecules, both neutral and charged, and yields high-efficiency separation in a short time with minimum amounts of sample and reagents. To improve the concentration sensitivity of detection, several on-line sample preconcentration techniques such as sweeping have been developed.

A capillary electrophoresis method using indirect UV detection has been applied to the determination of arsenate [As(V)], arsenite [As(III)], monomethylarsonic acid and dimethylarsinic acid. The arsenic species were successfully separated in a successive multiple ionic polymer layer coated capillary. On-line sample preconcentration of arsenic compounds were performed by employing field enhanced sample injection. A baseline separation was achieved in a basic background solution of 10 mM 2,6-pyridinedicarboxylic acid at pH 10.3. The precision of migration time was 1.2-2.4% RSD and peak height was 8.1-12.9% RSD. The limits of detection at a S/N ratio of 3 for the four arsenic compounds were found to be 20-70 ppb, which are comparable to other on-line preconcentration techniques. The enhancement factor was improved by 230-1,500-fold.

On-line preconcentration is one of the aspects of analytical method development using capillary electrophoretic techniques. The choice of the sample matrix alone can significantly alter both method sensitivity and separation efficiency. The recent trend to detect samples in narrower separation vessels also necessitates the need to improve detection sensitivity. The desire to detect very low levels of analytes using limited amounts of sample from biological specimens and the high separation efficiency obtainable using very large injections compared to classical small size injections also adds to this list. Indeed, one of the rich areas of research in the capillary electrophoresis field is on on-line sample preconcentration. More than 400 published research articles gathered from the http://www.webofscience.com from the year 2000 described a form of on-line preconcentration in capillary electrophoresis. This review provides a comprehensive table listing the applications of on-line preconcentration in capillary electrophoresis.

Capillary electrophoresis (CE) is a relatively new method of analytical separation having the advantages of high separation efficiency, requirement of a small sample amount, low operating cost, and fast separation time. CE is a separation method where the analyte migrates under an electric field due to a charge on the analyte. Hence, CE was unable to separate neutral analytes until the advent of micellar electrokinetic chromatography (MEKC). MEKC is performed with an addition of ionic micelles to an electrophoretic medium, where a portion of the analyte is incorporated into the micelle and has an apparent charge, which can be subject to electrophoretic separation. The migration velocity of the neutral analyte in MEKC depends on what portion of the analyte is incorporated into the micelle. Thus, the separation principle of MEKC is similar to that of chromatography, although the micelle corresponding to the stationary phase in chromatography is not stationary inside the capillary. The fundamental characteristics and theoretical treatments of the behavior of the analyte in MEKC were studied extensively by the author's group. MEKC has been established as one of the most popular separation modes in CE. This review describes how MEKC was developed and how it is useful as a method of analytical separation. © 2008 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 8: 291–301; 2008: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.20156

A general and simple implementation of simultaneous multiparametric sensing in a single microchip is presented by using a capillary-assembled microchip (CAs-CHIP) integrated with the plural different reagent-release capillaries (RRCs), acting as various biochemical sensors. A novel "drop-and-sip" technique of fluid handling is performed with a microliter droplet of a model sample solution containing proteases (trypsin, chymotrypsin, thrombin, elastase) and divalent cations (Ca2+, Zn2+, Mg2+) that passes through the microchannel with the aid of a micropipette as a vacuum pump, concurrently filling each RRC via capillary force. To avert the evaporation of the nanoliter sample volume in each capillary, PDMS oil is dropped on the outlet hole of the CAs-CHIP exploiting the capillary force that results in spontaneous sealing of all the RRCs. In addition, this high-speed sample introduction alleviates the possibility of protein adsorption and capillary cross-contamination, allowing a reliable and multianalyte determination of a sample containing many different proteases and divalent cations by using the fluorescence image analysis. Presented results suggested the possible application of this microchip in the field of drug discovery and systems biology.

A capillary-assembled microchip (CAs-CHIP), prepared by simply embedding square capillaries in a lattice polydimethylsiloxane (PDMS) channel plate with the same channel dimensions as the outer dimensions of the square capillaries, has been used as a diffusion-based pretreatment attachment in capillary electrophoresis (CE). Because the CAs-CHIPs employ square-section channels, diffusion-based separation of small molecules from sample solutions containing proteins is possible by using the multilayer flow formed in the square section channel. When a solution containing high-molecular-weight and low-molecular-weight species makes contact with a buffer solution, the low-molecular-weight species, which have larger diffusion coefficients than the high-molecular-weight species, can be collected in a buffer-solution phase. The collected solution containing the low-molecular-weight species is introduced into the separation capillary to be analyzed by CE. This type of system can be used for CE analysis in which pretreatment is required to remove proteins. In this work a fluorescently labeled protein and rhodamine-based molecules were chosen as model species and a feasibility study was performed.

Compared to chromatography-based techniques, the concentration limits of detection (CLOD) associated with capillary electrophoresis are worse, and these have largely precluded their use in many practical applications. To overcome this limitation, researchers from various disciplines have exerted tremendous efforts toward developing strategies for increasing the concentration sensitivities of capillary electrophoresis (CE) systems, via the so-called sample enrichment techniques. This review highlights selected developments and advances in this area as applied to the analyses of proteins and peptides in the last 5 years.

In this study, narrow pH cuts of carrier ampholytes have been used as buffers in CE for the analysis of protein tryptic digests. Their low conductivity allows very efficient separations under high electric field strength without inducing any significant Joule heating. In this study, the capabilities of narrow pH cuts of carrier ampholytes for the separation of protein tryptic digests have been assessed. Three proteins of different molecular masses have been studied: cytochrome C (horse heart), beta-lactoglobulin B (bovine) and human transferrin. Efficient, rapid and repeatable separations of the peptides resulting from the tryptic digestion have been achieved in this buffer. Moreover, the feasibility of the coupling of carrier ampholyte-based capillary electrophoresis with ESI-MS has been demonstrated through the study of the cytochrome C tryptic digest.

To increase the detection sensitivity of capillary electrophoresis (CE) and thereby widen its application to biomolecular analyses, we have developed a number of on-line preconcentration strategies, i.e., techniques to increase sample loading without compromising resolution and efficiency. Two such techniques, namely, dynamic pH junction and field-enhanced sample injection (FESI) are covered in this work. Dynamic pH junction is predicated on a sharp reduction in an analyte's migration velocity following a reversal of its electrophoretic direction from the acidic sample zone to the basic background solution (BGS) zone. FESI, on the other hand, depends on the retardation of analyte velocity as it transits from the low-conductivity sample zone to the high-conductivity milieu of the BGS zone. Their applications to high-sensitivity analyses of peptides and proteins are discussed.

Many researchers have invested considerable efforts toward improving capillary electrophoresis (CE)-mass spectrometry (MS) systems so they can be applied better to standard analyses. This review highlights the developments in CE-MS of proteins and peptides over the last five years. It includes the developments in interfaces, sample-enrichment techniques, microfabricated devices, and some applications, largely in capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF) and capillary isotachophoresis formats.

To improve the detection sensitivity of metal ions in capillary electrophoresis (CE), combination of on-line complexation and sweeping was explored using cyclohexane-1,2-diaminetetraacetic acid (CDTA). CDTA has two roles, as a dynamic complexation agent to form a UV-absorbing complex and a carrier for sweeping. Six metal ions were used as test analytes. The effect of pH, CDTA concentration, composition of BGS and sample matrix, injection length of sample solution were examined and optimized to obtain high performance of this method. Under optimized conditions, the technique was validated in terms of the limit of detection, repeatability, and sensitivity enhancement. Improved detection responses were 60- to 160-fold in terms of peak heights for metal ions in comparison with conventional CZE of precapillary complexed metal ions. The limits of detection were in the range of 0.4 – 4.2 × 10−7 M.

In this article, a microfluidic platform integrating capillary electrophoresis and bioluminescence (BL) detection that was fabricated in poly(dimethylsiloxane) (PDMS) with lab-on-a-chip technology was demonstrated for cellular metabolic analyses. A microchannels network, "cross combining with Y", was designed to perform on-chip sample preparation, separation, and BL detection of ATP and ATP-conjugated metabolites, using firefly luciferin-luciferase BL system. A dynamic modification of the channel wall of PDMS proved to be crucial to reverse the direction of electroosmotic flow (EOF), which was uniquely achieved by a prewash cycle with a cationic surfactant didodecyldimethylammonium bromide. The influences of surfactant on the EOF and BL reaction were also investigated. Quantitative analyses revealed a dynamic linear range over 2 orders of magnitude for ATP, with a detection limit down to submicromolar (midattomole). The method was validated by measuring cellular ATP of E. coli. with direct on-chip cell lysis. Further work was emphasized on ATP-conjugated metabolite analysis, using galactose as an example. Assays of galactose in human urine samples confirmed the reliability of the protocol, which revealed good prospect of this platform for ATP-conjugated submetabolomic profiling.

A capillary LC system was set up and optimized, in which a UV absorbance detector was used and a monolithic silica-ODS column as the separation column. Two on-line concentration techniques, namely, gradient elution mode and in-tube solid-phase ion-pair microextraction (SPIPME), were combined with the capillary LC system, which proved to be beneficial to enhance the concentration sensitivity by enabling the injection of large volumes of samples. The limits of detection at ppb levels for the flavins [riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD)] were achieved using the two techniques. For in-tube SPIPME, a monolithic silica-ODS column was employed as the extraction column, on which FAD and FMN were retained b y interaction with an ion-pair reagent, tetrabutylammonium phosphate, resulting in greater than 110-fold enhancement in their concentration sensitivities relative to conventional injection method. The reproducibility and linearity of the two methods were investigated. The two methods were applied to analyze trace amounts of flavins in bacterial Escherichia coli cell extracts and recombinant flavoenzymes.

A novel concept for assembling various chemical functions onto a single microfluidic device is proposed. The concept, called a capillary-assembled microchip, involves embedding chemically functionalized capillaries into a lattice microchannel network fabricated on poly(dimethylsiloxane) (PDMS). The network has the same channel dimensions as the outer dimensions of the capillaries. In this paper, we focus on square capillaries to be embedded into a PDMS microchannel network having a square cross section. The combination of hard glass square capillary and soft square PDMS channel allows successful fabrication of a microfluidic device without any solution leakage, and which can use diffusion-based two-solution mixing. Two different types of chemically modified capillaries, an ion-sensing capillary and a pH-sensing capillary, are prepared by coating a hydrophobic plasticized poly(vinyl chloride) membrane and a hydrophilic poly(ethyleneglycol) membrane containing functional molecules onto the inner surface of capillaries. Then, they are cut into appropriate lengths and arranged on a single microchip to prepare a dual-analyte sensing system. The concept proposed here offers advantages inherent to using a planar microfluidic device and of chemical functionality of immobilized molecules. Therefore, we expect to fabricate various types of chemically functionalized microfluidic devices soon.

Field-enhanced sample injection (FESI) was used to improve the concentration sensitivity of a capillary electrophoresis (CE)-mass spectrometry (MS) system with sheath flow configuration. Using some bioactive peptides, more than 3000-fold improvement in signal was obtained, permitting analysis in the low nM (fmol/microl) levels. The system was further evaluated for analysis of complex peptide mixtures by using low concentration tryptic digests of standard proteins. Rapid identification of the original protein was obtained by database searching using the observed molecular masses of the peptides, and by comparison of actual MS-MS spectra of selected peptides with the predicted fragmentation patterns.

Analyses of amino acids and peptides were performed using a quartz microchip and an interface for microchip electrophoresis-electrospray ionization mass spectrometry (MCE-ESI-MS). In MCE-ESI-MS, negative pressure caused by ESI increased band broadening and deteriorated separation. We tried to suppress the negative pressure and improve separation using a microchip with a long separation channel. Separations of peptide standards were compared using two microchips with long separation channel (58.9 mm) and short one (22.9 mm). Theoretical plate numbers and resolution were improved significantly using the former. The theoretical plate numbers of [Val4]angiotensin was 8600 using the former and 1700 using the latter. When background electrolytes of low pH were used in an uncoated quartz microchip, electrokinetic injection was difficult because of weak electroosmotic flow. The use of successive multiple ionic polymer layers coating of the microchip channel stabilized electrokinetic injection and permitted analysis of amino acids and peptides even under low pH conditions. Separation of amino acids was successfully performed using formic acid solution (pH 2.5) as background electrolyte.

In this article, it was demonstrated that a subsecond separation of cellular metabolites such as riboflavin (RF), flavin mononucleotide (FMN), and flavin-adenine dinucleotide (FAD) was achieved using microchip capillary electrophoresis with laser-induced fluorescence detection. The influences of crucial parameters that governed analysis time (e.g., channel length and electric field for separation) and separation resolution (e.g., sample size) were investigated, both in theoretical aspects and experimental practice. Quantitative analyses were performed that exhibited linear dynamic range of two orders of magnitude, with calculated detection limits of 34, 201, and 127 nM for RF, FAD, and FMN, respectively. To test the validity of the method, it was successfully applied to characterize several recombinant flavin-binding domains in a human neuronal nitric oxide synthase.

To improve detection sensitivity of cationic analytes, a dynamic pH junction technique was examined. Dynamic pH junction is an on-line focusing method in capillary electrophoresis (CE) based on the difference in the analyte’s mobility between the background electrolyte (BGE) and sample matrix. The effects of pH values and concentrations of the BGE and the sample matrix on dynamic pH junction were examined. Optimization of analyte focusing resulted in enhanced detection responses of about 100–160-fold in terms of peak heights for some anilines in comparison to conventional injections. In particular, the concentration limits of detection (LOD) (S/N=3) for the test anilines obtained with dynamic pH junction were from 1.9 to 3.7 ppb with UV detection without any pretreatment procedure.

Separation and on-line concentration of bisphenol A and three alkylphenols were investigated by micellar electrokinetic chromatography with the anionic surfactant, sodium dodecyl sulfate. The separation conditions were optimized by the simultaneous addition of the organic solvent and cyclodextrin to the running solution. The separation of hydrophobic analytes and 4-nonylphenol isomers was improved by the addition of 10% methanol and 5 mM beta-cyclodextrin to the running solution. When the sweeping with the running solution was used as the on-line concentration procedure, 69-, 48-, 55- and 41-fold increases in detection sensitivity were obtained for bisphenol A, 4-tert.-butylphenol and 4-(1,1,3,3-tetramethylbutyl)phenol, and the second peak of 4-nonylphenol isomers, respectively. The detection limits were 0.0071, 0.0065, 0.021 and 0.055 mg/l, respectively. These results were better than those with the cationic surfactant, tetradecyltrimethylammonium bromide.

Signal denoising and baseline correction using discrete wavelet transform (DWT) are described for microchip capillary electrophoresis (MCE). DWT was performed on an electropherogram describing a separation of nine tetramethylrohodamine-5-isothiocyanate labeled amino acids, following MCE with laser-induced fluorescence detection, using Daubechies 5 wavelet at a decomposition level of 6. The denoising efficiency was compared with, and proved to be superior to, other commonly used denoising techniques such as Fourier transform, Savitzky-Golay smoothing and moving average, in terms of noise removal and peak preservation by directly visual inspection. Novel strategies for baseline correction were proposed, with a special interest in baseline drift that frequently occurred in chromatographic and electrophoretic separations.

Complementary on-line preconcentration strategies are needed when analyzing different classes of solutes in real samples by capillary electrophoresis (CE) with UV detection. The performance of three different on-line preconcentration (focusing) techniques under alkaline conditions was examined in terms of their selectivity and sensitivity enhancement for a group of steroids, including classes of androgens, corticosteroids and estrogens. Electrokinetic focusing of large sample injection plugs (up to 28% of effective capillary length or 22.1 cm) directly on-capillary can be tuned for specific classes of steroids based on changes in their mobility (velocity) using a multi-section electrolyte system in CE. A dynamic pH junction was applied for the selective resolution and focusing of weakly acidic estrogens using borate, pH 11.0 and pH 8.0 in the background electrolyte and the sample, respectively. Sweeping, using an anionic bile acid surfactant and neutral gamma-cyclodextrin (gamma-CD) under alkaline conditions (pH 8), resulted in focusing and separation of the moderately hydrophobic (non-ionic) classes of steroids, such as androgen and corticosteroids. Optimal focusing and resolution of all test steroids under a single buffer condition was realized by a dynamic pH junction-sweeping format using borate, pH 11.0 and bile acid surfactant with gamma-CD in the BGE, whereas the sample is devoid of surfactant at pH 8.0. The design of selective on-line focusing strategies in CE is highlighted by the analysis of microgram amounts of ethynyl estradiol derived from a female contraceptive pill extract using the dynamic pH junction method, which resulted in over a 100-fold enhancement in concentration sensitivity.

A robust and simple interface for microchip electrophoresis-mass spectrometry (MCE-MS) was developed using a spray nozzle connected to the exit of the separation channel of the microchip. The spray nozzle was attached to the microchip using a polyether ether ketone screw without adhesive, thus allowing easy replaced. Sample injection and electrophoretic separation was performed by control of the voltage only. The analysis of a few basic drugs was performed using the optimized MCE-MS system. The separation was improved by using a high-viscosity separation buffer and a spray nozzle with a small bore size. This system was also applied to the separation of peptides and protein-trypsin digests. Sample adsorption was minimized by adding acetonitrile to the separation buffer when using a quartz microchip.

With a growing interest in metabolome analysis, there is a need for developing robust methods for analysis of intracellular metabolites profiles in real samples like e.g., bacteria cell. Due to their weak absorbance properties, tri- and dicarboxylic acids from TCA cycle (citric, isocitric, 2-oxoglutaric, succinic, fumaric, malic) as well as carboxylic acid metabolites from glycolysis pathway, urea cycle and metabolism of amino compounds (formic, pyruvic, lactic, acetic, glutamic) were analyzed by capillary electrophoresis (CE) with indirect UV detection. Using 4 mM 2,6-pyridinedicarboxylic acid as a highly UV absorbing carrier electrolyte, 0.2 mM cetyltrimethylammonium bromide, 10% ethylene glycol and 10% acetonitrile, pH 3.5, carboxylic acids metabolites were analyzed in Bacillus subtilis cell extract from two different cultures: glucose and malate. CE with an electrokinetic injection mode achieved limits of detection in the range of 13-54 ppb (1.12-10(-7) - 5.96-10(-7) M). The reproducibility and linearity of method was investigated with RSD for migration time less than 1.3% and acceptable correlation coefficients. The optimized CE method was used to compare metabolome content of cell extract derived from two different culture media containing either glucose or malate as a carbon source. The changes in carboxylic acid metabolites profile were observed depending from used culture medium. Carboxylic acid concentrations ranged: in cell extract from malate culture from 59 to 0.5 microM for lactate and citrate, respectively, and in cell extract from glucose culture from 133 to 0.5 microM for glutamate and citrate, respectively. Appropriate concentrations of carboxylic acid in the single bacterium cell were estimated at mM and sub-mM levels.

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.

Sensitive capillary electrophoresis (CE) methods are required for emerging areas of biochemical research such as the metabolome. In this report, dynamic pH junction-sweeping CE with laser-induced fluorescence (LIF) detection is applied as a robust single method to analyze trace amounts of three flavin derivatives, riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD), from several types of samples including bacterial cell extracts, recombinant protein, and biological fluids. Submicromolar amounts of flavin coenzymes were measured directly from formic acid cell extracts of Bacillus subtilis. Significant differences in flavin concentration were measured in cell extracts derived from either glucose or malate as the carbon source in the culture media. Quantitative assessment of FAD and FMN content from selected flavoenzymes was demonstrated after heat denaturation to release noncovalently bound coenzymes and deproteinization. This method was also applied to the analysis of free flavins in pooled human plasma and urine without the need for laborious off-line sample preconcentration. Picomolar detectability of flavins by CE-LIF detection was realized with on-line preconcentration (up to 15% capillary length used for injection) by dynamic pH junction-sweeping, resulting in a limit of detection (S/N = 3) of about 4.0 pM for FAD and FMN. This represents over a 60-fold improvement in concentration sensitivity compared to those of previous techniques using conventional injections. The method was validated in terms of reproducibility, sensitivity, linearity, and specificity. Flavin analysis by dynamic pH junction-sweeping CE-LIF offers a simple, yet sensitive way to analyze trace levels of flavin metabolites from complex biological samples.

Enantioselective separation of triadimenol, a component of systemic agricultural fungicide, by electrokinetic chromatography (EKC) using cyclodextrins (CDs) as chiral selectors was investigated. Both a neutral CD derivative, hydroxypropyl-gamma-CD (HP-gamma-CD), and an ionic one, heptakis-6-sulfato-beta-CD (HS-beta-CD), were employed as an additive in cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) and as a chiral pseudostationary phase in CDEKC, respectively. In each system, four stereoisomeric peaks were completely or partially separated from each other. To enhance the detectability or the concentration sensitivity, on-line preconcentration techniques were applied to both EKC systems. Sweeping was used in the CD-MEKC system under an acidic condition, whereas stacking with a reverse migrating pseudostationary phase (SRMP) in the CDEKC system. Around 10-fold increase in the detection sensitivity for each peak was attained with both sweeping and SRMP systems. Good repeatabilities in the migration time, corrected peak area, and peak height were recognized in terms of the relative standard deviation. The limit of detection for each peak in the SRMP-CDEKC system, calculated from the calibration curve, was found to be 0.8-3.8 ppm.

On-line preconcentration by sweeping in micellar electrokinetic chromatography using mixed micelles of sodium dodecyl sulfate (SDS)-SB-12 is presented. Because of their large micelle radius, they permit increased partitioning of hydrophobic analytes into the core. In addition, they also possess lower negative surface charge relative to pure SDS micelles so anionic analytes can be retained better due to decreased electrostatic repulsion. As the efficiency of sweeping is predicated on the magnitude of retention factors, these advantages translated to better focusing. As much as a 370-fold improvement in detector response, in terms of peak height, was obtained for some neutral steroids, while about a 360-fold improvement was obtained for some phenol derivatives, which were previously not amenable to sweeping by pure SDS micelles.

This review provides an overview as well as a practical understanding of on-line sample concentration techniques in micellar electrokinetic chromatography (MEKC). MEKC as well as other capillary electrophoretic modes suffer from low concentration sensitivity due to minute sample volume and limited optical pathlength for on-capillary photometric detection. Two on-line sample preconcentration techniques, sample stacking and sweeping are known to be effective techniques for enhancement of the concentration sensitivity in MEKC. Sample stacking occurs as ions cross a boundary that separates regions of the high electric field sample zone and the low electric field background solution zone. The difference in migration velocity of pseudostationary phases within the two zones is the key to achieving the focusing effect. Sweeping is defined as the picking and accumulating of analytes by the pseudostationary phase that penetrates the sample zone devoid of pseudostationary phase. In this review, several examples of the sample stacking and sweeping under different experimental conditions are given, besides many references to applications.

A simple and inexpensive approach to modify a commercial atmospheric pressure chemical ionization (APCI) interface was proposed for capillary electrophoresis–mass spectrometry (CE–MS). In order to accommodate lower flow rates in the range 1–10 μl/min, both sheath liquid and nebulizing gas were coaxially supplied to the nebulizer as an arrangement of pneumatically assisted electrospray interface. Since this paper focused on the primary study of the modified APCI interface, the performance of the interface was first evaluated by the direct infusion of a reserpine solution. Optimization of several APCI parameters, such as temperature of APCI vaporizer, nebulizing gas flow and APCI corona discharge current, were accomplished. The orifice dimension for the nebulizing gas flow was largely independent of the MS sensitivity when the nebulizing gas flow rate was ca. 0.4 l/min. A successful CE–APCI–MS separation is obtained using the modified APCI interface.

Emerging fields of biochemical research, such as metabolomics, present challenges to current separation technologies because of the large number of metabolites present in a cell and their often low (submicromolar) concentration. Although capillary electrophoresis (CE) holds great promise as the method of choice for high-resolution separations of biological samples, it suffers from poor concentration sensitivity, especially with the use of UV detection. In CE, sweeping and dynamic pH junction represent two complementary on-line focusing techniques that have been used for sensitivity enhancement of hydrophobic and weakly acidic analytes, respectively. However, the application of either the sweeping or dynamic pH junction technique alone might, in some cases, be less effective for the analysis of certain sample mixtures. Recent work in the development of a hyphenated dynamic pH junction-sweeping technique is presented as an effective on-line method of preconcentration suitable for both hydrophilic (anionic) and hydrophobic (neutral) analytes. Sensitive analyses of flavin metabolites by CE with laser-induced fluorescence (LIF) detection is demonstrated in various biological matrixes, including cell extracts of Bacillus subtilis, pooled human plasma, as well as heat-deproteinized flavoenzymes. Enhanced analyte band narrowing and improved sensitivity is achieved for flavins using dynamic pH junction-sweeping compared to either sweeping or dynamic pH junction alone. This results in over a 1200-fold improvement in sensitivity relative to conventional injection methods, giving a limit of detection (LOD, defined as S/N = 3) of about 4.0 × 10−12 M. Strategies for sensitive and more comprehensive analyses of other cell metabolites, including nucleotides, coenzymes, and steroids, are also discussed when using on-line focusing techniques in conjunction with multiplexed CE and UV detection. © 2002 The Japan Chemical Journal Forum and Wiley Periodicals, Inc., Chem Rec 2: 397–404; 2002: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.10041

Sweeping in capillary electrophoresis (CE) involves the interaction of a pseudostationary phase (PS) in the separation solution and a sample in the matrix that is free of the PS used. The PS includes not only the PSs employed in electrokinetic chromatography, but also complexation reagents such as borate. The sample matrix could have a lower, similar, or higher conductance than the separation solution. Thus, the basic condition for sweeping is a sample matrix free of the additive. The accumulation of analyte molecules during the interaction makes this interesting phenomenon very useful as an on-line preconcentration method for CE. Preconcentration occurs due to chromatographic partitioning, complexation, or any interaction between analytes and PS. Contact between analyte and PS is facilitated by the action of electrophoresis and is independent of electroosmosis. The analyte, PS, or both should have electrophoretic velocities when an electric field is applied. The extent of preconcentration is dictated by the strength of the interaction involved. From tens to several thousand-fold improvements in detector response for many neutral and charged analytes have been achieved with this technique, suggesting sweeping as a general approach to on-line preconcentration in CE. The mechanism and applications of the sweeping phenomenon under different experimental conditions are discussed in this review, with particular emphasis on a better understanding of the sweeping mechanism under reduced electric field (high conductivity) in the sample zone.

Simple yet effective methods to enhance concentration sensitivity is needed for capillary electrophoresis (CE) to become a practical method to analyze trace levels of analytes in real samples. In this report, the development of a novel on-line preconcentration technique combining dynamic pH junction and sweeping modes of focusing is applied to the sensitive and selective analysis of three flavin derivatives: riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Picomolar (pM) detectability of flavins by CE with laser-induced fluorescence (LIF) detection is demonstrated through effective focusing of large sample volumes (up to 22% capillary length) using a dual pH junction-sweeping focusing mode. This results in greater than a 1,200-fold improvement in sensitivity relative to conventional injection methods, giving a limit of detection (S/N = 3) of approximately 4.0 pM for FAD and FMN. Flavin focusing is examined in terms of analyte mobility dependence on buffer pH, borate complexation and SDS interaction. Dynamic pH junction-sweeping extends on-line focusing to both neutral (hydrophobic) and weakly acidic (hydrophilic) species and is considered useful in cases when either conventional sweeping or dynamic pH junction techniques used alone are less effective for certain classes of analytes. Enhanced focusing performance by this hyphenated method was demonstrated by greater than a 4-fold reduction in flavin bandwidth, as compared to either sweeping or dynamic pH junction, reflected by analyte detector bandwidths0.20 cm. Novel on-line focusing strategies are required to improve sensitivity in CE, which may be applied toward more effective biochemical analysis methods for diverse types of analytes.

A selective detection method for biogenic amines present in highly complex matrixes was devised by employing both electrokinetic injection and on-column-derivatization capillary electrochromatographic methods. The on-column derivatization capillary electrochromatography system was evaluated by use of a capillary column (total length of 45 cm, effective length of 25 cm) fabricated using a 100-mcirom (i.d.) fused-silica capillary tube packed with 5-microm (i.d.) ODS particles that were tolerant of an alkaline environment. The column was filled with a run buffer consisting of a derivatization reagent, o-phthalaldehyde/2-mercaptoethanol, in a mixture of borate buffer (pH 10). After electrokinetic injection of a mixture of five biogenic amines (histamine, serotonin, tyramine, putrescine, cadaverine) as a test sample, the free amines entered into the anodic site of the capillary column and started to travel along the column, during which time the analytes reacted with the derivatization reagent, separated out, and were detected with an absorbance at 340 nm when high voltage was applied to the column. When this system was applied to a mixture containing 5 biogenic amines and 17 amino acids, the 5 biogenic amines plus arginine selectively entered into the capillary with the electrokinetic injection and were observed on the electrochromatogram, but none of the amino acids lacking arginine were detected. The designated method was also tested for its ability to determine the presence of biogenic amines in the crude extracts obtained from two types of aged fish.

Capillary electrophoresis (CE) has become a useful technique for measuring binding constants. This review is focused on recent trends in the estimation of binding constants by affinity CE. First, we introduce several mathematical equations in which it is assumed that the stoichiometry of the binding between drug and protein is 1:1 as a simple model. In order to calculate accurate binding constants by affinity CE, several experimental considerations are described in this review. In addition, some recent methodologies, such as partial filling technique and multiple-step ligand injection method, are introduced. Among research publications within 3 years, recent applications for determining binding constants are reviewed.

On-line sample concentration by sweeping was investigated in microchip micellar electrokinetic chromatography (MEKC), By changing the distance between the injection cross and the detection points, the profile of the concentration process and the diffusion process in sweeping was elucidated. Rhodamine B injected for 4 s was best concentrated by sweeping at 9.4 mm from the injection cross and the enhancement factor was 450. At the longer distance from this point the peak of Rhodamine B was broadened and diluted by diffusion. The diffusion constant of Rhodamine B calculated from the experiment was 5.7 x 10(-6) cm2s(-1). The mixture of rhodamine B, sulforhodamine B, and cresyl fast violet was concentrated by sweeping and separated by MEKC at the same time.

Enantiomer separations by capillary electrochromatography (CEC) using a packed capillary were investigated. As a chiral stationary phase, an OD-type packing material of 3 microm particle diameter, which is a silica-gel based material coated with cellulose tris(3,5-dimethylphenylcarbamate), was employed. The chiral packing was packed into a 100 microm I.D. fused-silica capillary by a pressurized method. Several racemic enantiomers were successfully resolved with acidic or basic buffers containing acetonitrile as mobile phases. The separation efficiencies obtained in this chiral CEC system were superior to those in the previously reported chiral CEC system using 5 microm particles. The typical plate heights obtained for several enantiomers were 4.6-6.6 microm or reduced plate heights of 1.5-2.2. Good repeatabilities in the migration time, peak height, and corrected peak area were observed as well as for the plate number. As an application study of this CEC system, an optical purity test was carried out by using benzoin enantiomers. A 1% content of one enantiomer in the other enantiomer was successfully separated and detected.

Two on-line sample concentration techniques, sample stacking and sweeping, were evaluated using cationic surfactants as pseudostationary phases in micellar electrokinetic chromatography. As cationic surfactant micelles, tetradecyltrimethylammonium bromide and cetyltrimethylammonium chloride were employed. About 10-fold and 1000-fold increases in detection sensitivity in terms of peak heights were observed by sample stacking and sweeping, respectively, without suppression of the electroosmotic flow. In particular, the concentration limits of detection (S/N=3) for test naphthalenesulfonic acids obtained with sweeping were from 0.96 to 0.47 ppb with UV detection without any preconcentration procedure.

The electroosmotic flow was successfully suppressed even in the presence of cationic surfactants, when a polyacrylamide-coated capillary was employed. Two on-line sample concentration techniques of sample stacking and sweeping were evaluated in micellar electrokinetic chromatography (MEKC) using the polyacrylamide-coated capillary. Cationic surfactants were used as pseudostationary phases in MEKC. At least 60-fold and about 600-fold increases in detection sensitivity were obtained in terms of peak heights by sample stacking and sweeping, respectively.

The sweeping concept is extended to capillary zone electrophoresis (CZE) separation of neutral solutes involving complexation with borate. Analogous to the pseudostationary phase in electrokinetic chromatography (EKC), the complexing agent (borate) serves as carrier for sweeping and separation in CZE. Therefore, similar to the retention factor in EKC, the focusing effect in the present system is directly related to the association constant between the analyte and complexing agent. Theoretical and some preliminary experimental studies gerenally suggest that the electrophoretic mobility of the complex and the concentration of the complexing agent affect the resulting length of narrowed zones. Moreover, sweeping using borate is affected by pH since borate complexation is pH dependent. From around 10 to 40-fold improvement in peak heights has been observed experimentally for some neutral test analytes (monosaccharides, catechols, and nucleosides)

細胞内代謝中間産物の網羅的分析法開発にキャピラリー電気泳動の利用の可能性を評価するために行った予備実験結果の一部を報告する. PTHアミノ酸22種の一斉濃縮分離を酸性条件下MEKCにより行い, スウィーピング法により約5倍の試料濃縮ができた. 細胞内に代謝中間産物として含まれると考えられる芳香族カルボン酸6種についても濃縮分離を試みた. アルカリ性条件下スタッキングCZEにより25～30倍の濃縮が可能であった. 酸性条件下MEKCによる分離濃縮においてもほぼ同様の濃縮結果が得られた.

The behavior of charged species along concentration boundaries in capillary zone electrophoresis (CZE) that was first described in detail by Everaerts et al. in 1979 assured the possibility of concentrating charged solutes inside the capillary. The concentration effect is based on the sudden change in analyte electrophoretic velocity brought about by the difference in the magnitude of the electric field. Furthermore, this on-line method could be the needed solution to the problem of low concentration sensitivity in CZE. Sample stacking, which is now its well known name, has then found valuable use in applying CZE in many fields, especially after the in-depth studies performed in the early 90s by Chien and Burgi. This article reviews the theory and methodological developments of sample stacking developed for charged analytes in CZE and also in electrokinetic chromatography. A table conveying the reported applications especially in the biomedical and environmental fields is given. On top of this, other on-line concentration methods for charged species, namely, sample self-stacking, acetonitrile stacking, sweeping, cation selective exhaustive injection–sweeping, and use of a pH junction, are briefly discussed.

Three environmentally important phenylurea herbicides (monuron, isoproturon, diuron) were separated in reversed migration micellar electrokinetic chromatography (RM-MEKC) using 50 m M sodium dodecyl sulfate, 50 m M phosphoric acid, and 15 m M γ-cyclodextrin. Three on-line concentration techniques are then evaluated to increase the detection sensitivity of the RM-MEKC system. Stacking with reverse migrating micelles (SRMM, water as the sample solvent) provided the best results among the focusing techniques studied. Using a z-shaped detection cell, more than 500-fold increase in peak height is obtained. As a sample preparation and off-line concentration method, solid-phase extraction (SPE) that further improved detection sensitivity was used in the analysis of spiked tap and pond water. For example, 1 parts per billion of each herbicide spiked in tap or pond water was detected by MEKC after SPE and SRMM.

The applicability of capillary electrochromatography (CEC) using packed capillary column to enantiomer separations was investigated. As chiral stationary phases, OD type packing materials of 5 and 3 microm particle diameters, originally designed for conventional high-performance liquid chromatography (HPLC) were employed. The chiral packing materials were packed by a pressurized method into a 100 microm I.D. fused-silica capillary. Several racemic enantiomers, such as acidic, neutral and basic drug components, were successfully resolved, typically by using acidic or basic solutions containing acetonitrile as mobile phases. The separation efficiencies for some enantiomers in the chiral CEC system using the 5 microm OD type packing were superior to those obtained in HPLC using chiral packings. The plate heights obtained for several enantiomers were 8-13 microm or the reduced plate height of 1.6-2.6, which indicates the high efficiency of this chiral CEC system.

A review surveying enantiomer separations by micellar electrokinetic chromatography (MEKC) using chiral surfactants is described. MEKC is one of the most popular techniques in capillary electrophoresis, where neutral compounds can be analyzed as well as charged ones, and the use of chiral micelles enable one to achieve the enantioseparation. The chiral MEKC systems are briefly reviewed according to the types of chiral surfactants along with typical applications. As chiral micelles or pseudostationary phases in MEKC, various natural and synthetic chiral surfactants are used, including several low-molecular-mass surfactants and polymerized surfactants or high-molecular-mass surfactants. Cyclodextrin modified MEKC using chiral micelles is also considered.

The concept of sweeping neutral analytes using a high-conductivity matrix or under a reduced electric field in micellar electrokinetic chromatography (MEKC) using anionic micelles and in the presence of electroosmotic flow is presented. Three important processes are identified. First, stacking of the micelles at the cathodic interface between the sample solution (S) and background solution (BGS) zones is identified. This is then followed by the sweeping of analyte molecules by the stacked micelles that enter the S zone. Finally, the destacking of the stacked micelles at the anodic interface between the S and BGS zones occurs. The stacking of the micelles improves the focusing effect of sweeping by a factor approximately equal to the ratio of conductivities between the S and BGS zones (ratio = enhancement factor = γ‘). However, the destacking of the stacked micelles broadens the swept zones by a factor approximately equal to 1/γ‘. In effect, the focusing effect of sweeping using a matrix with equal or high...

A novel method that combines two on-line concentration techniques in capillary electrophoresis (CE), namely, sample stacking with electrokinetic injection (field-enhanced sample injection, FESI) and sweeping, afforded the detection of positively chargeable analytes in parts per trillion (ppt) levels. The main idea is to selectively introduce by FESI as many molecules of cationic analytes as possible from a very dilute sample solution and focus the resulting zone by sweeping. Limit of detection values (signal-to-noise ratio 3) of 4.1 and 8.0 ppt-the lowest concentration reported by direct UV detection in CE-with average plate numbers of 3.6 x 10(5) and 4.4 x 10(5) are obtained for laudanosine and naphthylamine (standard solutions), respectively. This translates to improvements in peak heights compared with usual injection approaching a million-fold. Optimization schemes and application to quantitative and qualitative analyses are also investigated.

A simple and effective way to improve detection sensitivity of positively chargeable analytes in capillary zone electrophoresis more than 100-fold is described. Cationic species were made to migrate toward the cathode even under reversed electroosmotic flow caused by a cationic surfactant by using a low pH run buffer. For the first time, with such a configuration, large volume sample stacking of cationic analytes is achieved without a polarity-switching step and loss of efficiency. Samples are prepared in water or aqueous acetonitrile. Aromatic amines and a variety of drugs were concentrated using background solutions containing phosphoric acid and cetyltrimethylammonium bromide. Qualitative and quantitative aspects are also investigated.

各種産地のウコン中のクルクミノイドを高精度かつ迅速に定量することを目的として,HPLC及びMEKCの測定条件を検討し,次の結果を得た.(1) ウコン乾燥物からの超音波によるクルクミノイド抽出には,溶媒として99.5%エタノールが最も適当であった.(2) HPLCによるクルクミノイドの分離は,グラジエント溶出による方法が最も短時間に分析できた.また,BBMAによるMEKCでは,更に短時間に分析することができた.定量においてもHPLC及びMEKC共に高い相関の検量線が得られた.(3) 産地の異なる8種のウコン乾燥物中のクルクミノイドについて定量した結果,MEKCはHPLCとほぼ同様の精度で分析することができた.MEKCはHPLCに比べ,短時間に,しかも少ない溶媒量で分析ができ,品質管理分析に適用できることを示した.

The important features of electrokinetic chromatography are critically reviewed. Special emphasis is given to systems using micelles as pseudostationary phase. Short and comprehensive overviews are given on the subjects of separation, comparison with capillary electrochromatography, on-line coupling with mass spectrometry, and developments that are expected in the future. A greater coverage on the subject of improvement of detection sensitivity, specifically by on-line concentration was also contributed.