Miniaturization of batch- and flow-type chemiluminescence detectors in capillary electrophoresis

Glass and PTFE tubes as detection cells were put in small light-tight boxes to achieve miniaturization of batch-and flow-type chemiluminescence detectors for capillary electrophoresis. These light-tight boxes which included a detection cell and a photosensor module were successfully designed. In the batch-type detector using a glass tube as a detection cell, the influences of a repeated injection of sample and a reagent volume of the detection cell on chemiluminescence intensity were examined in detail. By using 3.8 mm I.D. glass tube including 400 microl chemiluminescence reagent solution, the chemiluminescence peaks were reproducibly observed for the repeated injection experiment up to the eight injection with each run time of 3.0 min. Dansyl-Trp was determined over the range 3 x 10(-8)-1 x 10(-5) M with the detection limit of 0.43 fmol (S/N=3). In the flow-type detector using a PTFE tube as a detection cell, both ends of the PTFE tube were connected to three-way joints; a chemiluminescence reagent solution was delivered into the cell and a capillary was inserted through one of the joints while an electrode was inserted through the other one. Dansyl-Trp was determined over the range 1 x 10(-7)-1 x 10(-5) M with the detection limit of 1.3 fmol (S/N=3). By using the compact flow-type detector, a mixture of dansyl-amino acids was separated and detected in micellar electrokinetic chromatography mode.     

On-column catalysis of hydratization equilibria during liquid chromatographic analysis of acetaldehyde and formaldehyde

Summary The aldehydes were separated by reversed phase chromatography on a polymer-based support. Acid catalysis on the column was needed to obtain sufficiently fast equilibrium between acetaldehyde and its hydrate. Deuterium labeled acetaldehyde (D₃) yielded complete separation from the nonlabeled compound, probably due to a difference in hydratization. Fluorescent products were obtained by reaction between the aldehydes and a mixture of ammonia and dimedone (5.5-dimethyl-1.3-cyclohexanedione). The fluorescence was detected at 460 nm after excitation at 390 nm. A reaction time of 42 seconds at 90°C in a knitted teflon capillary produced detection limits of 0.5 and 1 ng for acetaldehyde and formeldehyde, respectively. A method for preparation of a stable reagent with low background fluorescence is described.     

Manipulation of protein fingerprints during on-column fluorescent labeling: protein fingerprinting of six Staphylococcus species by capillary electrophoresis

Bacterial proteomes were analyzed by use of electrophoretically mediated microanalysis (EMMA) and field-enhanced stacking. A water-soluble protein fraction was injected onto a capillary. Next, a fluorogenic reagent was injected and allowed to react with the protein mixture, producing fluorescent products that were separated by submicellar capillary electrophoresis and detected by laser-induced fluorescence. By use of a low-ionic strength sample buffer and a brief electrophoretic step, slow moving anionic proteins were stacked at the reagent-sample interface and were preferentially labeled. By reversing the order of sample injection and labeling reagent, fast moving cationic proteins were preferentially labeled. By adjustment of the sample buffer pH, proteins with different isoelectric points were selectively labeled. Electrophoresis fingerprints were generated for the water-soluble protein fraction from six Staphylococcus species. The protein patterns produced were species-specific and were used to construct a phylogenetic tree.     

Biomolecule analyses in an open-tubular capillary chromatography using ternary mixed carrier solvents with chemiluminescence detection

We examined the elution behavior of isoluminol isothiocyanate (ILITC)-labeled biomolecules (α-amino acids, peptides, and proteins) in an open-tubular capillary chromatography system using an untreated fused-silica capillary tube and a water-acetonitrile-ethyl acetate mixture carrier solution. Such an open-tubular capillary chromatography is called "tube radial distribution chromatography (TRDC)" for convenience. A mixture of ILITC and ILITC-labeled biomolecules was analyzed using TRDC with chemiluminescence detection that provided simple instrument without a light source and complex optical devises. The ILITC and the labeled twenty α-amino acids were separated, in this order or the reverse order, or not separated with an organic solvent-rich and water-rich carrier solution. Their elution behavior was considered to be of hydrophilic or hydrophobic nature of ILITC and the labeled α-amino acids. The ILITC and the labeled protein, alcohol dehydrogenase and bovine serum albumin, were separated in this order with an organic solvent-rich carrier solution, while they were eluted in the reverse order with a water-rich carrier solution, based on the TRDC separation performance. The TRDC system worked with the untreated open-tubular capillary tube not using any specific capillary tubes, such as coated, packed, or monolithic.     

Determination of carbohydrates as their 3-aminophthalhydrazide derivatives by capillary zone electrophoresis with on-line chemiluminescence detection

A method based on pre-capillary derivatization with luminol (3-aminophthalhydrazide) for carbohydrate analysis using capillary electrophoresis with on-line chemiluminescence (CL) detection was developed. The derivatives of seven monosaccharides were separated and detected by using 200 mM borate buffer containing 100 mM hydrogen peroxide at pH 10.0 as separation electrolyte and 25 mM hexacyanoferrate in 3 M sodium hydroxide solution as post-capillary chemiluminescence reagent with separation efficiencies ranging from 160,000 to 231,000 plates per metre. The minimum amount of carbohydrate derivatized was 2 pmol (corresponding to the concentration of 2 microM). The method also provided a linear response for glucose in the concentration range of 0.1-250 microM with a mass detection limit of 420 amol or a concentration detection limit of 0.1 microM. Preliminary work using the CE-CL format to determine glucose in a rat brain microdialysis sample is presented as a typical case.     

Low-cost, high-sensitivity laser-induced fluorescence detection for DNA sequencing by capillary gel electrophoresis.

A low cost, 0.75-mW helium neon laser, operating in the green region at 534.5 nm, is used to excite fluorescence from tetramethylrhodamine isothiocyanate-labelled DNA fragments that have been separated by capillary gel electrophoresis. The detection limit (3 sigma) for the dye is 500 ymol [1 yoctomole (1 ymol) = 10(-24) mol] or 300 analyte molecules in capillary zone electrophoresis; the detection limit for labeled primer separated by capillary gel electrophoresis is 2 zmol [1 zeptomole (1 zmol) = 10(-21) mol]. The Richardson-Tabor peak-height encoded sequencing technique is used to prepare DNA sequencing samples. In 6% T, 5% C acrylamide, 7 M urea gels, sequencing rates of 300 bases/hour are produced at an electric field strength of 200 V/cm; unfortunately, the data are plagued by compressions. These compressions are eliminated with addition of 20% formamide to the sequencing gel; the gel runs slowly and sequencing data are generated at a rate of about 70 bases/hour.     

Migration behavior of isoluminol isothiocyanate-labeled alpha-amino acids in capillary electrophoresis with an absorption/chemiluminescence dual detection system

In our previous study, we developed capillary electrophoresis with an ultraviolet absorption/chemiluminescence dual detection system. Here, to demonstrate one of the possible applications of the capable system as well as confirm the advantage, migration behavior of isoluminol isothiocyanate-labeled alpha-amino acids was examined in the capillary electrophoresis with a dual detection system. The labeled samples were first analyzed by absorption detection with an on-capillary, followed by chemiluminescence detection with an end-capillary. The system easily, rapidly, and simultaneously produced useful information concerning chemiluminescence quenching and amino group-labeling due to the presence of both absorption and chemiluminescence detections.     

A highly efficient and versatile microchip capillary electrophoresis method for DNA separation using gold nanoparticle as a tag

A highly efficient and versatile method for DNA separation using Au nanoparticles (Au NPs) as a tag based on microchip capillary electrophoresis (MCE) was developed. The thiol-modified DNA-binding Au NPs were utilized as a tag. Target DNA was sandwiched between Au NPs and probe DNA labeled with horseradish peroxidase (HRP). In electrophoresis separation, the difference in electrophoretic mobility between free probe and probe-target complex was magnified by Au NPs, which enabled the resulting mixture to be separated with high efficiency by microchip capillary electrophoresis. Horseradish peroxidase was used as a catalytic label to achieve sensitive electrochemical DNA detection via fast catalytic reactions. With this protocol, 27-mer DNA fragments with different sequences were separated with high speed and high resolution. The proposed method was critical to achieve improved DNA separations in hybridization analyses.     

Determination of chlorogenic acid and rutin in cigarettes by an improved capillary electrophoresis indirect chemiluminescence system

An improved capillary electrophoresis indirect chemiluminescence system was employed for the determination of chlorogenic acid and rutin in cigarette samples. After being separated by capillary electrophoresis, the analyte zones were determined by indirect chemiluminescence of luminol-potassium hexacyanoferrate. In this system, luminol was added into running buffer solution and introduced at the head of separation capillary, and potassium hexacyanoferrate was introduced at the end of the capillary. A high potential buffer reservoir was constructed from a running buffer cell and an electrode buffer one, which were jointed with a frit, in order to avoid luminol electrolysis in high potential reservoir and the excursion of chemiluminescence baseline. A low potential flow reservoir was used to prevent electrode buffer solution from the contamination of chemiluminescence waste. Therefore, the proposed capillary electrophoresis-chemiluminescence system can avoid the electrolysis of chemiluminescence reagent, retain the stability of chemiluminescence baseline and prolong the working time of running and electrode buffer solutions. In addition, the matrix of cigarette sample solutions has also an inhibitory effect on the chemiluminescence intensity in the indirect detection, whereas the influence was not observed in the separation of standard solutions. After the correction of matrix inhibition and the calibration with standard addition method, chlorogenic acid and rutin were determined in four cigarette samples by the improved capillary electrophoresis-chemiluminescence system.     

Analysis of antioxidants using a capillary electrophoresis with chemiluminescence detection system

We developed a capillary electrophoresis with chemiluminescence detection system using 2-methyl-6-p-methoxyphenylethynylimidazopyrazinone as a chemiluminescence reagent for determination of antioxidants of superoxide anions. 2-Methyl-6-p-methoxyphenylethynylimidazopyrazinone reacted with superoxide anions generated through the reaction of hypoxanthine and xanthine oxidase, and then emitted chemiluminescence. Suppression of the chemiluminescence in the presence of antioxidants for superoxide anions was introduced as a detection principle for antioxidants into the capillary electrophoresis with chemiluminescence detection system. After optimizing the analytical conditions, various antioxidants, such as superoxide dismutase, nitroblue tetrazolium, ascorbic acid, and catechin, were subjected to the present system. They gave negative peaks due to the quenching effect; the detection limits of superoxide dismutase, nitroblue tetrazolium, ascorbic acid, and catechin were 1, 100, 100, and 10 μM, respectively (S/N = 2). A model sample consisting of superoxide dismutase and nitroblue tetrazolium was satisfactorily separated and detected within ca. 10 min. We also applied the present system to analysis of catechin in green tea as a real sample.     

On-capillary chemiluminescence detection for capillary electrophoresis with a single capillary.

On-capillary chemiluminescence detection for capillary electrophoresis with a single capillary was reported. A hole (about 30 microm diameter) was made on the capillary wall at about 50.5 cm from the inlet end. Hydrogen peroxide solution could enter the capillary from the hole, and mixed with luminol and copper(II) to produce chemiluminescence. The chemiluminescence was detected by a PMT under the hole. Several factors that influenced chemiluminescence intensity were investigated. The detection limits for luminol and N-(4-aminolbutyl)-N-ethylisoluminol (ABEI) were 1 x 10(-11) and 2 x 10(-10) mol L(-1), respectively. The method features simple construction and no dead volume.     

Integration of a flow-type chemiluminescence detector on a glass electrophoresis chip

A glass electrophoresis microchip integrated a flow-type chemiluminescence (CL) detection cell has been developed and evaluated. The chip pattern is a double-T-type electrophoretic sample injection and separation combining with a Y-type chemiluminecent detector. The double-T geometry allows for high-efficiency sample injection and geometric definition of sample plug size. The branch of Y was used as CL reagent channel, and the CL reagent was delivered by a lab-made micropump. Bis[(2,4,6-trichlorophenyl)]oxalate-H₂O₂ CL system was employed to detect dansyl amino acids. On this microchip, dansyl-phenylalanine and -sarcosine were successfully separated by electrophoresis and detected within 250 s. The detection limits (S/N=3) of dansyl-phenylalanine and -sarcosine could reach to 2.8 and 3.2 μM, respectively, due to the vigorous dilution of sample with CL reagent and timely removal of the waste solution from reaction area.     

Derivatization, extraction and concentration of amino acids and peptides by using organic/aqueous phases in capillary electrophoresis with fluorescence detection

We report a novel method that facilitates sample pretreatment and detection in amino acid analysis by coupling solvent extraction with capillary electrophoresis. Amino acids and peptides were fluorescently labeled, concentrated into an organic solvent, and then separated by capillary zone electrophoresis with fluorescence detection. To achieve this, acetophenone was first employed to dissolve the derivatizing reagent, fluorescamine. The products, which possessed both hydrophilic and hydrophobic moieties, could be extracted and concentrated into the organic phase by suppressing the deprotonation of carboxyl groups, thus enhancing the hydrophobicity of the resulting molecules through pH modification in the aqueous solution. Furthermore, by fine-tuning the pH value, individual amino acids and short peptide molecules could be separated selectively from the sample bulk. This convenient, chemically controllable concentration technique may be useful in sample concentration and purification of biologically related samples such as amino acids and short peptides.     

Determination of different forms of human interferon-gamma in single natural killer cells by capillary electrophoresis with on-capillary immunoreaction and laser-induced fluorescence detection

A novel method for determining different forms of human interferon-gamma (IFN-gamma) in single natural killer cells was developed by capillary electrophoresis (CE) with on-capillary immunoreaction and laser-induced fluorescence (LIF) detection. Cells were perforated with digitonin and one single cell was electrokinatically introduced into the front end of a separation capillary. The monoclonal antibody labeled with fluorescein isothiocyanate of IFN-gamma was hydrodynamically injected into the front end of the capillary around the cell introduced. After the cell was lysed by ultrasonication, the front end of the capillary was used as a microreactor to allow different forms of IFN-gamma to process the immunoreaction with their labeled antibody. Finally, the complexes of different forms of IFN-gamma with their labeled antibody were separated and detected by CE with LIF detection with a limit of detection of zeptomoles (10(-21) mol).     

Capillary electrophoresis microchip coupled with on-line chemiluminescence detection

In the present work, chemiluminescence detection was integrated with capillary electrophoresis microchip. The microchip was designed on the principle of flow-injection chemiluminescence system and capillary electrophoresis. It has three main channels, five reservoirs and a detection cell. As model samples, dopamine and catechol were separated and detected using a permanganate chemiluminescent system on the prepared microchip. The samples were electrokinetically injected into the double-T cross section, separated in the separation channel, and then oxidized by chemiluminescent reagent delivered by a home-made micropump to produce light in the detection cell. The electroosmotic flow could be smoothly coupled with the micropump flow. The detection limits for dopamine and catechol were 20.0 and 10.0 μM, respectively. Successful separation and detection of dopamine and catechol demonstrated the distinct advantages of integration of chemiluminescent detection on a microchip for rapid and sensitive analysis.     

In-capillary derivatization and capillary electrophoresis separation of amino acid neurotransmitters from brain microdialysis samples

A new in-capillary derivatization method with naphtalene-2,3-dicarboxyaldehyde (NDA)/CN(-) has been developed for capillary electrophoresis with laser-induced fluorescence detection of brain microdialysate amino acids. Samples are sandwiched between two plugs of reagent mixture at the capillary inlet and subsequently separated. Highest derivatization yields are obtained by using a reagent to sample plug length ratio equal to 4, performing a first electrophoretic mixing followed by a zero potential amplification step before applying the separation voltage and using a NaCN to NDA concentration ratio equal to 1. This new single-step methodology allows the analysis of amino acid neurotransmitters in rat brain microdialysis samples.     

Microchip capillary electrophoresis using on-line chemiluminescence detection

Chemiluminescence detection was used in capillary electrophoresis integrated on a microchip. Quartz microchips have two main channels and four reservoirs. Dansyl-lysine and -glycine were separated and detected with bis[(2-(3,6,9-trioxadecanyloxycarbony)-4-nitrophenyl]oxalate as peroxyoxalate chemiluminescent reagent. These dansyl amino acids came into contact with the chemiluminescence reagent to produce visible light at the interface between the separation channel and chemiluminescence reagent-containing reservoir. The detection limit (S/N = 3) for dansyl-lysine was 1 x 10(-5) M, which corresponded to the very small mass detection limit of ca. 0.4 fmol. However, the concentration sensitivity in the present system was approximately two orders of magnitude lower than that in the conventional capillary electrophoresis-chemiluminescence detection system. The relative standard deviations of migration time and peak height for dansyl-lysine were 4.2 and 4.5%, respectively. A channel conditioning before every run and an appropriate control of voltages were needed for the reproducible results. The present system had advantages in rapid separation time (within 40 s), small (several 10 pI) and accurate sample injection method using a cross-shaped injector, and simplification and miniaturization of the detection device.     

Capillary electrophoretic system incorporating an UV/CL dual detector

We developed a capillary electrophoretic system incorporating an ultra-violet absorption (UV)/chemiluminescence (CL) dual detector, taking advantage of the CL reaction of luminol-hydrogen peroxide and the batch-type CL detection cell. UV detection was carried out using the on-capillary method while CL detection was performed using the end-capillary method. Examination of isoluminol isothiocyanate (ILITC) as a model sample revealed two main peaks with UV detection and one main peak with CL detection. The first peak in the UV detection data corresponded to the main peak in the CL detection data. We then determined that the ILITC sample included natural ILITC as well as an impurity that had absorption behavior but did not have CL properties and labeling ability. Furthermore, the components of a mixture containing glycine, glycylglycine and glycylglycylglycine, all labeled with ILITC, were well separated and detected using the present system. The present system easily, rapidly, and simultaneously produces useful information due to the presence of both UV and CL detectors.     

毛细管电泳分离和激光检测分析多糖胶

将多糖胶的混合物与荧光剂9-氨基芘-1,4,6-三磺酸(APTS)派生后再进行微量离心过滤分离。所得到的高分子部分采用毛细管电泳(CE)分离和激光诱导荧光(LIF)检测技术进行分析。缓冲溶液pH的调节和聚丙烯酰胺(PAA)涂层毛细管的使用有效地改善了多糖胶的分离效率和峰形。在优化条件下,iota、kappa角叉菜胶、藻胶、xanthan、carboxymethyl cellulose (CMC)等5种组分的混合物和阿拉伯树胶、刺梧桐树胶、CMC等3种组分的混合物分别在pH 3.2和7.8的缓冲溶液下得到了     

Michrochip chromatography using an open‐tubular microchannel and a ternary water–ACN–ethyl acetate mixture carrier solution

A capillary chromatography system has been developed using a ternary mixed‐solvents solution, i.e. water–hydrophilic/hydrophobic organic solvent mixture as a carrier solution. Here, we tried to carry out the chromatographic system on a microchip incorporating the open‐tubular microchannels. A model analyte solution of isoluminol isothiocyanate (ILITC) and ILITC‐labeled biomolecule was injected to the double T‐junction part on the microchip. The analyte solution was delivered in the separation microchannel (40 μm deep, 100 μm wide, and 22 cm long) with the ternary water–ACN–ethyl acetate mixture carrier solution (3:8:4 volume ratio, the organic solvent rich or 15:3:2 volume ratio, the water‐rich). The analyte, free‐ILITC and labeled BSA mixture, was separated through the microchannel, where the carrier solvents were radially distributed in the separation channel generating inner and outer phases. The outer phase acts as a pseudo‐stationary phase under laminar flow conditions in the system. The ILITC and the labeled BSA were eluted and detected with chemiluminescence reaction.