On-line sample preconcentration by LVSEP-FASI with simple voltage control on Y-channel chips.

To eliminate complicated voltage controls for highly sensitive microchip electrophoresis (MCE) analyses on the basis of combining two online sample preconcentration techniques, large-volume sample stacking with an electroosmotic flow (EOF) pump (LVSEP) and field-amplified sample injection (FASI), cross-channel microchips and a multichannel high-voltage power supply were replaced to Y-channel chips and a conventional power supply designed for capillary electrophoresis, respectively. By simple switching of the electric circuit after the LVSEP-FASI sample enrichments, the focused analytes could be separated during anodic migration in a separation channel. In the LVSEP-FASI analysis of fluorescein using the Y-channel microchip, the maximum sensitivity enhancement factor (SEF) of 7400 was achieved, resulting in a 30-fold detectability increase compared to the conventional LVSEP. The developed method was applied to the oligosaccharide analysis in MCE. As a result, the SEF for maltotriose was improved from 450 to 2300 and the baseline separation of the oligosaccharides was achieved without any complicated voltage control in LVSEP-FASI on the Y-channel chips.

A thin-layer solid-phase extraction-liquid film elution technique used for the enrichment of polycyclic aromatic hydrocarbons in water.

In this article, we propose a novel microsolid-phase extraction and elution technique, which we called the thin-layer solid-phase extraction-liquid film elution technique. The thin-layer solid-phase extraction phase is an octadecylsilylated sol gel- coated porous silica thin film prepared on the outer wall of a test tube, which has a larger surface area for the extraction of the target compounds compared to a conventional solid-phase microextraction phase. After optimization of the extraction procedure for five types of polycyclic aromatic hydrocarbons, the liquid film elution technique was investigated. Liquid film elution is an elution technique wherein the compounds extracted into the thin-layer solid-phase extraction phase are eluted using a small volume of solvent film formed around the extraction phase. The results show that the elution can be carried out using 150 µL of eluent. Enrichment factors between 20 and 34 were obtained for polycyclic aromatic hydrocarbons containing more than four aromatic rings in 10 mL aliquots of aqueous samples. Finally, recoveries of 85-112% were obtained for polycyclic aromatic hydrocarbons containing more than four aromatic rings from spiked natural water samples using the thin-layer solid-phase extraction-liquid film elution technique.

Nonaqueous capillary gel electrophoretic analysis of metal nanoclusters in polymeric-DMSO-Li+ systems.

In this study, we investigated a combination of nonaqueous CE with capillary gel electrophoresis to achieve highly efficient analysis of metal nanoclusters. In the nonaqueous capillary gel electrophoresis (NACGE), PVA and hydroxypropyl methylcellulose were dissolved in DMSO. In addition, to enhance the entanglement of the polymer chains, Li+ ions were also added. By employing the PVA-DMSO-Li+ solution, we studied the effects of the molecular weight, the degree of hydrolysis, and the concentration of the polymers and Li+ on the separation. As a result, good separations of standard mononuclear metal complexes and tetrairon nanoclusters were achieved by NACGE.

Highly Sensitive Analysis in Capillary Electrophoresis Using Large-volume Sample Stacking with an Electroosmotic Flow Pump Combined with Field-amplified Sample Injection.

To achieve highly sensitive analysis without labor-intensive experimental procedures in capillary electrophoresis (CE), large-volume sample stacking with an electroosmotic flow pump (LVSEP)-field-amplified sample injection (FASI) was combined with a dynamic coating technique. In this study, poly(vinyl pyrrolidone) (PVP) was employed for the dynamic coating additive. Since a standard fluorescent dye, fluorescein, was well concentrated in a conventional LVSEP, the PVP dynamically-coated capillaries can be also applied to the LVSEP-FASI analysis. In our home-made CE apparatus, however, current breakdown was often caused, especially at a longer electrokinetic injection time due to bubble formation. To avoid the interference of bubble formation, the distance between the tips of the electrode and the capillary in the vertical direction was changed from 0 to 2.5 cm under the magnetic stirring condition. This allowed for a long electrokinetic injection time of up to 20 min, resulting in a sensitive enhancement factor (SEF) of 34900 for fluorescein. The developed method was applied to the chiral analysis of amino acids in CE. As a result, leucine (Leu) was successfully separated in LVSEP-FASI with SEFs of 6420 and 4500 for the D- and L-Leu peaks, respectively.

On-line sample preconcentration by polarity switching in floating electrode-integrated microchannel.

In this study, we found that the polarity switching was effective to enrich and separate fluorescent analytes which have weakly-dissociated groups in a floating platinum electrode (width, 50 µm; thickness, 2.5 µm)-integrated straight-channel in microchip electrophoresis (MCE). In the straight channel filled with an Alexa Flour 488 (AF488) solution, a sharp peak was observed after the polarity inversion with a 530-fold enhancement of the sensitivity relative to the conventional MCE analysis. By using a fluorescent pH indicator, we verified that a sharp high-pH zone was generated nearby the floating electrode and moved toward the anode with maintaining the high pH, which induced the sample enrichment like a dynamic pH junction mechanism. In the floating electrode-embedded channel, the mixture of AF488-labeled proteins was also well concentrated and separated within 100 s.

Combination of large-volume sample stacking with an electroosmotic flow pump with field-amplified sample injection on cross-channel chips.

A combination of two online sample concentration techniques, large-volume sample stacking with an electroosmotic flow (EOF) pump (LVSEP) and field-amplified sample injection (FASI), was investigated in microchip electrophoresis (MCE) to achieve highly sensitive analysis. By applying reversed-polarity voltages on a cross-channel microchip, anionic analytes injected throughout a microchannel were first concentrated on the basis of LVSEP, followed by the electrokinetic stacking injection of the analytes from a sample reservoir by the FASI mechanism. As well as the voltage application, a pressure was also applied to the sample reservoir in LVSEP-FASI. The applied pressure generated a counter-flow against the EOF to reduce the migration velocity of the stacked analytes, especially around the cross section of the microchannel, which facilitated the FASI concentration. At the hydrodynamic pressure of 15 Pa, 4520-fold sensitivity increase was obtained in the LVSEP-FASI analysis of a standard dye, which was 33-times higher than that obtained with a normal LVSEP. Furthermore, the use of the sharper channel was effective for enhancing the sensitivity, e.g., 29 100-fold sensitivity increase was achieved with the 75-µm wide channel. The developed method was applied to the chiral analysis of amino acids in MCE, resulting in the sensitivity enhancement factor of 2920 for the separated d-leucine.

On-line coupling of sample preconcentration by LVSEP with gel electrophoretic separation on T-channel chips.

To achieve an on-line coupling of the sample preconcentration by a large-volume sample stacking with an electroosmotic flow pump (LVSEP) with microchip gel electrophoresis (MCGE), a sample solution, a background solution for LVSEP and a sieving solution for MCGE were loaded in a T-form channel and three reservoirs on PDMS microchips. By utilizing the difference in the flow resistance of the two channels, a low-viscosity sample and a viscous polymer solution were easily introduced into the LVSEP and MCGE channels, respectively. Fluorescence imaging of the sequential LVSEP-MCGE processes clearly demonstrated that a faster stacking of anionic fluorescein and successive introduction into the MCGE channel can be carried out on the T-channel chip. To evaluate the preconcentration performance, a conventional MCZE analysis of fluorescein on the cross-channel chip was compared with LVSEP-MCGE on the short T-channel chip, and as a result that the value of sensitive enhancement factor (SEF) was estimated to be 370. The repeatability of the peak height was good with the RSD value of 3.2%, indicating the robustness of the enrichment performance. In the successive LVSEP-MCGE analysis of φX174/HaeIII digest, the DNA fragments were well enriched to a sharp peak in the LVSEP channel, and they were separated in the MCGE channel, whose electropherogram was well-resembled with that in the conventional MCGE. The values of SEF for the DNA fragments were calculated to be ranging from 74 to 108. Thus, the successive LVSEP-MCGE analysis was effective for both preconcentrating and separating DNA fragments.

Simple and Rapid Immobilization of Coating Polymers on Poly(dimethyl siloxane)-glass Hybrid Microchips by a Vacuum-drying Method.

A simple and rapid vacuum-drying modification method was applied to several neutral and charged polymers to obtain coating layers for controlling electroosmotic flow (EOF) and suppressing sample adsorption on poly(dimethyl siloxane) (PDMS)-glass hybrid microchips. In the vacuum-dried poly(vinylpyrrolidone) coating, the electroosmotic mobility (µeo) was suppressed from +2.1 to +0.88 × 10(-4) cm(2)/V·s, and the relative standard deviation (RSD) of µeo was improved from 10.2 to 2.5% relative to the bare microchannel. Among several neutral polymers, poly(vinylalcohol) (PVA) and poly(dimethylacrylamide) coatings gave more suppressed and repeatable EOF with RSDs of less than 2.3%. The vacuum-drying method was also applicable to polyanions and polycations to provide accelerated and inversed EOF, respectively, with acceptable RSDs of less than 4.9%. In the microchip electrophoresis (MCE) analysis of bovine serum albumin (BSA) in the vacuum-dried and thermally-treated PVA coating channel, an almost symmetric peak of BSA was obtained, while in the native microchannel a significantly skewed peak was observed. The results demonstrated that the vacuum-dried polymer coatings were effective to control the EOF, and reduced the surface adsorption of proteins in MCE.

Effects of divalent cations on bovine testicular hyaluronidase catalyzed transglycosylation of chondroitin sulfates.

Glycosaminoglycans were prepared as salts of different divalent cations and tested as donors in bovine testicular hyaluronidase catalyzed transglycosylation reactions. All of the metal cations examined had similar binding efficiency of divalent cations to hyaluronan. However, cations bound with different efficiencies to chondroitin sulfate species and the differences were marked in the case of chondroitin 6-sulfate; the numbers of cations bound per disaccharide unit were estimated to be 0.075 for Mn, 1.231 for Ba, 0.144 for Zn, and 0.395 for Cu. While barium salt of chondroitin sulfates enhanced transglycosylation, the zinc salt of chondroitin sulfates inhibited transglycosylation. Therefore, by selecting the proper divalent cation salt of chondroitin sulfates as a donor in the transglycosylation reaction it is possible to improve the yields of the products.

Preparation of open tubular solid-phase extraction column with 5-amino-8-hydroxyquinoline-modified gold nanoparticle phase for the enrichment of heavy metal ions.

A 5-amino-8-hydroxyquinoline (AHQ)-modified gold nanoparticle (GNP) layer was fabricated on an inner wall of a silica capillary column by alternatively passing a citrate-stabilized GNP solution and an AHQ solution in a repeating fashion. The observations by a field emission scanning electron microscope showed that the thickness of the resulting GNP layer was about 0.15 microm. This column was then used as an open tubular solid-phase extraction column for cadmium, followed by electrothermal atomic absorption spectrometric determination. The detection limit of 0.009 ng ml(-1) was obtained.

Determination of tin by resin-suspension electrothermal atomic absorption spectrometry after enrichment as the complex with ammonium pyrrolidinedithiocarbamate.

A resin-phase extraction method has been optimized for the trace determination of tin(II) by ETAAS. Tin(II) was extracted on a finely divided anion exchange resin as the complex with ammonium pyrrolidinedithiocarbamate (APDC). The resin was collected on a membrane filter and then dispersed in 1.00 ml of 1 mol l(-1) nitric acid containing 100 microg of Pd(II) and 60 microg of Ni(II). The resulting resin suspension was subjected to GFAAS. The proposed method was applied to the determination of tin(II) in hydrochloric acid.

An enrichment method of molybdenum on a cellulose nitrate resin for the determination by electrothermal atomic absorption spectrometry coupled with the resin suspension injection.

Cellulose derivative resin (CDR) suspensions containing resin particles of cellulose nitrate (CDR(CN)), cellulose acetate (CDR(CA)), or cellulose triacetate (CDR(CTA)) were prepared as the sorbent for resin suspension injection (RSI) electrothermal atomic absorption spectrometry (ETAAS), in which fine resin particles holding a desired trace element were directly injected into the graphite tube as the suspension. To compare the sorption ability, the distribution ratios of the Mo(VI)-phenylfluorone complex were measured between the aqueous phase and the sorbents, including the CDRs mentioned above and the finely divided anion exchange resin (AR). The results showed that the sorption ability decreases in the following order: CDR(CN) > or = AR > CDR(CA) > or = CDR(CTA). It was concluded that CDR(CN) was able to extract more than 98% of Mo(VI), and was suitable for RSI-ETAAS as well as AR. CDR(CN) was used for the determination of Mo in NIES certified reference materials, No. 10 Rice flour-unpolished; the results showed fairly good agreements between the analytical values and the certified values.

Determination of thallium in potassium chloride and electrolyte replenishers by electrothermal atomic absorption spectrometry.

Thallium in potassium chloride and electrolyte replenishers was determined by electrothermal atomic absorption spectrometry (ETAAS) with direct injection of a resin suspension. Thallium(III) was extracted on fine particles of a cellulose nitrate resin (CNR) from dilute HCl (pH 1.6) in the presence of ammonium pyrrolidine-1-carbodithioate. The CNR particles were collected on a membrane filter by filtration under suction, suspended in 1.0 mL 10mM HNO3, and then delivered directly to ETAAS as the suspension. The effects of chloride ions were thoroughly investigated. The results showed that the addition of 0.5mM NaCl to the suspension (10mM HNO3) was recommended, after CNR and a membrane filter holding the CNR were washed thoroughly with 0.025M HCl, to eliminate interference from chloride ions. No chemical modifier was required. Extraction from the solution containing up to 2M chloride ion was allowable. The proposed method gave a concentration factor of 50 for a 50 mL sample volume. The detection limit (3sigma, n = 5) was 1 ng (20 pg/mL). The relative standard deviation was 4.9% (n = 5) at 30 ng level of thallium. The content of thallium in potassium chloride was 15.7-32.8 ng/g, and in electrolyte replenishers was 0.18-4.16 ng/mL.

A glycomic approach to proteoglycan with a two-dimensional polysaccharide chain map.

Glycosaminoglycan chains were liberated from proteoglycans (bovine lung, tracheal cartilage, and cerebrum) by successive digestion with actinase and with cellulase from Aspergillus niger, which has endo-beta-xylosidase activity. The glycosaminoglycan chains were fluorescence-labeled with 2-aminopyridine after digestion with Streptomyces hyaluronidase. The resulting pyridylamino-glycosaminoglycans, including heparan sulfate, chondroitin sulfate/dermatan sulfate, and heparin, were separated by high-performance liquid chromatography. Each separated fraction was analyzed by two types of high-performance liquid chromatography: gel-filtration chromatography and anion-exchange chromatography. The correlation between molecular weight and degree of sulfation could be shown on the two-dimensional polysaccharide chain map. Use of a commonly available cellulase with endo-beta-xylosidase activity together with the two-dimensional polysaccharide chain map allows easy analysis of various glycosaminoglycan chains and comprehensive comparison among the structures. These techniques will become useful tools in the further development of glycotechnology and glycome analysis.

Determination of V(IV) and V(V) by electrothermal atomic absorption spectrometry following selective solid-phase extraction and the study on the change in the oxidation state of vanadium species in seawater during the sample storage.

The changes in the oxidation state of vanadium in artificial and natural seawater samples were studied by electrothermal atomic absorption spectrometry (ETAAS) with a direct injection of a resin suspension. V(IV) and V(V) were extracted as the complex with Chromazurol B and with N-cinnamoyl-N-2,3-xylylhydroxylamine, respectively, using a suspension of an anion-exchange resin and determined by ETAAS independently. The detection limits of both methods were 0.02 ng ml(-1) for 40 ml of a sample solution. The recovery tests for an artificial seawater sample spiked with V(IV) and/or V(V) were carried out carefully. The results showed that the recoveries of V(IV) or V(V) were 99.2-109% and the standard deviations were 1-6%. The total V was also determined after V(V) was reduced by ascorbic acid. In artificial seawater at pH 7.8, V(V) was stable but V(IV) was oxidized rapidly. In acidified artificial seawater (pH 2.0), V(IV) was oxidized slowly but only a small tendency of such reduction of V(V) was observed. In a natural seawater sample, V(IV) was not detected. The acidification of the natural seawater sample resulted in the reduction of V(V).