A micro-flow separation system was developed using an open capillary, fused-silica or polyethylene tube, and an aqueous-organic mixture (water-acetonitrile-ethyl acetate mixture) as a carrier solution. A model analyte solution containing 2,6-naphthalenedisulfonic acid and 1-naphthol was injected into the capillary tube by a gravity method. The analyte solution was subsequently delivered through the capillary tube with the carrier solution by a micro-syringe pump; the system worked under laminar flow conditions. The analytes were separated through the capillary tube and detected on-capillary by an absorption detector. 2,6-Naphthalenedisulfonic acid and 1-naphthol were detected in this order with a carrier solution of water-acetonitrile-ethyl acetate (15:3:2 volume ratio), while they were detected in the reverse order with a carrier solution of water-acetonitrile-ethyl acetate (2:5:9 volume ratio) using a fused-silica capillary tube. Similar separation behavior, i.e., that the elution times of the analytes could be easily reversed by changing the component ratio of the solvents in the carrier solution, was observed with a polyethylene capillary tube.

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We have realized a cloned enzyme donor immunoassay (CEDIA) on a microchip in 96 s. CEDIA is a homogeneous immunoassay, based on the bacterial enzyme β-galactosidase, which was genetically engineered into two inactive fragments: an enzyme donor and an enzyme acceptor. A model analyte was theophylline, and the detectable concentration range was from 0 to 40 µg mL^-1. Our CEDIA using a microfluidic device was very simple and rapid, unlike microchip-based heterogeneous immunoassays and CEDIA on a well-type microchip.

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The protein-induced chemical structure in calcified organisms is an important factor contributing to their growth, but until now no direct evidence has been reported. Hence, we demonstrate a new analytical technique that combines an FTIR spectrometer and an apertureless-type near-field microscope with high spatial resolution. The complete structural composition of the biomineralized structure of marine organisms has been measured using this technique. The results show that -OH is distributed over the center of the mineral phase of small calcite, which was formed from interactions of matrix proteins.

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A fast Fourier transform (FFT) analyzer was employed to estimate the emission intensity from a radio-frequency (RF)-powered glow discharge plasma for atomic emission analysis. A bias current induced by a self-bias voltage in the RF plasma was pulsated to modulate the emission intensity, and then the modulated component was selectively detected with the FFT analyzer. This method greatly improved the data precision of the emission intensity, thus contributing to better detection ability. The emission intensity of the Mn I 403.08-nm line in an Fe-0.2 mass % Mn alloy sample could be determined with a relative standard deviation of 0.060%. The 3-sigma detection limit of manganese in Fe-based alloys could be obtained to be 1.6 × 10^-5 mass %, when Mn I 403.08 nm was selected as the analytical line.

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The analytical sensitivities of elements were improved by Ar-N₂ inductively coupled plasma (Ar-N₂ ICP) in axially viewing optical emission spectrometry (OES). A commercially available ICP-OES instrument was used, and only N₂ gas was added at flow rates of up to 1.0 L min^-1 into Ar plasma gas. A stable Ar-N₂ ICP was obtained, and the net signal intensities of elements were enhanced by factors of up to 25 compared to those observed for Ar ICP-OES. The detection limits evaluated were also improved by factors from 5 to 10, especially for elements with relatively higher ionization energies. Moreover, the analytical sensitivities for these elements observed in axially viewing Ar-N₂ ICP-OES were superior to those in radially viewing Ar-N₂ ICP-OES reported previously. The Ar-N₂ ICP is expected to be a useful alternative source for excitation and ionization in axially viewing ICP-OES.

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The hydration of ions in confined spaces, such as the interior of ion-exchange resins, micelles, and surface monolayers, is discussed on the basis of results obtained with X-ray absorption fine structure studies, electrophoresis, and ion-transfer voltammetry. The general trends are that anions are partly dehydrated therein, whereas cations are likely to keep their first hydration shells. For bromide ions, the hydration numbers under various circumstances have been determined. The extents of dehydration depend not only on the structure of the cationic sites electrostatically attracting bromide ions but also on whether the cationic sites are exposed to a solution or are effectively shielded from it. These findings will be useful for designing the systems for ionic recognition and separation.

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A liquid phase is coexistent with water-ice prepared from a brine solution at any temperature above the eutectic point of the system. Ice chromatographic measurements have provided information on the liquid phase in an ice particle prepared from NaCl. The growth of the liquid phase causes the alternation of the dominant separation mechanism from the adsorption on the ice surface to the partition into the liquid phase, resulting in an increase in the retention of a water-soluble probe. When the liquid phase is developed, the retention is well explained by the partition coefficient determined with bulk solvents, suggesting that all of the liquid pools in a water-ice particle are connected to each other. In contrast, only a part of the liquid pools act as a stationary phase when the volume of the liquid phase is small. A model, which assumes the homogenous distribution of a salt in an ice particle and spherical shapes of a liquid pool, has allowed the estimation of its size.

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Using techniques comprising laser trapping, microcapillary injection/manipulation, fluorescence microspectroscopy and electrochemistry of single microdroplets, we kinetically investigated the electron transfer (ET) reaction between decamethylferrocene in tributyl phosphate and hexacyanoferrate(III) in water. In the oil-microdroplet/water system, the overall ET reaction rate significantly depended on the droplet radius (r_d, 0.5 µm < r_d < 10 µm) and on the potential-determining ion concentration in the oil phase. The interfacial ET reaction rate constant determined in the water-microdroplet (r_d = 21 µm)/oil system agreed very well with that in the oil-microdroplet (r_d > 2 µm)/water system. The rate constant values were extremely small in the Gibbs free energy (ΔG) range of -10 to -25 kJ mol^-1, with ΔG consisting of the Galvani potential difference between the water and oil phases and the redox potential difference of the solutes. The characteristic ET reaction was discussed in terms of the ion transfer and the ET across the interfacial mixed layer with nanometer-sized thickness.

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Ion transport from one aqueous (W1) to another (W2) across bilayer lipid membranes (BLM) containing gramicidin A (GA) was investigated by recording current fluctuations, when various alkali metal chlorides and potassium salts were used as supporting electrolytes. The magnitude of the single-channel current at a given membrane potential depended on not only the cationic species, but also on the anionic species, and then it decreased with an increase in the diameter of the anion when the diameter of the anion was larger than the pore size of the GA channel. The baseline of the recording current, however, increased with an increase in the diameter of the anion, and its height depended on the concentration of GA in the BLM. The results indicate that GA serves as not only a channel-forming compound, but also as a carrier compound in the BLM.

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The enhanced effect of chemiluminescence (CL) of lucigenin (Luc²⁺) at a flat liquid/liquid interface and at O/W emulsions was demonstrated. The CL was emitted when Luc²⁺ was transferred from an organic phase to an aqueous one containing NaOH and H₂O₂. The CL intensity and emission spectrum depended strongly on the kind of organic solvent. We attempted to apply the CL reaction of Luc²⁺ in the O/W emulsion system to the analysis of H₂O₂. The available analytical conditions were discussed from the aspects of the kind of organic solvent, the reaction time and wavelength detected.

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The adsorption behavior of reverse micelles of cetyltrimethylammonium chloride (CTAC) onto porous silica gels of various pore sizes has been investigated. A decrease in the amount of CTAC in the reversed micellar bulk solution in chloroform/water added to the silica gel in a column was observed, indicating the CTAC adsorption onto the silica surface. The adsorption profile obtained showed a maximum of the adsorbed surfactant amount, where the point of surface saturation may be reached, beyond which the adsorption amount remained unchanged. The adsorption of CTAC was also dependent on the specific surface area of the silica gel used. These results suggest that a CTAC monolayer may be formed and completed on the silica surface. The adsorbed CTAC amount slightly decreased with increasing the water to surfactant molar ratio, R, in the reversed micellar bulk solution, although at R < 2 the CTAC adsorption remained constant with silica gels having larger pore diameters. Even in the presence of traces of water moisture in the bulk solution used for the CTAC adsorption, the adsorption of water on silica gel was also observed and the molar ratios, R_Γ, of the water to surfactant adsorbed on different silica gels were almost unity, implying that the silica gel may tend to hold some of water molecules originally adsorbed on its surface. The adsorption of CTAC molecules appears to occur through the adsorbed water molecules, probably polarized at the silica surface, and a CTAC-water-silica nanointerface seems to be formed. The adsorbed water amount or R_Γ also increased with an increase in R and on silica gels with larger pore diameters and a plateau was observed in the R range of 3 - 4, beyond which an increase in R_Γ occurred again. The water molecules present in the vicinity of the adsorbed CTAC and the silica surface may possess different properties at different R and R_Γ values.

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Traces of P(V) and As(V) in sample solutions were converted into heteropoly molybdic acids at pH 1.5 and collected onto polyoxyethylene(20)-4-isononylphenoxy ether-coated Amberlite XAD-4 particles. The desorption was carried out with 0.3 M tetramethylammonium hydroxide solution for the HPLC analysis. Molybdophosphoric and molybdoarsenic acids were separated from excess molybdate by ion-pair reversed phase HPLC, though the peaks of P(V) and As(V) could not be resolved. However, the selective decomposition of molybdoarsenic acid with citric acid allowed the differential determination of P(V) and As(V) at the ng mL^-1 level. The proposed method was applied to the analysis of high-purity iron for P and As.

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The peak parking (PP)-moment analysis (MA) method was applied to the measurement of surface diffusion coefficients (D_s) in reversed-phase liquid chromatography using a C₁₈-silica gel and an aqueous solution of methanol (70 vol%). The values of D_s measured by the PP-MA method were in agreement with those obtained by the pulse response (PR)-MA method, which is one of powerful strategies for the quantitative study on the mass transfer kinetics in columns and stationary phases. It was demonstrated that the PP-MA method is an alternative for the experimental measurement of D_s. The mechanism of surface diffusion was considered on the basis of molecular diffusion by introducing a restriction energy (E_r) for surface diffusion. Physical meanings of E_r were discussed by analyzing the correlation between the enthalpy change due to sample retention (Q_st) and D_s. It seems that surface diffusion of a sample molecule is restricted by its retention strength and by the solvent structure formation due to the hydrophobic solvation of the stationary phase surface.

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A new model was developed for explaining the retention behavior in reversed-phase liquid chromatography (RPLC) on the basis of two extrathermodynamic relationships, i.e., enthalpy-entropy compensation (EEC) and linear free energy relationship (LFER). Krug's four tests were used for analyzing the temperature dependence of the retention equilibrium constant normalized by alkyl ligand density. It was demonstrated that a real EEC takes place in the retention equilibrium. A new model based on the real EEC was developed to explain the LFER concerning the retention equilibrium. The model formulates the slope and intercept of the LFER with the compensation temperatures and molecular thermodynamic parameters relating to the changes in enthalpy and entropy. The values of the retention equilibrium constant under different RPLC conditions were estimated with a relative error of 6.9%. The new model provides an alternative approach for interpreting the retention equilibrium in RPLC from the viewpoints of thermodynamics and extrathermodynamics.

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A layered manganese oxide film grown electrochemically was applied to remove methylene blue (MB), a cationic dye, from an aqueous solution. A layered MnO_x film intercalated with tetraethylammonium (Et₄N) cations was deposited potentiostatically at +1.0 V (vs. Ag/AgCl) from an aqueous Mn²⁺ solution containing Et₄NCl. Et₄N cations were sandwiched between negatively charged MnO_x layers. When the Et₄N/MnO_x film was immersed in a solution containing MB alone, the intercalated Et₄N cations were replaced with MB in a solution phase by an ion-exchange mechanism. The uptake capacity of the Et₄N/MnO_x film for MB was estimated to be 45.3 mg per 1.0 g of MnO₂. In the presence of KCl, the MB sorption was seriously restricted because the interlayer space was occupied by K⁺ ions. However, the MnO_x film anodized at +0.8 V can selectively take up MB molecules from the KCl solution. This can be ascribed to an increase in the interlayer space available to the sorption of MB due to the extraction of K⁺ ions, where the MnO_x surface probably has a specific affinity toward MB molecules.

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In order to the increase sensitivity of enzyme-linked immunosorbent assay (ELISA) using micro-droplet reaction system formed by ink-jet, we employed small dots of two-dimensionally well-ordered structure of polystyrene (PS) microbeads (710 ± 25 µm in diameter) formed on the surface of polydimethyl siloxane (PDMS). An aqueous suspension of polystyrene microbeads (5 µm) was ejected on the PDMS plate with ink-jet. The PS beads were automatically assembled by capillary force accompanied with solvent evaporation. The evaporation rate was controlled by ambient relative humidity. The assembled beads were solidly immobilized on the surface of PDMS surface. The dots of well-ordered structure were stable against vigorous washing. ELISA using the structure as a reaction location was three times more sensitive than the material without the structure due to the increase of the surface area and consequent increase of the amount of antibody.

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Physicochemical analyses of Au nanoparticle monolayer formation on a glass surface functionalized with 3-aminopropyltrimethoxysilane (APTMS) and ethyltrimethoxysilane (ETMS) were performed for fabricating a nanoarchitecture of metal nanoparticles using self-assembly. The Au nanoparticle surface density on the functionalized glass surface correlated linearly with the optical response because of Au-nanoparticle localized surface plasmon resonance (LSPR). This relation enabled assessment of the Au nanoparticle deposition. A Frumkin isotherm showed the deposition of Au nanoparticles on the APTMS or APTMS/ETMS functionalized glass surface. For an APTMS-derivatized surface, the affinity between the Au nanoparticles and the surface decreased with the lowering of the APTMS surface coverage. The interaction parameter exhibited attractive interaction of 30-nm-diameter Au nanoparticles; 12 nm particles were repulsive. Adsorption isotherm experiments using Au nanoparticles on glass surfaces with an APTMS and ETMS mixed layer suggest that hydrophobic interaction between Au nanoparticles' bare gold surfaces caused the attractive interaction among 30-nm-diameter nanoparticles.

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A seed-mediated growth method for surface modification was applied to the attachment of gold nanoparticles (AuNPs) to glassy carbon (GC) surfaces. By simply immersing a GC plate at first into a seed solution containing 4 nm Au nano-seed particles and then into a growth solution containing HAuCl₄, ascorbic acid and cetyltrimethyammonium bromide, AuNPs could be successfully attached to the GC surface via the growth of nanoparticles. A possible control of the size and density of AuNPs on GC was examined by observing surface images with a field-emission scanning electron microscope (FE-SEM) after several preparations with different immersion times. Compared with previous results on the growth of AuNPs on indium tin oxide (ITO) surfaces, it was characteristic that the AuNPs attached to GC surfaces exhibited smaller size and higher density as well as a flatter and non-crystal-like morphology. In addition, for performing the dense attachment of regular nano-sized AuNPs on GC surfaces, immersion for 2 h into the growth solution was sufficient. Longer immersion for 24 h caused an irregular growth of bold Au micro-crystals, while 24 h was necessary in the case of AuNPs on ITO surfaces. Shorter seeding and growth times were found to be effective for a sparse attachment of smaller Au nanoparticles whose size was ca. 20 nm. It was clarified that the seed-mediated growth method for surface modification was valid for fabricating a nanointerface composed of AuNPs on GC surfaces.

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Gold nanorods (AuNRs), rod-like gold nanoparticles, show strong plasmon bands in the near-infrared region, based on the longitudinal oscillation mode of surface plasmons. In this research, we have coated AuNRs with silica or poly (sodium 4-styrenesulfonate) (PSS) in order to evaluate the effects of surface modifications as a sensing probe material of AuNR. Multilayered assemblies of the PSS-coated AuNRs have been successfully fabricated on the glass substrate by electrostatic layer-by-layer adsorption, and their plasmonic band profiles have been analyzed as a function of refractive index. The profiles showed satisfactory linear responses with the refractive indices. The effect of the thickness of silica layer on the shift of plasmon band with refractive index of the surrounding media has been investigated, along with, the effect of silica coating on the thermal stability of AuNR. The thermal stability of the silica-coated AuNR has also been investigated. The two types of the above-described coating approaches have been compared.

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We have prepared gold nanoparticles (GNPs) modified with a thiol compound that possesses a phenylurea moiety for the spectrophotometric sensing of hydrophilic anions, such as dihydrogen phosphate, based on changes in the surface plasmon absorption of the GNP. We examined the spectral change of phenylurea-modified GNP in dichloromethane upon the addition of various anions as tetrabutylammonium salts to the solution. The GNP showed increasing plasmon intensity with the concentration of dihydrogen phosphate. For a control experiment with an inactive hexanethiolate-modified GNP, such an ion-selective change in the plasmon band was not observed. Furthermore, in order to realize the spectrophotometric detection of hydrophilic anions in water using GNP with the urea functionality, we attempted to prepare bifunctional GNP modified with both the phenylurea derivative and a water-soluble thiol (e.g., L-cysteine). The resulting bifunctional GNP showed anion-selective changes in the plasmon band accompanied by increasing absorbance at a longer wavelength due to GNPs aggregation.

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A colorimetric change of gold nano-composites conjugated with a thermo-responsive copolymer has been applied to a colorimetric assay to quantify the activity of aminopeptidase N (APN). Heating a solution of assembled gold nano-composites provokes the gold nano-composites to disassemble, leading to a colorimetric change in the solution from blue-purple to red. The disassembly is inhibited by cysteine, but not by cysteinylglycine, thus allowing us to monitor the progress of an enzymatic decomposition of cysteinylglycine into cysteine and glycine with APN. The activity of APN is estimated through a colorimetric change in a solution of gold nano-composites from blue-purple to red, after being heated (98°C for 30 min), followed by being cooled. A good relationship between the a^* value in L^*a^*b^* color coordinates, which quantified the color of the solution, and the activity of APN was obtained in 3 h of the incubation time, indicating the potential of a colorimetric assay of APN activity.

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Nanoparticles are a key material in nanoscience and nanotechnology due to their unique physicochemical properties, so an analytical method is increasingly required. In the present research, we developed a method for individual nanoparticle detection by thermal lens microscopy and microfluidic chips. Pulsed signals were clearly observed, as nanoparticles were passing through the detection volume. The scale of the microfluidic channel was reduced from 100 to 1 µm to improve the detection efficiency. As a result, a detection efficiency of 100% was demonstrated.

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An electric field parallel to electrode arrays, which can be realized by switching DC electric fields, allows the electrophoretic levitation and focusing of negatively charged particles at the height of the anodes in the electrode arrays. Electric field calculations predict that charged particles will migrate parallel to the electrode array. The trajectories of particles well reflect the calculated electrical field. The present principle is also applied to the focusing and sweeping of the particles. Results obtained with varying experimental variables indicate that an increase in applied voltage results in a linear increase in an initial focusing rate but lowers the focusing efficiency. Also, the focusing efficiency is enhanced with decreasing intervals of field-switching. These results strongly suggest that the elimination of the large lateral movements of particles is important to attain high focusing efficiency.

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Realisation of a device intended for the manipulation and detection of bead-tagged DNA and other bio-molecules is presented. Acoustic radiation forces are used to manipulate polystyrene micro-beads into an optical evanescent field generated by a laser pumped ion-exchanged waveguide. The evanescent field only excites fluorophores brought within ∼100 nm of the waveguide, allowing the system to differentiate between targets bound to the beads and those unbound and still held in suspension. The radiation forces are generated in a standing-wave chamber that supports multiple acoustic modes, permitting particles to be both attracted to the waveguide surface and also repelled. To provide further control over particle position, a novel method of switching rapidly between different acoustic modes is demonstrated, through which particles are manipulated into an arbitrary position within the chamber. A novel type of assay is presented: a mixture of streptavidin coated and control beads are driven towards a biotin functionalised surface, then a repulsive force is applied, making it possible to determine which beads became bound to the surface. It is shown that the quarter-wave mode can enhance bead to surface interaction, overcoming potential barriers caused by surface charges. It is demonstrated that by measuring the time of flight of a microsphere across the device the bead size can be determined, providing a means of multiplexing the detection, potentially detecting a range of different target molecules, or varying bead mass.

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A measurement cell for DNA migration was fabricated by agarose gel on a glass plate for the purpose of in situ fluorescence microscopic measurements of the electrophoretic behaviors of single giant DNA (T4GT7DNA, 165600 base pairs) molecules. The cell was connected to an original circuit that can supply a constant electric field strength to a DNA-migration-space of the cell. Conformation and electrophoretic velocity of single DNA molecules were measured at the same time as a function of acid or spermidinium concentration. As a result, the conformational change from the random-coiled to globular states and reduced electrophoretic velocities were induced by an addition of acid or spermidinium. The change of the electrophoretic velocity was quantitatively attributable to the partial neutralization of anionic phosphate groups of DNA molecules by H⁺ or tricationic spermidinium. The acid-dissociation constant of the conjugate acid of phosphate group of the DNA molecules (pK_a) and the association constant of the phosphate groups of DNA molecule with tricationic spermidinium (K_ass) were determined to be 2.0 and 5.5 × 10³ dm³ mol^-1, respectively.

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Hydrodynamic chromatography of silica colloids with 5 - 78 nm was investigated for spherical nonporous silica packed columns. Use of phosphate buffer as the eluent improved peak shape of silica colloids and allowed elution of the analytes at times relating to their sizes. The larger the diameter of the silica colloids, the faster the elution time was. Elution volumes of analytes were slightly increased with decreasing eluent flow rate, which may be due to more diffusion to narrower interstitial channels at lower flow rates. The separation column packed with 1.9-µm nonporous silica achieved slightly better resolution than that with 3.1-µm nonporous silica. Silica colloids were monitored based on turbidity using a UV detector, and larger signals were observed for larger silica colloids.

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The mesostructure of nanoparticle stabilized interfacial films, in systems such as particle stabilized emulsions, plays a key, yet little understood, role. We have studied a system of gold nanoparticles being formed and aggregating at a planar oil/water interface using optical microscopy. A rapid “nucleation” step of meso (micron) scale aggregates was observed. Aggregation of the meso aggregates to form a percolating structure is seen to switch from a slow to a fast aggregation regime. Aggregation in the slow regime appears to be reaction limited. We tentatively attribute the fast aggregation regime to a capillary attraction.

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A microscopic measurement system of circular dichroic spectra of second harmonic generation, which is applicable to detect the chirality of micro-region of molecular aggregates, has been investigated. The performance of the system was demonstrated by the discrimination of chirality of molecular aggregates of porphyrin on a glass plate.

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