Tetsuo Okada

Enhanced kinetics of pseudo first-order hydrolysis in liquid phase coexistent with ice.

The reaction rate of the hydrolysis of fluorescein diacetate (FDA) is several times larger in the frozen state than that in the unfrozen solution of the same composition at the same temperature. The freeze concentration of reactants in the liquid phase expelled form ice crystals cannot explain the kinetic enhancement of pseudo first order reactions such as the FDA hydrolysis. However, the reaction rate increases as the freeze concentration ratio becomes larger at a constant temperature. Direct pH measurements have revealed that the basicity of the liquid phase is unchanged at any concentration ratio, suggesting that the reactivity enhancement is not caused by increased basicity. The reaction rate enhancement is clearly related to the size of the space in which the liquid phase is confined upon freezing. The ice wall itself or the water structure formed near the wall should thus be responsible for this kinetic enhancement.

Fluidic Grooves on Doped-Ice Surface as Size-Tunable Channels

We propose a new principle for fabrication of size-tunable fluidic nano- and microchannels with a ubiquitous green material, water. Grooves filled with a solution are spontaneously formed on the surface of ice when an appropriate dopant is incorporated. Sucrose doping allows the development of grooves with lengths of 300u2009μm along the boundaries of ice crystal grains. This paper focuses on controlling the size of the liquid-filled groove and reveals its applicability to size-selective differentiation of nano- and micromaterials. The width of this groove can be varied in a range of 200u2009nm to 4u2009μm by adjusting the working temperature of the frozen platform. The channel dimension is reproducible as long as the same frozen condition is employed. We demonstrate the size-selective entrapment of particles as well as the state evaluation of DNA by controlling the physical interference of the ice wall with the electrophoretic migration of particles.

Freeze sample enrichment highly adaptable to capillary electrophoresis

We propose a novel sample enrichment scheme particularly suitable for capillary electrophoresis (CE). This method is based on the principle of freeze concentration. When an aqueous solution is frozen, solutes are expelled from the ice crystal phase. When an inert dopant is added to a sample, an unfrozen dopant solution is accumulated along ice grain boundaries. Analytes are also enriched in the liquid phase during this process. The liquid phase volume and in turn the enrichment factor can be controlled by adjusting the dopant concentration in an original sample solution and freezing temperature. The liquid volume is, in general, so small that it is difficult to separate it from the ice matrix for the following analysis. However, its application to CE is possible because a sample volume of nL is large enough for this method. Glycerol is a suitable dopant for the present purpose. Although disagreements between theoretical and experimental enrichment factors have been found, the addition of a low concentration of LiCl as a supporting electrolyte allows better predictions of enrichment factors. The enrichment by a factor of 1000 is possible by simple freezing of samples, and in addition, the simultaneous concentrations of anions and cations are also demonstrated.

Microfluidic protein crystallisation controlled using spontaneous emulsification

Microdroplet-based protein crystallisation using spontaneous emulsification is proposed and demonstrated. The dependence of water transport on the concentration of Span 80 was investigated. Crystallisation of the lysozyme was then demonstrated. Based on the results obtained, the dependency of the crystal number in a single microdroplet on the surfactant concentration was discussed.

Freeze Enrichment Protocol Based on Voltammetric Probing of Liquid-Phase Growth in Frozen Aqueous Electrolyte Solutions

Voltammetry of the reversible redox couple, Fe(CN)6(4-)/Fe(CN)6(3-), using a microdisc electrode, is employed to probe the dynamic process of the growth of the liquid phase (LP) in frozen aqueous NaCl solutions. Critical factors are the temperature history of a frozen sample and a freeze-concentration ratio. When the temperature is directly set to the working temperature after freezing at -25.0 °C, CV measurements are often impossible at high concentration ratios. Even if measurements are possible, the shapes of the CVs and the currents change with time over 3 h. Combined measurements of CV and chronoamperometry clearly indicate that the growth of the LP near the working electrode is responsible for these phenomena. The development of an LP along the electrode surface leads to a change in the shape of the CV due to an increase in the contribution from the radial diffusion. When the LP mainly grows orthogonal to the electrode surface, the peak-shaped CV representing linear diffusion is maintained over several hours. In contrast, when the temperature of a frozen sample increases up to -2.0 °C for annealing, reproducible cyclic voltammograms (CVs) are always measured at temperatures higher than -18 °C. This procedure allows us to handle frozen solutions as a deterministic system rather than a stochastic one. The present results strongly suggest that annealing of frozen samples is critical for successful uses of the freeze enrichment of trace solutes. Up to 1000-fold enrichments for voltammetric measurements are demonstrated by the proposed procedure involving an annealing step.

Chiral resolution with frozen aqueous amino acids

Frozen aqueous amino acids are screened to determine their chiral resolution using ice chromatography. Freezing allows convenient preparation of functional solid materials without organic syntheses. Frozen proline and leucine resolve the enantiomers of 1,1′-bi-2-naphthol (BINOL), while other amino acids tested do not show chiral selectivity. The effects seen when changing the pH of an amino acid solution before freezing suggest that the amino group of the amino acid plays an important role as a hydrogen bond acceptor. The molecular mechanism of chiral resolution is examined using Gaussian 9.0. A single proline molecule does not show chiral resolution capability. In contrast, two proline molecules fixed on the ice surface through the carboxyl groups show preferable interaction with the R enantiomer of BINOL. In the optimized structure of the Pro molecules on the ice surface, the distance between carboxyl oxygen and water oxygen on the ice surface ranges from 0.2533 to 0.2594 nm. The amino nitrogen atoms interact with the OH groups in BINOL with the distances of 0.2668 and 2.770 nm for the R-enantiomer and 0.2711 and 0.2717 nm for S-enantiomer. The preferable interaction with the R-enantiomer of BINOL is in accordance with the ice chromatography results, which reveal a stronger retentivity for the R enantiomer. These results strongly suggest that amino acids are expelled from the ice phase upon freezing, and form aggregates that provide multiple hydrogen bonding sites to enhance chiral resolution.

Enhanced chiral recognition by β-cyclodextrin at liquid/liquid interfaces as revealed by chromatographic and interfacial tension measurements

The chiral selectivity of β-cyclodextrin (β-CD) in the water/hexane (3.0% THF) two-phase system is studied with chromatography and interfacial tension measurements. Chromatography using silica gel impregnated with aqueous β-CDas the stationary phase reveals that the chiral selectivity of this system is higher than that predicted from the β-CD complexation that occurs in bulk water. The retention of the solutes and chiral selectivity can be explained by the adsorption of β-CD at the interface between the aqueous phase (AP) and hexane (3.0% THF) phase. The interfacial tension measurements suggest that β-CD molecules forms a monolayer at this interface. The interfacial complexation constants are larger than the corresponding bulk water constants by at least one order of magnitude. The β-CD molecules adsorbed at the interface are preferably oriented for the formation of inclusion complexes, and thereby solute molecules are directly accommodated in the CD cavity at the interface without partitioning into the AP. The present chromatography-based method probes the interfacial phenomena that are not accessible by other methods. Hence, the liquid/liquid interface is a new field for molecular recognition that does not occur in bulk solution phases.

Number Density of Liquid Inclusions Formed in Frozen Aqueous Electrolyte

Frozen aqueous chlorides (≤50 mM) are characterized by using confocal fluorescence microscopy and small angel X-ray scattering (SAXS). The former method allows us to determine the size of a liquid inclusion formed in the ice matrix at temperatures above the eutectic point of the system (t(eu)). Isolated liquid inclusions of a uniform size are formed when the temperature of a frozen electrolyte increases past t(eu). The size of the liquid inclusions depends on the observation temperature as well as on the concentration (c(salt)) and type of salt dissolved in the original unfrozen solution. However, the number density of liquid inclusions is almost constant and independent of these experimental parameters, particularly when an electrolyte is frozen in liquid nitrogen. Salt accumulation can then occur at the imperfections of the ice crystals. The occurrence probability of the imperfections is independent of the nature of an incorporated salt. The amount of a salt confined in each inclusion ranges from 7 to 240 fmol, depending on c(salt). SAXS measurements provide information on the size of individual salt crystals formed at temperatures below t(eu). The radius of gyration of a salt crystal ranges from 2 to 2.8 nm, and does not depend significantly on c(salt). Thus, each inclusion is formed from 10(6)-10(9) nanocrystals, which can act as seeds. When doped ice is prepared at higher temperatures, for example -16u2009°C, the isolation of liquid inclusions is not sufficient and coalescence occurs more easily upon an increase in temperature or cs(alt). However, when c(salt) is lower than 10 mM, the number density of liquid inclusions is almost constant, irrespective of the freezing temperature.

Freezing-Facilitated Dehydration Allowing Deposition of ZnO from Aqueous Electrolyte

When an aqueous NaCl solution with low concentrations of transition metal ions is frozen, most of the solutes are concentrated in the freeze concentrated solutions (FCS). However, Zn2+ is often accumulated at sites where other elements are not detected. X-ray absorption near-edge structure spectroscopy at the Zn K-edge indicates the formation of ZnO. A possible mechanism for the ZnO formation is discussed.

Zeta potential determination with a microchannel fabricated in solidified solvents

This paper proposes a simple and versatile method for the determination of the zeta potential of a channel wall and discusses the values measured for the surface of frozen solvents, which are not only of scientific interest but also of potential use for microfluidic platforms. The zeta potential of the solid surface is an important parameter for discussing its electrokinetic properties, the distribution and reaction of ions in an electric double layer, and the fluidic behavior in the space surrounded by the surface. While the zeta potential of colloidal matters can be determined from their electrophoretic mobility, it is often difficult to determine that of a bulk material. In this paper, the zeta potential of a microchannel fabricated in a frozen solvent is determined by measuring the apparent mobility of microparticles as the probe. The electrophoretic mobility of the microparticles has been measured in advance using microchip electrophoresis under various conditions. This approach allows us to determine the zeta potential of water-ice and frozen cyclohexane. We discuss the pH dependence of the zeta potential of ice and also effects of the NaCl concentration on that of ice and frozen cyclohexane.