Thin Layer Chromatography-Freeze Surface-Enhanced Raman Spectroscopy: A Powerful Tool for Monitoring Synthetic Reactions.

Thin layer chromatography (TLC) is widely used to confirm the formation of the target compound in chemical synthesis. The key issue in TLC is spot identification as it primarily relies on retention factors. Coupling of TLC with surface-enhanced Raman spectroscopy (SERS), which gives direct molecular information, is an appropriate choice, to overcome this challenge. However, interference from the stationary phase and impurities on the nanoparticles added for SERS measurements significantly degrades TLC-SERS efficiency. It was found that freezing effectively eliminates such interferences and dramatically improves the performance of TLC-SERS. In this study, TLC-freeze SERS is applied to the monitoring of four chemically important reactions. The proposed method can identify the product and side-products of similar structures, detect compounds with high sensitivity, and provide information of quantities that allows the reliable determination of the reaction time based on kinetic analysis.

Freeze Surface-Enhanced Raman Scattering Coupled with Thin-Layer Chromatography: Pesticide Detection and Quantification Case.

Thin-layer chromatography (TLC) is widely used in various branches of chemical science to separate components in complex mixtures because of its simplicity. In most cases, analyte spots are visually detected by fluorescence, and the retention factor (Rf) is determined from the distance traveled by the analyte. Further characterizations are often necessary to identify separated chemicals because molecular information other than Rf is not available. Surface-enhanced Raman scattering (SERS) has been coupled with TLC to complement molecular information. In previously reported TLC-SERS, metal nanoparticle suspension was dropped onto analyte spots to obtain SERS spectra. This approach is simple and efficient for SERS measurements on the TLC plate but has limited sensitivity for several reasons, such as the low solubility of analytes in the dropped solution, difficult control of nanoparticle aggregation, and interference from the stationary phase. We recently showed that freezing enhances SERS sensitivity by a factor of ∼103. Freezing simultaneously concentrates analytes and silver nanoparticles (AgNPs) in a freeze concentrated solution, where aggregation of AgNPs is facilitated, allowing sensitive freeze SERS (FSERS) measurements. Here, we discuss FSERS measurements on TLC plates to demonstrate the superiority of this combination, i.e. TLC-FSERS. Freezing enhances SERS sensitivity by freeze concentration and facilitated aggregation of AgNPs and, in addition, eliminates interference from the stationary phase. Under the optimized condition, TLC-FSERS enables the on-site detection of pesticides at the nM level. The use of the SERS signal from adenine added as the internal standard allows us to quantify pesticides. Applications to a commercial green tea beverage are also demonstrated.

Aqueous-nonaqueous solvent-switching ion chromatography of halide impurities in ionic liquids.

We here propose an efficient solvent-switching preconcentration method for the ion-chromatographic (IC) determination of halide impurities contained ionic liquids (ILs). Because halide impurities strongly affect the physicochemical properties of ILs, their analysis is an important task for the successful utilization of ILs. Although IC is an efficient method for this purpose, its application still involves significant challenges. The major halide impurities, such as F- and Cl-, show much smaller retention in aqueous anion-exchange chromatography than IL component anions. Therefore, if an IL sample is directly analyzed by IC with aqueous mobile phases, the halide impurities are eluted earlier, whereas the IL component anion is hardly eluted and gives a large peak once eluted. Thus, the introduction of the IL component anions into the IC separation column should be avoided for efficient analyses and also for preventing the degradation of the column by the accumulation of the IL anions in it. This problem, which arises from the ion-exchange selectivity in aqueous media, is solved by a solvent switching preconcentration method. The anion-exchange selectivity in aqueous media is reversed by a use of an aprotic solvent, such as acetonitrile (MeCN). Hence, we have come up with the idea of preconcentrating anions in MeCN and stripping them with an aqueous mobile phase for IC analysis. The introduction of the IL component anions into the IC separation column is substantially reduced while maintaining high sensitivity for the halide impurities. Sub µM impurities are detectable in the mM level of ILs.

Surface-enhanced Raman scattering of DNA bases using frozen silver nanoparticle dispersion as a platform.

Raman spectroscopy is a powerful method to characterize molecules in various media. Although surface-enhanced Raman scattering (SERS) is often employed to compensate for the intrinsically poor sensitivity of Raman spectroscopy, there remain serious tasks, such as simple preparations of SERS substrates, sensitivity control, and reproducible measurements. Here, we propose freezing as an efficient way to overcome these problems in SERS measurements using DNA bases as model targets. Solutes are expelled from ice crystals and concentrated in the liquid phase upon freezing. Silver nanoparticles (AgNPs) are also concentrated in the liquid phase to aggregate with Raman target analytes. The SERS signal intensity is maximized when the AgNP concentration exceeds the critical aggregation value. Freezing allows up to 5000 times enhancements of the SERS signal. Thus, an efficient SERS platform is prepared by simple freezing. The simultaneous detection of four DNA bases effectively eliminates variations of signal intensities and allows the reliable determination of concentration ratios.

Microscale pH inhomogeneity in frozen NaCl solutions.

When an aqueous solution freezes at temperatures above the eutectic point, a freeze concentrated solution (FCS) is separated from the ice phase. Reactions of environmental importance often occur in the FCS and, in some cases, are accelerated compared to those in solution conditions. The pH of the FCS is an essential factor governing the thermodynamics and kinetics of the reactions occurring therein. It is known that freezing of aqueous NaCl causes an increase in the FCS pH, which arises from the difference in the partition to the ice phase between Na+ and Cl-. It has also been shown that H+ and other ions show surface-specific behaviors on ice. Although the details are not known, the ice/FCS interface can also affect the behaviors of ions. In this study, the pH distribution in the FCS is evaluated using ratiometric fluorescence microscopy, and the pH inhomogeneity is confirmed for frozen aqueous NaCl. However, interestingly, buffered solutions and frozen aqueous glycerol result in a uniform pH value. The pH in frozen NaCl is always higher near the ice/FCS interface than in the middle of the FCS vein.

Quantification using statistical parameters derived from signal intensity distributions in surface enhanced Raman scattering (SERS).

Raman spectroscopy is a powerful method, which provides information on molecular structures, conformations, interactions etc. However, its applications are severely restricted because of low sensitivity. Although surface enhanced Raman scattering (SERS) significantly enhances sensitivity and enables single-molecular detection, quantification by this method is still challenging because of large signal fluctuations. In the present study, the signal intensity distributions (SIDs) in SERS of adenine and thymine on the silver nanoparticle (AgNP) platform are analyzed based on more than 10000 spectra to pursue the possibility of SERS quantification. The signals always involve large fluctuations but show statistically relevant patterns. SIDs are well represented by the exponentially modified Gaussian function, which is characterized by reproducible parameters. Thus, robust quantification is feasible using the parameters derived from the SIDs. At least 200 spectra for a given concentration are necessary to derive reproducible parameter values from the SID. The mean signal intensity determined from the SIDs is proportional to the adenine concentration in the range of 10-75 µM. However, this parameter becomes independent of the adenine concentration in the lower concentration range. In such concentrations, minor events, which give distinct SERS spectra, occasionally occur but have only marginal impacts on the mean signal intensity. The corrected standard deviation of the SID, which is estimated from the complementary error function, well represents the minor events and provides a clear correlation with the concentration in the range of 0.5-7.5 µM. Furthermore, the quantification in the nanomolar range is made possible by the incorporation of sample freezing, which enables to enrich target analytes and AgNPs in a liquid phase confined by ice.

Structures of ions accommodated in salty ice Ih crystals.

Frozen aqueous electrolytes are ubiquitous and involved in various phenomena occurring in the natural environment. Although salts are expelled from ice during freezing of aqueous solutions, minor amounts of the constituent ions are accommodated in the crystal lattice of ice. This phenomenon was associated with the generation of the Workman-Reynolds freezing potential. Molecular simulations also confirmed the ion incorporation in the crystal lattice of ice Ih upon freezing of aqueous electrolytes and identified possible local structures of the ions. However, no experimental information is available on the structure of ions accommodated in the crystal lattice of ice Ih. In this work, we use X-ray absorption fine structure (XAFS) to study the local structures of K+ and Cl- accommodated in ice Ih single crystals. Previous molecular simulations predicted that ions are trapped in the hexagonal cavities of the ice structure or replace two water molecules in the crystal lattice. Four possible configurations are considered and optimized by the calculations using ONIOM (QM/QM/QM). The results are evaluated in terms of the agreement between the experimental XAFS spectra and those simulated from the optimized structures. The spectra are most reasonably interpreted by assuming that K+ replaces one water molecule in the ice crystal lattice and is accommodated in a tetrahedral coordination cage. Similarly, Cl- probably adopts the same configuration, because it explains the coordination number better than other structures, such as that assuming the replacement of two water molecules belonging to the same hexagonal planes.

Directional Supramolecular Polymerization in a Dynamic Microsolution: A Linearly Moving Polymer's End Striking Monomers.

Although directional chain reactions are common in nature's self-assembly processes and in covalent polymerizations, it has been challenging to perform such processes in artificial one-dimensional self-assembling systems. In this paper, we describe a system, employing perylene bisimide (PBI) derivatives as monomers, for selectively activating one end of a supramolecular polymer during its growth and, thereby, realizing directional supramolecular polymerization. Upon introduction of a solution containing only a single PBI monomer into the microflow channel, nucleation was induced spontaneously. The dependency of the aggregation efficiency on the flow rate suggested that the shear force facilitated collisions among the monomers to overcome the activation energy required for nucleation. Next, by introducing a solution containing both monomer and polymer, we investigated how the shear force influenced the monomer-polymer interactions. In situ fluorescence spectra and linear dichroism revealed that growth of the polymers was accelerated only when they were oriented under the influence of shear stress. Upon linear motion of the oriented polymer, polymer growth at that single end became predominant relative to the nucleation of freely diffusing monomers. When applying this strategy to a two-monomer system, the second (less active) monomer reacted selectively at the forward-facing terminus of the first polymer, leading to the creation of a diblock copolymer through formation of a molecular heterojunction. This strategy-friction-induced activation of a single end of a polymer-should be applicable more generally to directional supramolecular block copolymerizations of various functional molecules, allowing molecular heterojunctions to be made at desired positions in a polymer.

Hydration of bromide at reverse micelle interfaces studied by X-ray absorption fine structure.

Nanoconfined water exhibits various interesting properties, which are not only of fundamental importance but also of practical use. Because reverse micelles (RMs) provide versatile ways to prepare nanoconfined water, the understanding of their physicochemical properties is essential for developing efficient applications. Although the water properties in the RMs could be affected by its interaction with the RM interface, the details have not been well understood. This study focuses on the local structures of Br- in hexadecyltrimethylammonium bromide (HTAB) RMs formed in chloroform and 10% hexanol/heptane. The dependence in Br- hydration on the molar ratio of water to HTAB (w) is investigated using X-ray absorption fine structure (XAFS). These systems cover a wide range of w values (0-30) and allow us to study the impact of this parameter on the local structure of Br- at the RM interface, which comprises water, surfactant headgroups, and organic solvent components. The presence of multiple scattering paths complicates the XAFS spectra and makes it difficult to analyze them using standard fitting methods. The linear combination of the spectra corresponding to the individual scattering paths captures the molecular processes that occur at the RM interface upon increasing w. The maximum hydration number of Br- is found to be 4.5 at w > 15, suggesting that although most of the ions remain at the interface as partly hydrated ions, some of them dissociate as completely hydrated ones.

Controlling water transport between micelles and aqueous microdroplets during sample enrichment.

Droplet microfluidics technologies have advanced rapidly, but enrichment in droplets has still been difficult. To deterministically control the droplet enrichment, the water transport from an aqueous microdroplet in organic continuous phase containing span 80 micelles was investigated. Organic phase containing Span-80-micelles contacted a NaCl aqueous solution to control hydration degree of the micelles, prior to being used in the microfluidic device. Then, the organic phase was continuously applied to the microdroplets trappled in microwells. Here, water was transported from the microdroplet to the organic phase micelles. This spontaneous emulsification process induced the droplet shrinkage and stopped when the microdroplet reached a certain diameter. The micelle hydration degree correlated well with the final water activity of droplets. The enrichment factor can be determined by the initial microdroplet salt concentration and by the micelle hydration degree. As a proof-of-concept experiment, enrichment of fluorescent nanoparticles and dye was demonstrated, and fluorescent resonance energy transfer was observed as expected. Another demonstration of bound-free separation was performed utilizing the avidin-biotin system. This technique has the potential to be a powerful pretreatment method for bioassays in droplet microfluidics.

Particle Manipulation with External Field; From Recent Advancement to Perspectives.

Physical forces, such as dielectric, magnetic, electric, optical, and acoustic force, provide useful principles for the manipulation of particles, which are impossible or difficult with other approaches. Microparticles, including polymer particles, liquid droplets, and biological cells, can be trapped at a particular position and are also transported to arbitrary locations in an appropriate external physical field. Since the force can be externally controlled by the field strength, we can evaluate physicochemical properties of particles from the shift of the particle location. Most of the manipulation studies are conducted for particles of sub-micrometer or larger dimensions, because the force exerted on nanomaterials or molecules is so weak that their direct manipulation is generally difficult. However, the behavior, interactions, and reactions of such small substances can be indirectly evaluated by observing microparticles, on which the targets are tethered, in a physical field. We review the recent advancements in the manipulation of particles using a physical force and discuss its potentials, advantages, and limitations from fundamental and practical perspectives.

Multiphase Behavior of Tetraphenylethylene Derivatives with Different Polarities at High Pressures.

Although both pressure and temperature are essential parameters governing thermodynamics, the effects of the pressure on solution-phase equilibria have not been well studied compared to those of temperature. Here, we demonstrate the interesting pressure-dependent behavior of tetraphenylethylene (TPE) derivatives in multiphase systems composed of an organic phase and an aqueous phase in the presence and absence of γ-cyclodextrin (γ-CD). In this system, tetraphenylethylene monocarboxylic acid (TPE1H) and its dicarboxylic acid (TPE2H2) are distributed in the aqueous phase and dissociated into the corresponding anions, that is, TPE1- and TPE22-, when the pH is sufficiently high. The distribution ratios of TPE1H/TPE1- and TPE2H/TPE22- show opposing pressure dependencies: the distribution of the former in the organic phase increases with increasing pressure, whereas that of the latter decreases. The 1:1 complexation constants of TPE1- and TPE22- with γ-CD, which can be determined from the distribution ratios in the presence of γ-CD, also show opposing pressure dependencies: the former shows a positive pressure dependence, but the latter exhibits a negative one. These pressure effects on the distribution and complexation of TPE derivatives can be interpreted based on the differences in the molecular polarity of these solutes. The water permittivity is enhanced at high pressure, thus stabilizing the more polar TPE22- in the aqueous phase to a larger extent than TPE1- and, as a result, reducing its distribution in the organic phase, as well as its complexation with γ-CD. Fluorescence spectra in the aqueous phase suggest that the TPE derivatives form aggregates with γ-CD molecules, as detected by the specific fluorescence. In addition, the fluorescence intensities of the γ-CD complexes are enhanced at high pressures because of the restricted rotation of the phenyl rings in the TPE molecules. This study provides new perspectives for multiphase partitioning and an attractive alternative to conventional extraction methods.

Reverse micelle chromatography for evaluation of partition of organic solutes to micellar pseudophases.

The evaluation of solute partition from an organic solvent to a reverse micelle (RM) is critical for designing effective reaction systems and for synthesizing functional materials using the nano water-phase in the RM. Although spectroscopic methods have been extensively employed for determining the partition constants of solutes in the RM systems, their applications are limited to the case, in which the background absorption is low and the spectroscopic features of the solute are effectively varied by its partitioning to RMs. This paper proposes a novel chromatographic method to overcome this limitation of conventional methods. In the present system, a size-exclusion chromatographic column is used with RM solutions as mobile phases. The RM is excluded from the stationary phase and, therefore, is eluted first. A solute is eluted with the retention volume determined by its affinities to the stationary phase and the RM. The elution volumes of a solute measured by varying the concentrations of RM in the mobile phase allow us to determine its partition constant to the RM. The partition of phenols in hexadecyltrimethylammonium chloride and bromide RMs in chloroform is successfully evaluated without interferences from the UV-absorption of the media. The larger partition constant is confirmed for a smaller water/surfactant molar ratio (w) and for chloride than bromide as the RM counterion. This suggests that the nature of water is more strongly influenced by confinement in the RM cores for smaller w and the chloride counterion because water molecules are strongly imbibed by the interface.

Ice Confinement-Induced Solubilization and Aggregation of Cyanonaphthol Revealed by Fluorescence Spectroscopy and Lifetime Measurements.

When an aqueous salt solution freezes, a freeze-concentrated solution (FCS) separates from the ice. The properties of the FCS may differ from those of a supercooled bulk solution of the same ionic strength at the same temperature. The fluorescence and lifetime characteristics of 6-cyano-2-naphthol (6CN) were studied in frozen NaCl solutions in order to provide insight into the solution properties of the FCS. While the photoacidity of 6CN in an FCS is similar to that in solution, several anomalous behaviors are observed. Fluorescence spectra indicate that the solubility of 6CN is significantly enhanced in the FCS (50 mM or higher) compared to that in the bulk NaCl solution where the solubility limit is 250 µM. The high solubility induces the aggregation of 6CN in the FCS, which is not detected in bulk solutions. This trend becomes marked as the initial NaCl concentration decreases and the FCS is confined in a small space. The fluorescence lifetimes of 6CN in the FCS support the spectroscopy results. In addition to the species identified by fluorescence spectroscopy, excimers are assigned from lifetime measurements in the FCS. The excimer formation is also a result of the enhanced solubility of 6CN in the FCS.

Interaction between antifreeze protein and ice crystal facet evaluated by ice-channel electrophoretic measurements of threshold electric field strength.

The chemical interaction between antifreeze proteins (AFPs) and ice crystals is evaluated via electrophoresis of AFP-anchored microparticles in fluidic channels formed in frozen aqueous sucrose. Straight fluidic channels are created in a flat glass chamber connecting two Ag/AgCl electrodes. This configuration allows us to estimate an electric field strength exerted on probe particles migrating along the channel. When the channel width is comparable to the particle size, the particle is immobile because of the resistance force induced by the interaction with the ice wall. However, when the overall electrophoretic force surpasses the resistance force, the microsphere starts to migrate. From the threshold electric field strengths determined for unmodified and AFP-modified particles, the resistance forces for the chemical interaction between AFPs and ice wall are estimated.

Aptamer-Based Sensing of Small Organic Molecules by Measuring Levitation Coordinate of Single Microsphere in Combined Acoustic-Gravitational Field.

We present aptamer-based sensing using a coupled acoustic-gravitational (CAG) field, which transduces a change in the density of a microparticle (MP) to a change in the levitation coordinate. A large density of the MP is initially induced by the binding of gold nanoparticles (AuNPs) on the MP through sandwich hybridization with aptamer DNA molecules. Targets added to the system interact with the aptamer DNA molecules to form complexes, and the duplex between the aptamer and the probe DNA molecules is dissociated. This leads to the release of AuNPs from the MP and a decrease in its density. As the target concentration increases, the levitation coordinate of the MP increases. From the levitation coordinate shift, we can determine the target concentration. The detection limits for adenosine triphosphate, dopamine, and ampicillin as test targets are 9.8 nM, 17 nM, and 160 pM, respectively. The dissociation constants for the aptamer-target complexes are quantitatively determined from the dependence of the levitation coordinate on the target concentration. This scheme is a useful analytical tool not only for the trace analyses of targets but also for the evaluation of aptamer-target interactions.

Space Size-Dependent Transformation of Tetraphenylethylene Carboxylate Aggregates by Ice Confinement.

Tetraphenylethylene carboxylate (TPEC) aggregates are transformed by ice confinement, which is controlled by the initial concentration of sucrose employed as a cryoprotectant and temperature. The freezing of aqueous sucrose leads to the formation of micro- or nanoliquid phase confined in ice. Aggregation-induced emission (AIE) of tetraphenylethylene carboxylate (TPEC) in the ice-confined space is explored using fluorescence spectroscopy and lifetime measurements. The characteristics of AIE in the ice-confined space strongly depend on the initial sucrose concentration and temperature, which determine the size of the liquid phase. The AIE of TPEC in the ice-confined space can be classified into three regimes in terms of spectroscopic features. Loosely packed J aggregates of TPEC are formed in the microliquid phase (>2 µm). The fluorescence intensity increases, and the wavelength is hypsochromically shifted with a decrease in the size of the space, indicating that the molecular arrangement in the aggregate depends on the space size. The fluorescence lifetimes indicate polydisperse, loosely packed aggregation. No further change in aggregate structure is observed once the liquid phase size is decreased to ∼2 µm, and a spectroscopically identical structure is maintained upon further reduction of the space size to ∼0.5 µm. The molecular arrangement in the aggregate is independent of the space size in this regime. However, when the size of the space becomes smaller than ∼0.5 µm, the aggregate structure again starts to change into a more tightly packed aggregate and a hypsochromic shift of the fluorescence wavelength occurs again. The fluorescence lifetime indicates monodispersed aggregation in this submicrospace.

Frozen Solution-Mediated Asymmetric Synthesis: Control of Enantiomeric Excess.

Asymmetric List-Mannich reactions were carried out in the frozen state to afford optically active adducts in moderate-to-good chemical yields and enantiomeric excesses (ee). The frozen solution exerts critical control of ee via entropy changes, in sharp contrast to the enthalpy-driven asymmetric reactions typically observed in homogeneous solvents. This study provides new perspectives for asymmetric syntheses and an attractive alternative to conventional media.

Hydrostatic Pressure-Induced Spectral Variation of Reichardt's Dye: A Polarity/Pressure Dual Indicator.

The famous solvatochromic Reichardt's dye was applied to quantify hydrostatic pressure in media. The UV/vis spectra of the dye in various organic solvents are shifted bathochromically or hypsochromically at the shorter- or longer-wavelength band, respectively, upon hydrostatic pressurization. The E T value, determined by an absorption maximum, in ethyl acetate increases from 38.5 kcal mol-1 at 0.1 MPa to 39.2 kcal mol-1 at 300 MPa, which is mostly equal to the one in chloroform at 0.1 MPa. These spectroscopic origins were supported by the time-dependent density functional theory (TD-DFT) calculations. The concept and approach proposed in this paper, i.e., a dual indicator, should attract the attention of a broad spectrum in multidisciplinary science.

Size-Tunable Micro-/Nanofluidic Channels Fabricated by Freezing Aqueous Sucrose.

Upon freezing aqueous sucrose at temperatures higher than the eutectic point (-14 °C in this case), two phases, that is, ice and freeze concentrated solution (FCS), are spontaneously separated. FCS forms through-pore fluidic channels when thin ice septum is prepared from aqueous sucrose. Total FCS volume depends on temperature but is independent of the initial sucrose concentration. This allows us to control the size of the FCS channels simply by changing the initial sucrose concentration as long as temperature is kept constant. In this paper, we show that the size of the channel, which has a layered structure, can be controlled in a range from 50 nm to 3 µm. Thus, the FCS channel is suitable for size-sorting of micro- and nanoparticles. We discuss the size-sorting efficiency of the channel and demonstrate the separation of particles with different sizes.