Kazuhiko Tsukagoshi’s research while affiliated with Doshisha University and other places

A novel HPLC system utilizing a phase-separation multiphase flow as the eluent has been developed, referred to as phase-separation mode. This research explores the influence of the porous structure in an octadecyl-modified silica (ODS) column (with a pore diameter of 12 nm) on chromatographic outcomes under the phase-separation mode in HPLC. The chromatograms obtained from the porous ODS column were compared to those generated with a non-porous ODS column. In preliminary experiments, twenty-four mixed solutions, comprising combinations of water/acetonitrile/ethyl acetate and water/acetonitrile, were introduced as eluents at a column temperature of 20 °C. A model mixture of 2,6-naphthalenedisulfonic acid (2,6-NDS) and 1-naphthol (1-NA) was injected into the system, with separation achieved in most solutions except for some highly organic solvent-rich solutions where 2,6-NDS eluted faster than 1-NA, indicating reverse-phase mode operation. Subsequently, the separation of the model mixture was assessed at 0 °C, and four specific ternary mixtures were analyzed in detail at both 20 °C and 0 °C. These ternary mixtures, defined by their volume ratios, exhibited a two-phase separation, establishing a phase-separation multiphase flow. Consequently, the solution flow was homogeneous at 20 °C and heterogeneous at 0 °C. For instance, solutions with water/acetonitrile/ethyl acetate ratios of 20:60:20 (organic solvent-rich) and 70:23:7 (water-rich) were introduced as eluents at both 20 °C and 0 °C. At 0 °C in the organic solvent-rich eluent, 1-NA eluted faster than 2,6-NDS, characteristic of the phase-separation mode. In contrast, the water-rich eluent resulted in faster elution of 2,6-NDS at both temperatures. The porous ODS column displayed improved separation efficiency at 0 °C compared to the non-porous column, which can be attributed to the porous effect under phase-separation conditions.

We developed a new type of HPLC system that uses phase-separation multiphase flow as an eluent. The separation mode is called phase-separation mode. A HPLC system with a packed separation column filled with non-porous octadecyl-modified silica (ODS) particles was used in the previous study. This study examined the effects of porous structure of the ODS column on chromatograms obtained by phase-separation mode in HPLC using porous ODS packed column (pore diameter, 12 nm). The obtained chromatograms were compared with those obtained by non-porous ODS column before. First, as preliminary experiments, twenty-four kinds of mixed solutions of water/acetonitrile/ethyl acetate and water/acetonitrile were supplied to the system to act as eluents at a columns temperature of 20°C. 2,6-Naphthalenedisulfonic acid (2,6-NDS) and 1-naphthol (1-NA) mixture was used as a model and mixed analyte was injected into the system. They were separated in each solution except for several highly organic solvent-rich solutions, in which 2,6-NDS eluted faster than1-NA. This means that HPLC worked under a reverse-phase mode for separation at 20°C. Next, the separation of the mixed analyte was examined on HPLC at a column temperature of 0°C, and then after judging the results four kinds of ternary mixed solutions were evaluated in detail as eluents on HPLC at 20°C and 0°C. Based on their volume ratio, the ternary mixed solutions acted as a two-phase separation mixed solution, leading to a phase-separation multiphase flow. Consequently, the solutions flowed homogeneously and heterogeneously in the column at 20°C and 0°C, respectively. For example, the ternary mixed solutions containing water/acetonitrile/ethyl acetate at volume ratios of 20:60:20 (organic solvent-rich) and 70:23:7 (water-rich) were delivered into the system as eluents in the column at 20°C and 0°C. In the organic solvent-rich eluent, the mixture of 2,6-NDS and 1-NA was separated at 0°C, the elution of 1-NA being faster than 2,6-NDS (phase-separation mode). In the water-rich eluent, the mixture of analytes was separated at both 20°C and 0°C, the elution of 2,6-NDS being faster than 1-NA. The separation at 0°C with porous ODS column was more effective than that with non-porous ODS column. These separation performance and elution order can be attributed to porous effect in the phase-separation mode at 0°C.

We have been developing an HPLC system that uses a phase-separation multiphase flow as the eluent. This separation mode is called phase-separation mode. In this study, we used a phase-separated multiphase flow as the eluent for the first time in an HPLC system equipped with a column packed with porous ODS-modified particles and investigated the effect of the pore size on the new separation mode. Porous ODS columns with pore sizes of 7 and 30 nm were used. A water/acetonitrile/ethyl acetate mixed solution was used as the two-phase separation mixed solution to separate and detect the model samples, 2,6-naphthalenedisulfonic acid (NDS) and 1-naphthol (NA). At a column temperature of 20 °C, the eluent did not separate into two phases, and NDS and NA were separated and detected in this order on both porous ODS columns (reversed phase mode). Next, the analysis was performed using two solutions with different composition ratios, an organic solvent-rich and a water-rich solution, as eluents at a column temperature of 0 °C to create a phase-separated multiphase flow within the column. In the organic solvent-rich solution, in both porous ODS columns, NA and NDS were separated and detected in opposite order at 20 °C (phase separation mode). However, differences in NA and NDS elution times were observed for columns with pore sizes of 7 and 30 nm. In the water-rich solution, NDS and NA were detected in both porous ODS columns in the same order as at 20 °C, but the elution time of NA was delayed. With the 7 nm pore size column, the delay in elution time of NA was only affected by temperature change, and no effect of phase-separation mode was observed. In the 30 nm pore size column, not only the effect of temperature change but also the effect of phase-separation mode was observed. The difference in separation behavior between porous ODS columns with pore diameters of 7 and 30 nm was discussed from the perspective of phase separation multiphase flow. Although this is a qualitative discussion, the experimental results show that the organic solvent-rich phase (organic solvent-rich cluster) generated in the phase-separation multiphase flow would more easily enter into pore size of 30 nm in the ODS-modified particles than that of 7 nm.

We have developed a HPLC system where phase-separation multiphase flow works in the separation column as an eluent. We call the novel separation mechanism a phase-separation mode. The ternary mixed solution of water/acetonitrile/ethyl acetate, which is one of the two-phase-separation mixed solutions, caused the phase-separation multiphase flow via phase change from homogeneous to heterogeneous with the temperature effect (from 20 to 0 °C). In this study, we tried to perform phase-separation multiphase flow with the pressure effect instead of the temperature one. The fused-silica capillary tube (50 cm length and 50 µm inner diameter) was allied to the downstream of the column to apply the pressure of 5.5 MPa to the system. Model analytes of 2,6-naphthalenedisulfonic acid (2,6-NDS) and 1-naphthol (1-NA) were examined. For example, solutions (the volume% of water/acetonitrile/ethyl acetate; 20:55:25, organic-rich) and (60:30:10, water-rich) were used as eluent. The model analytes were not separated with both solutions at the pressure of 1.5 MPa and 20 °C. But with the organic-rich solution, 1-NA and 2,6-NDS were separated in this order and with the water-rich solution, they were separated in the reverse order at the pressure of 5.5 MPa and 20 °C. The phase-separation mode could be performed at the high pressure even at the room temperature.

We developed a novel HPLC device where the phase-separation multiphase flow worked as the eluent in the separation column by using a water/acetonitrile/ethyl acetate triple mixed solution as a dual-phase-separation solution. Dual-phase-separation solutions form a phase-separation multiphase flow in a microscopic space. The new separation mechanism in the HPLC is called phase-separation mode. In this study, we used water and acetonitrile with NaCl mixed solution as a dual-phase-separation solution instead of the triple mixed solution. Octadecylsilyl (ODS)-modified particle- and porous silica particle-packed separation columns were united with the HPLC device for phase-separation mode caused by phase-separation multiphase flow. NA (1-naphthol) and NDS (2,6-naphthalenedisulfonic acid) were analyzed by the device as model sample. Using the water and acetonitrile with NaCl mixed solution at the solvent volume ratio of 5:5, NA and NDS were not separated on either column at 25 °C. On the other hand, they were separated with the order NDS and NA on the ODS column and separated with the order NA and NDS on the silica column in phase-separation mode at 0 °C. We discuss the separation mechanism of phase-separation mode using the water and acetonitrile with NaCl mixed solution at 0 °C.

A two-phase separation mixed solution can undergo phase separation from one phase to two phases (i.e., upper and lower phases) in a batch vessel in response to changes in temperature and/or pressure. This phase separation is reversible. When the mixed solution undergoes a phase change while being fed into a microspace region, a dynamic liquid–liquid interface is formed, leading to a multiphase structure. This flow is called a phase-separation multiphase flow. Annular flow in a microspace, which is one such phase-separation multiphase flow, is interesting and has been applied to chromatography, extraction, reaction fields, and mixing. Here, research papers related to phase-separation multiphase flows—ranging from the discovery of the phenomenon to basic and technical research from the viewpoint of analytical science—are reviewed. In addition, the development of a new separation mode in a high-performance liquid chromatography system based on phase-separation multiphase flow is introduced. Graphical abstract

The study reports the development of a high-performance liquid chromatography (HPLC) system in which a phase-separation multiphase flow as the eluent and a silica-particle based packed column as the separation column were combined to form the phase separation mode. Twenty-four types of mixed solutions of water/acetonitrile/ethyl acetate and water/acetonitrile were applied as eluents to the system at 20 °C. 2,6-Naphthalenedisulfonic acid (NDS) and 1-naphthol (NA) were injected as model analytes into the system. They showed separation tendency in organic solvent-rich eluents in normal-phase mode and NA was detected earlier than NDS. Subsequently, seven types of the ternary mixed solutions were examined as eluents in the HPLC system at 20 °C and 0 °C. These mixed solutions worked as a two-phase separation mixed solution, providing a phase-separation multiphase flow at 0 °C in the separation column. In the organic solvent-rich eluent, the mixture of analytes was separated at both 20 °C (normal-phase mode) and 0 °C (phase-separation mode), with NA being detected earlier than NDS. The separation at 0 °C was more efficient than at 20 °C. In the water-rich eluent, the mixture of NDS and NA was not separated at 20 °C but was separated at 0 °C (phase-separation mode), with NDS being detected earlier than NA. We also discussed the separation mechanism of phase-separation mode in HPLC together with the computer simulation for the multiphase flow in the cylindrical tube having sub-μm inner diameter.

To understand the mechanism of antibacterial activity of bulk Cu, N2-atomized Cu powder has been used instead. It was heated from 425 to 673 K in air for 4.2 • 10² s and measured by XRD and Chemi-luminescence (CL) to determine the contents of Cu2O and CuO, and reactive oxygen species (ROS), respectively; ROS being much affected by Cu2O and CuO can play a role to kill bacteria. Cu2O increased from 3.3 mass% (R.T.) to around 20% (673 K), however, CuO was almost constant (1.2~3.5%). High CL intensity was observed for Cu powders heated at 598~673 K. Then, a small amount of anatase TiO2 (a-TiO2) was added and heated at 673 K for 4.2 • 10² s in air, 1% O2-99% N2, and N2. The powder heated under 1% O2 showed the high CL intensity summation (∑CL), especially, 8.0 mol% a-TiO2 added and heated sample (“8.0Ti powder”) gave the about 4.7 times higher ∑CL than the starting material. ESR results using the spin-trap method showed that they contained hydroxyl radical •OH and its intensity increased from starting material to “8.0Ti powder”. However, bio-test proved that these samples revealed the same antibiotic activity. Formation mechanism of ROS (•OH and super oxide radical •O2⁻) on the surfaces of Cu/Cu2O/CuO has been proposed.

We developed a new type of HPLC system that uses phase-separation multiphase flow as an eluent. A commercially available HPLC system with a packed separation column filled with octadecyl-modified silica (ODS) particles was used. First, as preliminary experiments, 25 kinds of mixed solutions of water/acetonitrile/ethyl acetate and water/acetonitrile were supplied to the system to act as eluents at 20 °C. 2,6-Naphthalenedisulfonic acid (NDS) and 1-naphthol (NA) mixture was used as a model and mixed analyte was injected into the system. Roughly speaking, they were not separated in organic solvent-rich eluents and well separated in water-rich eluents, in which NDS eluted faster than NA. This means that HPLC worked under a reverse-phase mode for separation at 20 °C. Next, the separation of the mixed analyte was examined on HPLC at 5 °C, and then after judging the results, four kinds of ternary mixed solutions were in detail as eluents on HPLC at 20 °C and 5 °C. Based on their volume ratio, the ternary mixed solutions acted as a two-phase separation mixed solution, leading to a phase-separation multiphase flow. Consequently, the solutions flowed homogeneously and heterogeneously in the column at 20 °C and 5 °C, respectively. For example, the ternary mixed solutions containing water/acetonitrile/ethyl acetate at volume ratios of 20:60:20 (organic solvent-rich) and 70:23:7 (water-rich) were delivered into the system as eluents at 20 °C and 5 °C. In the organic solvent-rich eluent, the mixture of NDS and NA was not separated at 20 °C but was separated at 5 °C, the elution of NA being faster than the one of NDS (phase-separation mode). In the water-rich eluent, the mixture of analytes was separated at both 20 °C and 5 °C, the elution of NDS being faster than the one of NA. The separation at 5 °C was more effective than at 20 °C (reverse-phase mode and phase-separation mode). This separation performance and elution order can be attributed to the phase-separation multiphase flow at 5 °C.