Development of an HPLC device with water/acetonitrile with NaCl mixed solution that induces a phase-separation multiphase flow as the eluent in the separation column.

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.

Phase-separation multiphase flow: preliminary application to analytical chemistry.

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.

Development of a HPLC system using a phase-separation multiphase flow as an eluent coupled to a silica-particle packed column.

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.

Development of HPLC system that uses phase-separation multiphase flow as an eluent.

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.

Observation of the phase-separation multiphase flow using a polyethylene glycol/phosphate mixed solutions and the aqueous two-phase distribution of red blood cells in the flow system.

Phase-separation multiphase flow at a liquid-liquid interface was successfully formed in an aqueous two-phase system of polyethylene glycol/phosphate mixed solutions when fed into a microchannel (100 µm wide and 40 µm deep) on a microchip and a fused-silica capillary tube (100 µm ID). As one example, tube radial distribution flow (annular flow) was observed when 10.0 wt% polyethylene glycol 6000 and 8.5 wt% dipotassium hydrogen phosphate aqueous solution containing 1.0 mM Rhodamine B was fed at 40 â„ƒ, recorded by bright field microscopy. It exhibited a dipotassium hydrogen phosphate-rich inner phase and polyethylene glycol-rich outer phase. Effects of conditions including composition, flow rate, viscosity, and contact angle on tube radial distribution flow were analyzed. It was found out that although the viscosity of PEG-rich solution was much higher than that of phosphate-rich one, the phase configuration in tube radial distribution flow did not necessarily obey the viscous dissipation law in untreated microchannel and capillary tube, as well as for all the types of PEG/phosphate mixed solution the PEG-rich solution occupied the outer phase near the ODS-treated inner wall of both microchannel and capillary tube against the law. To assess the use of microfluidic flow in applications, we examined the distribution of red blood cells in the inner and outer phases fed into double capillary tubes with different inner diameters. Cell distribution was found to concentrate in the inner (dipotassium hydrogen phosphate-rich) phase compared to the outer (polyethylene glycol-rich) phase at a ratio of 1.8.

Novel separation mode of HPLC based on phase-separation multiphase flow.

A novel separation mode for high-performance liquid chromatography (HPLC) is proposed based on phase-separation multiphase flow. A commercially available HPLC system was used with a packed-separation column of octadecyl-silica (ODS)-modified particles. Water/acetonitrile/ethyl acetate ternary mixed solutions, (a) 1:8:1, (b) 1:3:1, and (c) 16:3:1 (v/v/v), were delivered into the system as an eluent at 20 and 5 °C. The ternary mixed solution acted as a two-phase separation solution leading to phase-separation multiphase flow. The solution flowed in the column homogeneously and heterogeneously at 20 and 5 °C, respectively. 1-Naphthol (NA) and 2,6-naphthalenedisulfonic acid (NDS) were injected into the system as model analytes. At 20 °C, the analyte mixture did not separate in solutions (a) and (b) while it separated in solution (c) with the elution order of NDS followed by NA. At 5 °C, it did not separate in solution (a), while it separated in solution (b) with elution order of NA followed by NDS and solution (c) with elution order of NDS followed by NA more effectively than 20 °C. The separation behavior and elution order are possibly caused by the phase-separation multiphase flow.

Microfluidic behavior of ternary mixed solutions of water/acetonitrile/ethyl acetate through experiments and computer simulations.

When ternary mixed solutions of water/acetonitrile/ethyl acetate are delivered into a microspace under laminar flow conditions, the solvent molecules show specific microfluidic flows, such as microfluidic inverted flow and tube radial distribution flow, which have been applied to novel analytical methods. In this paper, inverted flow was examined using various Y-type microchannels that had mixing angles of 0°, 90°, 180°, and 270°. Inverted flow was experimentally observed and the trigger phenomenon was also successfully expressed through computer simulations. Tube radial distribution flow, that is, annular flow, in a capillary tube is reported to cause exchange of the inner and outer phases based on the solvent composition of the ternary mixed solution. Tube radial distribution flow for an organic solvent-rich inner and a water-rich outer phases, as well as for a water-rich inner and an organic solvent-rich outer phases, could be well recreated by computer simulations for a ternary mixed solution. This highlights the effectiveness of computer simulations for such flow scenarios and will allow optimization of the operating conditions and design of microfluidic analytical devices.

Consecutive Sample Injection Analysis in Tube Radial Distribution Chromatography.

Tube radial distribution chromatography based on the tube radial distribution flow, or annular flow, in an open-tubular capillary has been reported, where the annular flow is created through phase-separation multiphase flow. We have proposed the first-ever procedure for consecutive sample injection analysis using chromatography. In basic terms, a commercially available HPLC system could be used with a sample injector (0.2 µL volume) and a fused-silica capillary tube (250 cm long) as a separation column instead of a normal packed one, while the built-in detection cell was replaced by improved on-capillary detection. A ternary mixed solution of water/acetonitrile/ethyl acetate (3:8:4 volume ratio) was delivered into the capillary tube as an eluent at a flow rate of 2.0 µL min-1. Model sample solutions of 1-naphthol and 2,6-naphthalenedisulfonic acid were consecutively analyzed by the present chromatography with a processing rate of 6 samples per hour. Simple and rapid consecutive analysis could be performed because washing and initialization of the separation tube was no longer necessary. The obtained results provide clues to developing new methodologies which combine features of both chromatography (separation) and the flow injection method (consecutive analysis).

Phase Separation and Collection of Annular Flow by Phase Transformation.

A polyethylene glycol/citrate mixed solution was fed into a single channel of a Y-type micro-channel on a microchip as an aqueous two-phase system. A phase separation multi-phase flow with a liquid-liquid interface was generated due to a phase transformation. An annular flow, one of the flow types in the phase separation multi-phase flow, was observed through bright-field microscopy. The flow consisted of citrate-rich inner and polyethylene glycol-rich outer phases. We attempted to separate and collect the two phases in the single channel into two separate Y-type channels. When the pressure losses for the separated channels were not very different, we observed symmetric flow in the Y-type channel. When the pressure losses were quite different, the polyethylene glycol-rich phase with higher viscosity was selectively distributed to the separated channel with lower pressure loss. Thus, the polyethylene glycol-rich phase was successfully and intentionally collected from the chosen Y-type channel via the creation of annular flow in the single channel.

Development of Tube Radial Distribution Chromatography Based on Phase-Separation Multiphase Flow Created via Pressure Loss.

A tube radial distribution chromatography (TRDC) method based on phase-separated multiphase flow created through phase transformation via temperature change has been developed. These systems typically required a temperature-controlling device containing a water bath and a stirrer. Herein, we proposed a novel TRDC system without a cooling device, where the phase transformation was achieved via pressure loss in a capillary tube of 50 µm inner diameter and 550 cm length. Model analytes were successfully separated with the developed TRDC system, which provided a simplified platform and helped to clarify the operating principle of TRDC based on phase transformation in a capillary tube.

Microfluidic Inverted Flow of Ternary Water/Hydrophilic/Hydrophobic Organic Solvent Solution in a Y-Type Microchannel and a Proposal of the Response Microfluidic Analysis through the Experiment.

Two solutions that are individually fed at the same flow rate into two separate microchannels of a microchip, combine to form a single channel (a Y-type microchannel). This flow is either parallel for immiscible solutions or initially parallel, but then becomes homogeneous through diffusion, for miscible solutions. However, a new type of microfluidic behavior in a Y-type microchannel that was neither parallel nor homogeneous flow has been observed using, for example, water/acetonitrile (3:4.5, v/v) and acetonitrile/ethyl acetate (3.5:4, v/v) mixed solutions. Each mixed solution was marked with distinctive dyes and delivered at the same flow rate into a Y-type microchannel under laminar flow conditions. In the single channel, the two phases were initially observed to flow in parallel, but then apparently swapped to flow on the opposite wall while retaining parallel flow with a slight change in the components of the two phases. We have named this type of laminar flow "microfluidic inverted flow" for ternary water/hydrophilic/hydrophobic organic solvent mixed solutions. The inverted flow of a ternary water/acetonitrile/ethyl acetate system was examined in detail under various flow conditions. We also proposed a concept of response microfluidic analysis based on such microfluidic inverted flow.

Phase Separation Multi-phase Flow Using an Aqueous Two-phase System of a Polyethylene Glycol/Dextran Mixed Solution.

Polyethylene glycol/dextran mixed solution as an aqueous two-phase system was fed into a fused-silica capillary tube under different conditions, resulting in phase transformation leading to phase separation multi-phase flow through/along a liquid-liquid interface. As one flow-type example, when 6.4 wt% polyethylene glycol and 9.7 wt% dextran aqueous solution containing 1.0 mM Rhodamine B was fed into the capillary tube at 3°C, tube radial distribution flow (annual flow) was observed through bright-field microscopy. Tube radial distribution flow consisted of a dextran-rich inner phase and polyethylene glycol-rich outer phase. We also examined the distribution of proteins, such as bovine serum albumin, hemoglobin, and lysozyme, in the inner and outer phases through use of double capillary tubes with different inner diameters. The protein distribution was greater in the inner (dextran-rich) phase than the outer (polyethylene glycol-rich) phase. The distribution ratios of the three proteins (ratio of the inner/outer protein concentration) were 2.3, 4.2, and 1.8, respectively. The proteins concentrated in the dextran-rich phase through tube radial distribution flow of a polyethylene glycol/dextran mixed solution.

Tube radial distribution chromatography system developed by combining commercially available HPLC system and open-tubular capillary tube as separation column.

Tube radial distribution chromatography based on tube radial distribution flow, or annular flow, in an open-tubular capillary has been reported, where the annular flow is created through phase separation multiphase flow. The chromatographic system requires some specific instruments and treatments for microfluidic flow in the capillary tube. In this study, we developed a new set-up for tube radial distribution chromatography by combining a commercially available HPLC system with an open-tubular capillary tube (with an inner diameter of 100 µm) as a separation column instead of a conventional packed column. The analyte solution was injected with an injection valve (2 µL volume) and a ternary solution of water/acetonitrile/ethyl acetate (3:8:2 vol ratio) was delivered as the eluent to the capillary tube at a flow rate of 8.6 µL min-1. The chromatographic system, that is, the HPLC system equipped with the open-tubular capillary tube, could successfully separate the model analytes, 1-naphthol, 1-naphthalenesulfonic acid, and 2,6-naphthalenedisulfonic acid, with base-line separation. The inner and outer phases in the annular flow worked as the mobile and pseudo-stationary phases, respectively, in the tube radial distribution chromatography system. The experimentally obtained elution times of the analytes were compared with their corresponding theoretical values calculated using their capacity factors for the inner and outer phases and the linear flow velocities of the respective phases.

Implementation of Tube Radial Distribution Chromatography by Using a Commercially Available HPLC System.

Tube radial distribution chromatography based on tube radial distribution flow, or annular flow, in an open-tubular capillary has been reported. The chromatographic system requires specific instruments and treatments for microfluidic flow in the capillary tube. In this study, we have developed a new model of tube radial distribution chromatography, which is comprised of a commercially available HPLC system without any packed separation columns. Separation is performed in an open-tubular pipe (100-µm inner diameter and 350-cm length; temperature, 5°C) connected between the pump and the detector in the HPLC system. An analyte solution is introduced with a sample injector (2-µL volume) and a ternary water/acetonitrile/ethyl acetate mixed solution (volume ratio of 3:8:2) is delivered as an eluent solution into the pipe at a flow rate of 10-µL min-1. Fused silica and stainless pipes can separate 1-naphthol and 2,6-naphthalenedisulfonic acid, but a polyetheretherketone pipe cannot. The obtained data provides an important clue to practical developments in separation science.

Protein separation through preliminary experiments concerning pH and salt concentration by tube radial distribution chromatography based on phase separation multiphase flow using a polytetrafluoroethylene capillary tube.

Protein mixtures were separated using tube radial distribution chromatography (TRDC) in a polytetrafluoroethylene (PTFE) capillary (internal diameter=100µm) separation tube. Separation by TRDC is based on the annular flow in phase separation multiphase flow and features an open-tube capillary without the use of specific packing agents or application of high voltages. Preliminary experiments were conducted to examine the effects of pH and salt concentration on the phase diagram of the ternary mixed solvent solution of water-acetonitrile-ethyl acetate (8:2:1 volume ratio) and on the TRDC system using the ternary mixed solvent solution. A model protein mixture containing peroxidase, lysozyme, and bovine serum albumin was analyzed via TRDC with the ternary mixed solvent solution at various pH values, i.e., buffer-acetonitrile-ethyl acetate (8:2:1 volume ratio). Protein was separated on the chromatograms by the TRDC system, where the elution order was determined by the relation between the isoelectric points of protein and the pH values of the solvent solution.

A poly(dimethylsiloxane) microfluidic sheet reversibly adhered on a glass plate for creation of emulsion droplets for droplet digital PCR.

A PDMS microfluidic chip with T-junction channel geometry, two inlet reservoirs, and one outlet reservoir was reversibly adhered on a glass plate through the viscoelastic properties of PDMS. This formed a detachable microfluidic device for creation of water-in-oil emulsion droplets that were used as discrete reaction compartments for the droplet digital PCR. The PDMS/glass device could continuously produce monodisperse droplets without leakage of fluids using a vacuum-driven autonomous micropumping method. This droplet preparation technique only required evacuation of air dissolved in the PDMS before loading of oil and aqueous phases into separate inlet reservoirs. Degassing of the PDMS chip at approximately 300 Pa for 1.5 h in a vacuum desiccator gave 40 000 droplets in 80 min, which corresponded to a generation frequency of up to nine droplets per second. Over multiple runs the droplet creation was very reproducible, and the size reproducibility of generated droplets (polydispersity of up to 4.1%) was comparable to that acquired using other microfluidic droplet preparation techniques. Because the PDMS chip can be peeled off the glass plate, blocked channels can easily be fixed when they arise, and this extends the lifetime of the chip. Single DNA molecules partitioned into the droplets were successfully amplified by PCR. In addition, the droplet digital PCR platform allowed absolute quantification of low copy numbers of target DNA, and was robust against instrumental variance.

Tube Radial Distribution Flow Separation in a Microchannel Using an Ionic Liquid Aqueous Two-Phase System Based on Phase Separation Multi-Phase Flow.

Ionic liquid aqueous two-phase systems were delivered into a capillary tube to achieve tube radial distribution flow (TRDF) or annular flow in a microspace. The phase diagram, viscosity of the phases, and TRDF image of the 1-butyl-3-methylimidazolium chloride and NaOH system were examined. The TRDF was formed with inner ionic liquid-rich and outer ionic liquid-poor phases in the capillary tube. The phase configuration was explained using the viscous dissipation principle. We also examined the distribution of rhodamine B in a three-branched microchannel on a microchip with ionic liquid aqueous two-phase systems for the first time.

Consideration of Inner and Outer Phase Configuration in Tube Radial Distribution Phenomenon Based on Viscous Dissipation in a Microfluidic Flow Using Various Types of Mixed Solvent Solutions.

When mixed solvent solutions, such as ternary water-hydrophilic/hydrophobic organic solvents, water-surfactant, and water-ionic liquid, are delivered into a microspace under laminar flow conditions, the solvent molecules radially distribute in the microspace, generating inner and outer phases. This specific fluidic behavior is termed "tube radial distribution phenomenon", and has been used in separation technologies such as chromatography and extraction. The factors influencing the configuration of the inner and outer phases in "tube radial distribution phenomenon" using the above-mentioned mixed solvent solutions were considered from the viewpoint of viscous dissipation in fluidic flows. When the difference in the viscosity between the two phases was large (approximately >0.73 mPa·s), the phase with the higher viscosity formed as an inner phase regardless of the volume ratio. The distribution pattern of the solvents was supported by the viscous dissipation principle. Contrarily, when the difference was small (approximately <0.49 mPa·s), the phase with the larger volume formed as the inner phase. The distribution pattern of the solvents did not always correspond to the viscous dissipation principle. The current findings are expected to be useful in analytical science including microflow analysis research.

Improvement of the Mutation-Discrimination Threshold for Rare Point Mutations by a Separation-Free Ligase Detection Reaction Assay Based on Fluorescence Resonance Energy Transfer.

We previously developed a separation-free ligase detection reaction assay based on fluorescence resonance energy transfer from a donor quantum dot to an acceptor fluorescent dye. This assay could successfully detect one cancer mutation among 10 wild-type templates. In the current study, the mutation-discrimination threshold was improved by one order of magnitude by replacing the original acceptor dye (Alexa Fluor 647) with another fluorescent dye (Cyanine 5) that was spectrally similar but more fluorescent.

Tube Radial Distribution Chromatography on a Microchip Incorporating Microchannels with a Three-to-One Channel Confluence Point.

We developed a capillary chromatography system using a phase-separated solvent mixture as a carrier solution--i.e., a water-hydrophilic/hydrophobic organic solvent mixture--which we call "tube radial distribution chromatography" (TRDC). Here, we attempted to apply the TRDC system to a microchip incorporating microchannels with a double T-junction for injection of analyte solution and a three-to-one, narrow-to-wide channel confluence point for tube radial distribution phenomenon (TRDP) at room temperature. A ternary mixed solvent of water, acetonitrile and ethyl acetate was used as a carrier solution. TRDP in the wide microchannel was examined using various flow rates, temperatures, and component solvent ratios. Successful observation was carried out using a fluorescence microscope-CCD camera. Model analytes perylene (hydrophobic) and Eosin Y (hydrophilic) were separated by flowing through the microchannel, without any treatment such as packed columns or coating, at room temperature (25°C).