Classification method and field flow fractionation device

Abstract

A classification method according to the present invention comprises: a first step for focusing on target particles in a separation channel; a second step for subjecting the target particles to a cross flow flowing in a first direction, and then distributing the target particles in the first direction in the separation channel; a third step for distributing the target particles after the second step in the longitudinal direction in the separation channel such that a first particle group and a second particle group included in the target particles can flow out, in that order, from an outlet port of the separation channel; a fourth step for subjecting the second particle group after the third step to a reversed cross flow flowing in a second direction, which is the direction opposite to the first direction, thereby moving the second particle group in the second direction from the side of a membrane in the separation channel; a fifth step for causing the first particle group and the second particle group to flow out, in that order, from the outlet port to outside of the separation channel; and a sixth step for detecting, using a detector, the second particle group that has been made to flow out through the outlet port.

Description

Classification method and field-flow fractionation device

[0001] The present invention relates to a classification method and a field-flow fractionation device.

[0002] A cross-flow type field flow fractionation (FFF) device is known as a field flow fractionation device (hereinafter also referred to as an “FFF device”) that classifies particles contained in a liquid (for example, JP 2020-124662 A (Patent Document 1)).

[0003] An FFF device has a separation channel whose wall is partially constituted by a membrane. In an FFF device, when particles flowing through the separation channel are pushed toward the membrane by crossflow, relaxation occurs, in which the particles diffuse in a direction away from the membrane. At this time, the particles diffuse while forming a distribution according to differences in particle properties such as particle size, mass, and interaction between the particles and the membrane. The liquid flowing through the separation channel becomes a parabolic flow with a parabolic flow velocity distribution (flow velocity profile). Therefore, if particles diffuse while forming a distribution as described above, the particles can be classified based on differences in particle properties.

[0004] Japanese Patent Application Laid-Open No. 2020-124662

[0005] When particle distribution occurs as described above, particles present near the membrane are less likely to flow out of the separation channel, and it can take a long time to classify the particles in the sample.

[0006] An object of the present invention is to provide a classification method for efficiently classifying target particles in a sample using a field-flow fractionation device, and a field-flow fractionation device for the classification method.

[0007] A first aspect of the present invention is a classification method for classifying target particles in a sample using a field flow fractionation device, wherein the field flow fractionation device comprises: a separation channel having a longitudinal direction and an outlet port; a membrane arranged along the longitudinal direction and constituting a part of the wall surface of the separation channel; a discharge chamber arranged along the longitudinal direction so as to face the separation channel and whose wall surface is constituted by the membrane; and a detector for detecting the target particles flowing out of the outlet port, wherein the target particles include a first particle group and a second particle group that differ from each other in at least one property selected from the group consisting of particle diameter, mass, and strength of interaction with the membrane, and the classification method comprises: a first step of supplying a liquid and the sample into the separation channel and focusing the target particles in the separation channel; and a second step of applying a cross flow that flows in a first direction from the separation channel side toward the discharge chamber via the membrane to the target particles after focusing, and then distributing the target particles in the first direction in the separation channel according to the property. a third step of distributing the target particles in the longitudinal direction within the separation channel after the second step by supplying a liquid into the separation channel to form a parabolic flow that flows in the longitudinal direction and has a parabolic flow velocity distribution in the first direction, thereby allowing the first particle group and the second particle group to flow out of the outlet port in this order; a fourth step of applying a reverse cross flow that flows in a second direction opposite to the first direction to the second particle group after the third step, thereby moving the second particle group from the membrane side within the separation channel in the second direction; a fifth step of causing the first particle group and the second particle group to flow out of the separation channel from the outlet port in this order; and a sixth step of detecting the second particle group flowed out of the outlet port in the fifth step with the detector.

[0008] a flow rate regulator for regulating the flow rate of liquid discharged from the discharge chamber; a second liquid supply unit for supplying liquid to the discharge chamber, or between the discharge chamber and the flow rate regulator, for supplying liquid to the discharge chamber without passing through the separation channel; a detector for detecting the target particles flowing out from the outlet port; and a controller for controlling at least the first liquid supply unit, the flow rate regulator, the second liquid supply unit, and the detector. the target particles include a first particle group and a second particle group that differ from each other in at least one property selected from the group consisting of particle diameter, mass, and the magnitude of interaction with the membrane; and under the control of the control unit, in a first operation, the sample supplied from the sample supply unit and the liquid supplied from the first liquid supply unit are supplied into the separation channel so as to focus the target particles; and in a second operation, a cross flow that flows in a first direction from the separation channel side toward the discharge chamber through the membrane acts on the target particles after focusing, and then liquid is supplied from the first liquid supply unit into the separation channel so as to distribute the target particles in the first direction in the separation channel according to the property, and liquid is discharged from the discharge chamber at a flow rate adjusted by the flow rate adjuster to form the cross flow; in a third operation, after the second operation, a liquid is supplied from the first liquid supply unit into the separation channel to distribute the target particles in the longitudinal direction within the separation channel so that the first particle group and the second particle group can flow out of the outlet port in this order, thereby forming a parabolic flow that flows in the longitudinal direction and has a parabolic flow velocity distribution in the first direction;a fourth operation, in which a liquid is supplied from the second liquid supply unit into the discharge chamber so as to move the second particle group after the third operation from the membrane side within the separation channel in a second direction opposite to the first direction, thereby forming a reverse crossflow flowing in the second direction; a fifth operation, in which a liquid is supplied from the first liquid supply unit into the separation channel so that the first particle group and the second particle group flow out of the separation channel from the outlet port in this order; and a sixth operation, in which the second particle group flowed out of the outlet port in the fifth operation is detected by the detector.

[0009] According to the present invention, target particles in a sample can be efficiently classified using a field-flow fractionation device.

[0010] FIG. 1 is a schematic diagram showing the configuration of an FFF device according to one embodiment of the present invention. FIG. 2 is a schematic perspective view showing a separation channel of the FFF device shown in FIG. 1. FIG. 3 is a block diagram showing the configuration of a control unit and each unit controlled by the control unit of the FFF device shown in FIG. 1. FIG. 4 is a diagram showing a schematic explanation of an example of a first operation of an FFF device according to one embodiment of the present invention. FIG. 5 is a diagram showing a schematic explanation of an example of an operation that may be included in the first operation or the second operation of an FFF device according to one embodiment of the present invention. FIG. 6 is a diagram showing a schematic explanation of an example of a third operation of an FFF device according to one embodiment of the present invention. FIG. 7 is a diagram showing a schematic explanation of an example of a fourth operation of an FFF device according to one embodiment of the present invention. FIG. 8 is a flowchart showing a classification method according to one embodiment of the present invention. FIG. 9 is a schematic diagram showing the configuration of an FFF device according to another embodiment of the present invention. FIG. 10 is a graph showing changes in flow rate of crossflow and reverse crossflow in a comparative example and a reference example. Chromatograms obtained in a comparative example and a reference example.

[0011] Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

[0012] <FFF Apparatus (Field Flow Fractionation Apparatus)> Fig. 1 is a schematic diagram showing the configuration of an FFF apparatus according to one embodiment of the present invention. Fig. 2 is a schematic perspective view showing a separation channel of the FFF apparatus shown in Fig. 1. Fig. 3 is a block diagram showing the configuration of a control unit and each unit controlled by the control unit of the FFF apparatus shown in Fig. 1.

[0013] The FFF apparatus 1 of this embodiment is an apparatus for classifying target particles in a sample. The FFF apparatus 1 may be a so-called asymmetric-flow field flow fractionation (AF) apparatus.

[0014] The sample contains target particles. The sample is usually a dispersion liquid in which target particles are dispersed, and may also contain particles other than the target particles. The target particles are particles intended to be classified using the FFF device 1. The target particles are two or more types of particles that differ from each other in at least one property selected from the group consisting of particle diameter, mass, and the magnitude of interaction with the membrane (hereinafter sometimes referred to as "particle property"). The target particles include a first particle group and a second particle group that differ from each other in particle property.

[0015] The target particle is not particularly limited, and may be, for example, one or more particles selected from the group consisting of proteins, DNA, oligomers, dendrimers, polymers, gels, agglutinating particles, liposomes, lipid nanoparticles (LNPs), exosomes, emulsions, bacteria, viruses, algae, and protozoans.

[0016] As shown in FIG. 1 , the FFF apparatus 1 may include a separation channel 10, a membrane 11, a discharge chamber 20, a sample supply unit 31, a first liquid supply unit, a flow rate regulator 43, a second liquid supply unit, a detector 42, and a control unit 5 ( FIG. 3 ). The first liquid supply unit is configured to supply liquid to the separation channel 10 without passing through the discharge chamber 20. The second liquid supply unit is configured to supply liquid to the discharge chamber 20 without passing through the separation channel 10. The FFF apparatus 1 may further include pumps 32 and 34, a switching valve 33, a container 35, an on-off valve 41, and pipes p31 to p37 and p41 to p43. The separation channel 10 may include a first inlet port 13, a second inlet port 14, an outlet port 15, and the like. The discharge chamber 20 may include a porous support 21, a discharge port 22, an introduction port 23, and the like.

[0017] The separation channel 10 is formed, for example, in a cylindrical shape with both ends closed and has an internal space. A portion of the wall of the separation channel 10 is formed by a membrane 11 ( FIGS. 1 and 2 ). The internal space of the separation channel 10 is formed along the membrane 11 and serves as a flow path for a sample or liquid. The separation channel 10 communicates with a discharge chamber 20 via the membrane 11. The separation channel 10 may have a first inlet port 13 formed at a first end, which is one end in the longitudinal direction L, an outlet port 15 formed at a second end, which is the other end in the longitudinal direction L, and a second inlet port 14 formed between the first inlet port 13 and the outlet port 15. The first inlet port 13, the second inlet port 14, and the outlet port 15 communicate the internal space of the separation channel 10 with the outside without passing through the discharge chamber 20.

[0018] As shown in Fig. 2, the separation channel 10 has a polygonal shape in plan view, with the length in the longitudinal direction L being greater than the width of the separation channel 10. The width of the separation channel 10 is the length in the direction perpendicular to the longitudinal direction L in plan view. The width of the separation channel 10 is formed to have a portion that narrows continuously (linearly) from the first inlet port 13 side toward the outlet port 15 side in the longitudinal direction L. As shown in Fig. 2, the width of the separation channel 10 may be formed to have a portion on the first end side (first inlet port 13 side) that widens continuously in the longitudinal direction L, and to have a portion that narrows continuously from that portion.

[0019] As shown in FIG. 1 , the first inlet port 13 is connected to a sample supply unit 31 via, for example, a pipe p31. The sample supply unit 31 is connected to the discharge side of a pump 32 via a pipe p32. The suction side of the pump 32 is connected to a pipe p33 disposed in a container 35 that contains a liquid (eluent) flowing through the flow path of the separation channel 10. The pump 32 draws the liquid contained in the container 35 through the pipe p33 and supplies it to the separation channel 10 via the pipe p32, the sample supply unit 31, the pipe p31, and the first inlet port 13. When a sample is supplied from the sample supply unit 31, the sample is also supplied into the separation channel 10 through the first inlet port 13 together with the liquid flowing through the separation channel 10.

[0020] The sample supply unit 31 is configured to supply a sample into the separation channel 10. When the sample is automatically supplied to the FFF apparatus 1, the sample supply unit 31 may be an autosampler, and when the sample is manually supplied to the FFF apparatus 1, the sample supply unit 31 may have a sample supply port or may be a sample supply port.

[0021] The second inlet port 14 is connected to a switching valve 33 via, for example, a pipe p34. The switching valve 33 is connected to the discharge side of a pump 34 via a pipe p36. The suction side of the pump 34 is connected to a pipe p37 disposed in a container 35 containing a liquid (eluent). The pump 34 sucks the liquid contained in the container 35 through the pipe p37 and supplies it to the switching valve 33 via the pipe p36. The switching valve 33 selectively switches between a path (1) that supplies the liquid to the separation channel 10 without passing through the discharge chamber 20 and a path (2) that supplies the liquid to the discharge chamber 20 without passing through the separation channel 10. In the FFF apparatus 1, the path (1) is connected to the second inlet port 14, and the path (2) is connected from the switching valve 33 via a pipe p35 to the introduction port 23 of the discharge chamber 20.

[0022] When the switching valve 33 is switched to a state in which liquid is supplied to the separation channel 10 without passing through the discharge chamber 20, the liquid sucked by the pump 34 is supplied into the separation channel 10 through the pipe p36, the switching valve 33, the pipe p34, and the second inlet port 14. When the switching valve 33 is switched to a state in which liquid is supplied to the discharge chamber 20 without passing through the separation channel 10, the liquid sucked by the pump 34 is supplied into the discharge chamber 20 through the pipe p36, the switching valve 33, the pipe p35, and the introduction port 23.

[0023] The outlet port 15 is connected to an on-off valve 41 via, for example, a pipe p41. The on-off valve 41 is connected to a detector 42 via a pipe p42. When the on-off valve 41 is switched to an open state, the liquid flowing through the separation channel 10, the target particles, etc. can be discharged to the outside of the separation channel 10 through the outlet port 15.

[0024] The detector 42 detects the target particles that have flowed out of the separation channel 10 through the outlet port 15. The detector 42 is not particularly limited as long as it can detect the target particles. The detector 42 may be, for example, one or more detectors selected from the group consisting of an ultraviolet absorbance detector, a photodiode array detector, a fluorescence detector, and a multi-angle light scattering detector.

[0025] The membrane 11 is not particularly limited as long as it is impermeable to the target particles. The membrane 11 may be permeable to liquids and may also be permeable to particles other than the target particles. The membrane 11 may be selected depending on the particle characteristics of the target particles. The membrane 11 may be a porous membrane formed of one or more materials selected from the group consisting of regenerated cellulose, polyethersulfone, and polycarbonate.

[0026] The membrane 11 can have a flat membrane shape as shown in Figures 1 and 2. The membrane 11 may be supported by a porous support 21 (Figure 1). The porous support 21 may be arranged on the discharge chamber 20 side of the membrane 11, may be arranged inside the discharge chamber 20, or may be arranged so as to fill the entire internal space of the discharge chamber 20, for example. The porous support 21 has porosity that allows the components that have permeated the membrane 11 to pass through. The porous support 21 may be a porous sintered body (porous frit) or a mesh-structured sieve.

[0027] The discharge chamber 20 is disposed opposite the separation channel 10 along the longitudinal direction L, and the membrane 11 forms part of the wall surface. The internal space of the discharge chamber 20 and the internal space of the separation channel 10 can face each other via the membrane 11. The discharge chamber 20 has an internal space. Components of the sample and liquid flowing through the separation channel 10 that have permeated the membrane 11 can be introduced into the discharge chamber 20. Liquid drawn from a container 35 by a pump 34 can also be introduced into the discharge chamber 20 through an introduction port 23. Once the liquid introduced into the discharge chamber 20 permeates the membrane 11, it is introduced into the separation channel 10. As described above, a porous support 21 may be disposed within the discharge chamber 20. The discharge chamber 20 can have a discharge port 22 and an introduction port 23.

[0028] The discharge port 22 communicates the internal space of the discharge chamber 20 with the outside without passing through the separation channel 10. The discharge port 22 is connected to a flow rate regulator 43, for example, via a pipe p43. The flow rate regulator 43 regulates the flow rate of the liquid discharged from the discharge chamber 20 through the discharge port 22 and the pipe p43. The flow rate regulator 43 is, for example, a mass flow controller or a flow rate adjustment valve.

[0029] The introduction port 23 communicates between the outside and the inside of the discharge chamber 20 without passing through the separation channel 10. The introduction port 23 is connected to the switching valve 33, for example, via a pipe p35. As described above, when the switching valve 33 is switched to a state in which liquid is supplied to the discharge chamber 20 without passing through the separation channel 10, the liquid sucked by the pump 34 is supplied into the discharge chamber 20 through the switching valve 33, the pipe p35, and the introduction port 23.

[0030] The control unit 5 controls the pumps 32, 34, the switching valve 33, the on-off valve 41, the detector 42, the flow rate regulator 43, and the like ( FIG. 3 ). When the sample supply unit 31 is an autosampler, the control unit 5 also controls the sample supply unit 31 ( FIG. 3 ). The control unit 5 includes a CPU (central processing unit), a ROM (read-only memory), a RAM (random access memory), and the like. The ROM can store in advance programs and the like for the FFF apparatus 1 to perform classification operations including the first to seventh operations described below. The CPU executes the programs stored in the ROM on the RAM, thereby performing the classification operations by the FFF apparatus 1.

[0031] 1, the pumps 32 and 34, the pipes p31 to p34 and p36, the switching valve 33, the first inlet port 13, and the second inlet port 14 are examples of a first liquid supply unit. In the FFF apparatus 1, the sample supply unit 31 also constitutes a part of the first liquid supply unit. In the FFF apparatus 1 shown in FIG. 1, the pump 34, the pipes p34 to p36, and the introduction port 23 are examples of a second liquid supply unit.

[0032] <Operation of FFF Apparatus> Fig. 4 is a diagram schematically illustrating an example of a first operation of the FFF apparatus according to one embodiment of the present invention. Fig. 5 is a diagram schematically illustrating an example of an operation that may be included in the first or second operation of the FFF apparatus according to one embodiment of the present invention. Fig. 6 is a diagram schematically illustrating an example of a third operation of the FFF apparatus according to one embodiment of the present invention. Fig. 7 is a diagram schematically illustrating an example of a fourth operation of the FFF apparatus according to one embodiment of the present invention. In Figs. 4 to 7, the left side in the longitudinal direction L is the first inlet port 13 side, and the right side in the longitudinal direction L is the outlet port 15 side.

[0033] The FFF apparatus 1 performs a classification operation to classify target particles contained in a sample. The classification operation includes first to sixth operations and is controlled by the control unit 5. The classification operation may also include a seventh operation. The following describes an example in which the sample supply unit 31 is an autosampler and the control unit 5 controls the operation of the sample supply unit 31. In cases such as when a sample is manually supplied from the sample supply unit 31, the control unit 5 may not control the sample supply unit 31.

[0034] In the first operation, the sample supplied from the sample supply unit 31 and the liquid supplied from the first liquid supply unit are supplied into the separation channel 10 so as to focus the target particles within the separation channel 10. Focusing the target particles means gathering the target particles near the position where the carrier flow Fc and the focus flow Ff formed within the separation channel 10 collide, as will be described later.

[0035] In the first operation, the controller 5 controls at least the first liquid supply unit and the sample supply unit 31. In the first operation, the controller 5 may control, for example, the pumps 32 and 34, the sample supply unit 31, and the switching valve 33. In the first operation, the on-off valve 41 may be open or closed. When the controller 5 operates the pump 32, the liquid contained in the container 35 is aspirated through the pipe p33 and supplied into the separation channel 10 via the pipe p32, the sample supply unit 31, the pipe p31, and the first inlet port 13. This forms a carrier flow Fc flowing in the longitudinal direction L through the separation channel 10 from the first inlet port 13 side toward the outlet port 15 side ( FIG. 4 ). At this time, by supplying a sample from the sample supply unit 31 under the control of the controller 5, the sample is introduced into the liquid introduced to the sample supply unit 31, and the liquid and the sample are supplied into the separation channel 10.

[0036] In parallel with the operation of the pump 32, the control unit 5 switches the switching valve 33 so that liquid is supplied to the separation channel 10 without passing through the discharge chamber 20. When the control unit 5 operates the pump 34 in this state, the liquid contained in the container 35 is sucked through the pipe p37 and supplied into the separation channel 10 via the pipe p36, the switching valve 33, the pipe p34, and the second inlet port 14. This forms a focus flow Ff that flows in the separation channel 10 in a direction opposite to the carrier flow Fc ( FIG. 4 ). The direction opposite to the carrier flow Fc is the direction of flow in the longitudinal direction L through the separation channel 10 from the second inlet port 14 side toward the first inlet port 13 side.

[0037] When the carrier flow Fc and the focus flow Ff are formed in the separation channel 10, the target particles in the sample are gathered near the position where the carrier flow Fc and the focus flow Ff collide (the target particles are focused), as shown in Figure 4. In this way, the target particles are focused by supplying a sample into the separation channel 10 and supplying a liquid into the separation channel 10 so as to form the carrier flow Fc and the focus flow Ff in the separation channel 10.

[0038] In the first operation, a cross flow F1 may be formed in the separation channel 10 in addition to the carrier flow Fc and the focus flow Ff ( FIG. 4 ). As described below, the cross flow F1 is a flow that flows in a first direction D1 from the separation channel 10 through the membrane 11 toward the discharge chamber 20. The cross flow F1 can be formed, for example, by supplying liquid from the first liquid supply unit and discharging the liquid from the discharge chamber 20 by adjusting the flow rate of the flow rate regulator 43 using the control unit 5. By forming the cross flow F1 in the first operation, target particles that have gathered in the longitudinal direction L can also be moved in the first direction D1 toward the membrane 11 in the separation channel 10 ( FIG. 5 ). When the cross flow F1 is formed in the first operation, it is preferable that the flow rate of the cross flow F1 is constant.

[0039] In the second operation, a crossflow F1 flowing in a first direction D1 is applied to the target particles after focusing in the first operation. Liquid is then supplied from the first liquid supply unit into the separation channel 10 so as to distribute the target particles in the first direction D1 within the separation channel 10 according to the particle characteristics of the target particles. Liquid is also discharged from the discharge chamber 20 at a flow rate adjusted by the flow rate adjuster 43, forming the crossflow F1. The process of diffusing the target particles, which have been focused and moved toward the membrane 11 by the action of the crossflow F1, in the first direction D1 to form a distribution according to the particle characteristics of the target particles is generally referred to as relaxation. As described below, in relaxation, the target particles that have moved toward the membrane 11 within the separation channel 10 are distributed in the first direction D1, and therefore the target particles diffuse in a direction (second direction D2) from the membrane 11 toward the separation channel 10.

[0040] In the second operation, the control unit 5 controls at least the first liquid supply unit and the flow rate adjustment unit 43. In the second operation, the control unit 5 may control, for example, the pumps 32 and 34, the sample supply unit 31, the flow rate adjustment unit 43, and the switching valve 33. In the second operation, the on-off valve 41 may be in an open state or a closed state.

[0041] The second operation can be performed following the first operation. In the second operation, the controller 5 stops the sample supply unit 31, which was operating in the first operation, and maintains the pumps 32 and 34 and the switching valve 33 in the states they were in in the first operation, thereby adjusting the flow rate of the flow rate adjuster 43. The flow rate of the flow rate adjuster 43 is adjusted so that a cross flow F1 is generated in the separation channel 10. As a result, a carrier flow Fc, a focus flow Ff, and a cross flow F1 are formed in the separation channel 10.

[0042] In the second operation, when the cross flow F1 acts on the target particles focused in the first operation, the target particles are subjected to a force in the flow direction (first direction D1) of the cross flow F1, and move toward the membrane 11 (FIG. 5). This operation may be performed in the first operation if the cross flow F1 is formed in the first operation described above.

[0043] In the second operation, the cross flow F1 is applied to the target particles that have moved toward the membrane 11 so that they diffuse (relaxation) in a direction (second direction D2) from the membrane 11 toward the separation channel 10. The flow rate of the cross flow F1 during relaxation is preferably reduced compared to the flow rate of the cross flow F1 when moving the target particles toward the membrane 11.

[0044] The target particles that have moved toward the membrane 11 include particles (first particles and second particles) with different particle properties. Particles with different particle properties have different diffusibility in the liquid in the separation channel 10. Therefore, as shown in FIG. 5 , among the target particles that have moved toward the membrane 11, the relatively easily diffusible particle group is distributed farther from the membrane 11 and closer to the center of the separation channel 10 in the first direction D1 (or the second direction D2) than the relatively less diffusible particle group. For example, if the first particle group is more diffusible than the second particle group during relaxation, the second operation distributes the first particle group closer to the center of the separation channel 10 in the first direction D1 than the second particle group, and the second particle group is distributed closer to the membrane 11 in the first direction D1 than the first particle group. In this way, the second operation can be performed by applying the crossflow F1 to the focused target particles and then diffusing the target particles so that they are distributed in the separation channel 10 from the membrane 11 side in the first direction D1 according to their particle properties.

[0045] When the target particles include two or more particle groups with different particle sizes, the particle groups with relatively small particle sizes tend to diffuse more easily than the particle groups with relatively large particle sizes. When the target particles include two or more particle groups with different masses, the particle groups with relatively small masses tend to diffuse more easily than the particle groups with relatively large masses. When the target particles include two or more particle groups with different strengths of interaction with the membrane, the particle groups with relatively small interaction with the membrane tend to diffuse more easily than the particle groups with relatively large interaction with the membrane.

[0046] In the third operation, after the second operation, liquid is supplied from the first liquid supply unit into the separation channel 10 to form a parabolic flow Fp that flows in the longitudinal direction and has a parabolic flow velocity distribution in the first direction D1 ( FIG. 6 ). As shown in FIG. 6 , the parabolic flow Fp has a flow velocity that varies depending on the position in the first direction D1 and has a parabolic flow velocity distribution that is convex in the longitudinal direction of the separation channel 10 in the direction in which the liquid flows. The third operation is generally part of an operation called elution. This distributes the target particles in the longitudinal direction L within the separation channel 10 so that the first particle group and the second particle group contained in the target particles can flow out of the outlet port 15 in this order ( FIG. 6 ). As described above, the first particle group is more likely to diffuse during relaxation than the second particle group and is distributed farther from the membrane 11 in the first direction D1 than the second particle group. The second particle group is a particle group that is less likely to diffuse than the first particle group during relaxation and is distributed closer to the membrane 11 side than the first particle group in the first direction.

[0047] In the third operation, the control unit 5 controls at least the first liquid supply unit. In the third operation, the control unit 5 may control, for example, the pumps 32 and 34, the switching valve 33, and the flow rate regulator 43. In the third operation, the on-off valve 41 may be in an open state or a closed state.

[0048] The third operation can be performed following the second operation. In the third operation, the controller 5 stops the pump 34, which was operating in the second operation, for example. In the third operation, the controller 5 may maintain the states of the components other than the pump 34 in the states in the second operation. In the third operation, the controller 5 may adjust the flow rate of the flow rate adjuster 43. As a result, a focus flow Ff is not formed in the separation channel 10, and a parabolic flow Fp and a cross flow F1 are formed ( FIG. 6 ).

[0049] The parabolic flow Fp formed by the third operation may be the carrier flow Fc formed by the first and second operations. The carrier flow Fc is a flow that flows through the separation channel 10 and has a flow velocity distribution (parabolic flow Fp) in which the flow velocity is low near the wall surface and high near the center away from the wall surface, as shown in FIG. 6 . As a result, within the separation channel 10, among the target particles after the second operation, the first particle group that is distributed toward the center of the separation channel 10 in the first direction D1 is distributed closer to the outlet port 15 in the longitudinal direction L than the second particle group ( FIG. 6 ). By forming such a distribution, the first particle group and the second particle group can flow out of the outlet port 15 in this order.

[0050] In the third operation, it is sufficient that a distribution of target particles is formed in the longitudinal direction L within the separation channel 10 so that the first particle group and the second particle group can flow out of the outlet port 15 in this order. In the third operation after such a distribution is formed (before the fourth operation), the first particle group may not flow out of the outlet port 15 and be present within the separation channel 10, or may be present within the separation channel 10. The second particle group does not flow out of the outlet port 15 during the third operation, and remains within the separation channel 10 until the fourth operation is performed.

[0051] In the fourth operation, a liquid is supplied from the second liquid supply unit into the discharge chamber 20 so as to move the second particle group after the third operation from the membrane 11 side in the separation channel 10 in a second direction D2 opposite to the first direction D1, thereby forming a reverse crossflow F2 flowing in the second direction D2 ( FIG. 7 ). As in the second operation, the second particle group is a particle group distributed closer to the membrane 11 side in the separation channel 10 in the first direction D1 (second direction D2) than the first particle group.

[0052] In the fourth operation, the control unit 5 controls at least the second liquid supply unit. In the fourth operation, the control unit 5 may control, for example, the pumps 32 and 34, the switching valve 33, and the flow rate regulator 43. In the fourth operation, the on-off valve 41 may be in an open state or a closed state.

[0053] The fourth operation can be performed following the third operation. In the fourth operation, the controller 5 switches the switching valve 33 so that the liquid is supplied to the discharge chamber 20 without passing through the separation channel 10. When the controller 5 operates the pump 34 in this state, the liquid contained in the container 35 is aspirated through the pipe p37 and supplied into the discharge chamber 20 via the pipe p36, the switching valve 33, the pipe p35, and the introduction port 23. In the fourth operation, the controller 5 adjusts the flow rate of the flow rate adjuster 43 to be smaller than the flow rate of the liquid supplied from the second liquid supply unit. The flow rate of the flow rate adjuster 43 may be adjusted to zero. The flow rate of the liquid supplied from the second liquid supply unit is, for example, the flow rate of the liquid supplied from the introduction port 23 into the discharge chamber 20 by operation of the pump 34 described above. In the fourth operation, the controller 5 may maintain the states of the pump 32 and the sample supply unit 31 in the states in the third operation. As a result, in the fourth operation, the focus flow Ff and the cross flow F1 are not formed in the separation channel 10, but the parabolic flow Fp and the reverse cross flow F2 are formed.

[0054] Target particles that have been present on or near the membrane 11 in the separation channel 10 after the first to third operations tend to remain within the separation channel 10 even when a parabolic flow Fp is formed, and may be difficult to discharge from the separation channel 10 through the outlet port 15. This is because target particles that are present on or near the membrane 11 are subjected to the force of the flow of the slow flow velocity portion of the parabolic flow Fp having a flow velocity distribution, and move in the longitudinal direction L within the separation channel 10. If the target particles are difficult to move in the longitudinal direction L within the separation channel 10 and difficult to discharge from the outlet port 15, the time required to classify the target particles increases.

[0055] In the FFF device 1, because the reverse crossflow F2 is formed in the fourth operation, target particles present on or near the membrane 11 can be moved from the membrane 11 in the second direction D2 within the separation channel 10, i.e., toward the center of the separation channel 10. As a result, target particles that have moved from on or near the membrane 11 toward the center of the separation channel 10 are subjected to the force of the flow of the portion of the parabolic flow Fp with the higher flow velocity, and can move in the longitudinal direction L within the separation channel 10, making them more likely to flow out of the outlet port 15. Therefore, in the FFF device 1, the time required to classify target particles in a sample can be reduced, and the target particles can be efficiently classified.

[0056] In the fourth operation, it is sufficient that at least the second particle group is present in the separation channel 10. Particle groups such as the first particle group distributed in the longitudinal direction L within the separation channel 10 may be present in the separation channel 10 in the fourth operation so as to be able to flow out of the outlet port 15 before the second particle group, but it is preferable that they flow out of the outlet port 15 and are not present in the separation channel 10. In the fourth operation, if the first particle group is present in the separation channel 10, the reverse crossflow F2 may act on the first particle group. In the fourth operation, a particle group distributed in the longitudinal direction L within the separation channel 10 (hereinafter sometimes referred to as the "third particle group") is present in the separation channel 10 so as to be able to flow out of the outlet port 15 after the second particle group. In the fourth operation, the reverse crossflow F2 acts on the third particle group, and the third particle group may move toward the center of the separation channel 10 in the first direction D1. This makes it easier for the third particle group to move toward the outlet port 15 in the longitudinal direction L within the separation channel 10 due to the parabolic flow Fp, and makes it easier for the third particle group to flow out of the outlet port 15 .

[0057] In the fifth operation, a liquid is supplied from the first liquid supply unit into the separation channel 10 so that the first particle group and the second particle group flow out of the separation channel 10 from the outlet port 15 in this order. The fifth operation is generally part of an operation called elution. In the fifth operation, the control unit 5 controls at least the first liquid supply unit. In the fifth operation, the control unit 5 may control, for example, the opening / closing valve 41, the pump 32, the flow rate regulator 43, and the switching valve 33. In the fifth operation, the control unit 5 controls the opening / closing valve 41 to an open state.

[0058] The fifth operation may be performed in parallel with the third operation or subsequent to the third operation, and may also be performed subsequent to the fourth operation. In this case, the first particle group may be discharged during or after the third operation, and the second particle group may be discharged after the fourth operation. Alternatively, the fifth operation may be performed in parallel with the fourth operation or subsequent to the fourth operation. In this case, the first particle group may be discharged during or after the fourth operation, and the second particle group may be discharged after the fourth operation. When the first particle group is discharged in parallel with at least one of the third operation and the fourth operation, the control unit 5 controls the on-off valve 41 to an open state during the operation of discharging the first particle group, and in the fifth operation, the control unit 5 may control the states of the pumps 32, 34, the switching valve 33, and the flow rate regulator 43 to the states in the third operation or the fourth operation.

[0059] In the fifth operation, the third particle group may flow out of the separation channel 10 from the outlet port 15 following the first particle group and the second particle group.

[0060] In the sixth operation, the second particle group flowed out from outlet port 15 in the fifth operation is detected by detector 42. In the sixth operation, the first particle group flowed out from outlet port 15 in the fifth operation may further be detected by detector 42. If the third particle group is flowed out from outlet port 15 in the fifth operation, the sixth operation may be detected by detector 42.

[0061] In the sixth operation, the controller 5 controls at least the detector 42. In the sixth operation, the controller 5 may operate the detector 42 and maintain the states of the pumps 32, 34, the switching valve 33, the flow rate regulator 43, and the on-off valve 41 in the states in the fifth operation. In the sixth operation, a parabolic flow Fp may be formed in the separation channel 10, and the parabolic flow Fp and the reverse cross flow F2 may be formed.

[0062] The sixth operation is preferably performed in parallel with the fifth operation of causing the second particle group to flow out from the outlet port 15. When the fifth operation causes at least one of the first particle group and the third particle group to flow out and the sixth operation detects the particle group, it is preferable to perform the flow of the particle group in the fifth operation and the detection of the particle group in the sixth operation in parallel.

[0063] In the seventh operation, the liquid in the separation channel 10 is caused to flow out from the outlet port 15 while forming the reverse crossflow F2. The controller 5 controls the seventh operation to be performed at least either before the first operation or after the sixth operation. The reverse crossflow F2 can be formed by the method described in the fourth operation. The method of causing the liquid to flow out from the outlet port 15 can be performed by the method of causing the first particle group and the second particle group to flow out from the outlet port 15 described in the fifth operation. Therefore, in the seventh operation, the controller 5 controls at least the first liquid supply unit, the second liquid supply unit, and the on-off valve 41. In the seventh operation, for example, the controller 5 may control the pumps 32 and 34, the switching valve 33, the on-off valve 41, and the flow rate regulator 43. In the seventh operation, the controller 5 controls the on-off valve 41 to be open, and the carrier flow Fc is formed by supplying the liquid from the first liquid supply unit, and the reverse crossflow F2 is formed by supplying the liquid from the second liquid supply unit.

[0064] Some of the components in the sample may adhere to the surface of the membrane 11 or be captured in the pores of the membrane 11, resulting in some of the components remaining in the sample remaining on the membrane 11 even after the first to sixth operations. The components remaining on the membrane 11 (hereinafter sometimes referred to as "residual components") may contaminate new samples supplied to the separation channel 10 and reduce the detection accuracy of target particles by the detector 42. On the other hand, when the seventh operation is performed, the reverse crossflow F2 passes through the membrane 11, thereby causing the remaining components to be separated from the membrane 11 in the second direction D2, i.e., toward the center of the separation channel 10. In the seventh operation, the reverse crossflow F2 is formed and the liquid in the separation channel 10 is caused to flow out of the outlet port 15. Therefore, the remaining components separated from the membrane 11 can be discharged from the outlet port 15 together with the liquid flowing in the longitudinal direction L through the separation channel 10.

[0065] In this way, by performing the seventh operation before and after the classification operation that classifies the target particles in the sample, i.e., before and after performing the first to sixth operations, the remaining components can be detached from the membrane 11 and removed by flowing them out of the separation channel 10. This allows the first to sixth operations to be performed with reduced contamination of the membrane 11, thereby improving the detection accuracy of the target particles by the detector 42. The remaining components flowed out from the outlet port 15 in the seventh operation do not need to be detected by the detector 42, but may be detected by the detector 42.

[0066] The FFF apparatus 1 can perform the first, second, third, and fourth operations in this order under the control of the control unit 5. As shown in FIGS. 4 to 6 , in the classification operation performed by the FFF apparatus 1, a crossflow F1 may be formed during the first to third operations, and the flow rate of the crossflow F1 in the first operation may be gradually reduced to zero during the second and third operations. Then, in the fourth operation, the flow rate of the reverse crossflow F2 may be gradually increased from zero to a preset flow rate. The flow rate of the crossflow F1 may be gradually reduced, and when the flow rate of the crossflow F1 reaches zero, a reverse crossflow F2 may be generated and its flow rate gradually increased. After the flow rate of the reverse crossflow F2 reaches a preset flow rate, that flow rate may be maintained.

[0067] The flow rate of the crossflow F1 may be decreased linearly (continuously), in a stepwise manner, or in a combination thereof. "In a stepwise manner," as used herein, refers to decreasing the flow rate while providing a period during which the flow rate remains constant. As shown in FIG. 10 (described later), the flow rate of the crossflow F1 may be suddenly decreased when switching from the first operation to the second operation, and then may be decreased continuously.

[0068] The flow rate of the reverse cross flow F2 may be increased linearly (continuously), in stages, or in a combination thereof. Increasing the flow rate in stages means increasing the flow rate while providing a period during which the flow rate remains constant.

[0069] The flow rates of the cross flow F1 and the reverse cross flow F2 can be adjusted, for example, by adjusting the flow rate of the flow rate adjuster 43 using the control unit 5. By performing the classification operation in the FFF device 1 while controlling the flow rates of the cross flow F1 and the reverse cross flow F2 as described above, the target particles in the sample can be efficiently classified in the FFF device 1.

[0070] <Classification method> Figure 8 is a flowchart illustrating a classification method according to one embodiment of the present invention. The classification method of this embodiment is a method for classifying particles in a sample using an FFF device. The FFF device may be the FFF device 1 shown in Figure 1. Below, a classification method using the FFF device 1 will be described as an example, and components described in the FFF device 1 will be assigned the same reference numerals and detailed description thereof will be omitted.

[0071] 8 , the classification method includes a first step of focusing target particles within the separation channel 10, a second step of distributing the target particles in a first direction D1 within the separation channel 10 according to particle characteristics, a third step of distributing the target particles in the longitudinal direction L of the separation channel 10, a fourth step of moving second particles within the separation channel 10 from the membrane 11 side in a second direction D2, a fifth step of causing the first particle group and the second particle group to flow out of the outlet port 15 in this order, and a sixth step of detecting the second particle group with a detector 42. The classification method may further include a seventh step of forming a reverse crossflow F2 and causing the liquid within the separation channel 10 to flow out of the outlet port 15.

[0072] In the first step, a liquid and a sample are supplied into the separation channel 10, and target particles are focused within the separation channel 10. In the first step, for example, the first operation of the FFF apparatus 1 can be executed, and each component of the FFF apparatus 1 can be controlled as described in the first operation. In the first step, target particles in the sample are gathered near the position where the carrier flow Fc and the focus flow Ff collide (FIG. 4).

[0073] In the first step, in addition to the carrier flow Fc and the focus flow Ff, a cross flow F1 flowing in the first direction D1 may be formed in the separation channel 10. This allows target particles gathered at the collision position between the carrier flow Fc and the focus flow Ff to be moved in the first direction D1 toward the membrane 11 in the separation channel 10 ( FIG. 5 ). In the first step, the flow rate of the cross flow F1 is preferably constant.

[0074] The second step is a step in which a crossflow F1 flowing in a first direction D1 from the separation channel 10 through the membrane 11 toward the discharge chamber 20 acts on the target particles focused in the first step, and then the target particles are distributed in the first direction D1 within the separation channel 10 according to their particle characteristics. In the second step, for example, the second operation of the FFF apparatus 1 can be performed, and each part of the FFF apparatus 1 can be controlled as described in the second operation. In the second step, the target particles that have been focused and moved toward the membrane 11 diffuse within the separation channel 10 so as to be distributed in the first direction D1 according to their particle characteristics.

[0075] The second step can be performed following the first step. In the second step, a crossflow F1 is applied to the target particles focused in the first step, causing the target particles to move toward the membrane 11 (FIG. 5). This step may be performed by forming the crossflow F1 in the first step described above. In the second step, the target particles that have moved toward the membrane 11 diffuse in the first direction D1 (second direction D2) within the separation channel 10, forming a distribution according to the particle characteristics (relaxation). For example, if the first particle group is more diffusible than the second particle group during relaxation, in the second step, the first particle group will be distributed closer to the center of the separation channel 10 than the second particle group in the first direction D1, and the second particle group will be distributed closer to the membrane 11 than the first particle group in the first direction D1.

[0076] In the second step, a carrier flow Fc and a focus flow Ff are usually formed in addition to the cross flow F1 in the separation channel 10 (FIG. 5).

[0077] The third step is a step of distributing the target particles that have been relaxed in the second step in the longitudinal direction L within the separation channel 10 (elution). In the third step, after the second step, a liquid is supplied into the separation channel 10 to form a parabolic flow Fp that flows in the longitudinal direction L and has a parabolic flow velocity distribution in the first direction D1, thereby distributing the target particles after the second step in the longitudinal direction L within the separation channel 10 so that the first particle group and the second particle group can flow out from the outlet port 15 in this order. In the third step, for example, a third operation of the FFF device 1 can be performed, and the various components of the FFF device 1 may be controlled as described for the third operation.

[0078] The third step can be performed following the second step. In the third step, when the parabolic flow Fp formed in the separation channel 10 acts on the target particles after the second step, the flow velocity distribution of the parabolic flow Fp causes the first particle group, which is distributed toward the center of the separation channel 10 in the first direction D1, to be distributed closer to the outlet port 15 in the longitudinal direction L than the second particle group ( FIG. 6 ).

[0079] In the third step, a cross flow F1 is typically formed in addition to the parabolic flow Fp in the separation channel 10. In the third step, the first particle group may not flow out from the outlet port 15 and may be present in the separation channel 10, or may be present in the separation channel 10. The second particle group does not flow out from the outlet port 15 during the third operation and remains in the separation channel 10 until the fourth step is performed.

[0080] In the fourth step, a reverse crossflow F2 flowing in a second direction D2 opposite to the first direction D1 is applied to the second particle group after the third step, thereby moving the second particle group from the membrane 11 side to the second direction D2 within the separation channel 10. In the fourth step, for example, a fourth operation of the FFF apparatus 1 can be executed, and each part of the FFF apparatus 1 may be controlled as described in the fourth operation.

[0081] The fourth step can be performed following the third step. In the fourth step, when the reverse cross flow F2 is applied to the second particle group after the third step, the second particle group moves in the first direction D1 in a direction away from the membrane 11, i.e., toward the center of the separation channel 10 in the first direction D1. This makes the second particle group more susceptible to the force of the flow in the part of the parabolic flow Fp where the flow velocity is high, and the second particle group moves more easily within the separation channel 10 toward the outlet port 15 in the longitudinal direction L. This makes it easier for the second particle group to flow out of the outlet port 15, allowing the target particles in the sample to be efficiently classified.

[0082] In the fourth step, a parabolic flow Fp and a reverse cross flow F2 are typically formed in the separation channel 10, and a focus flow Ff and a cross flow F1 are not formed. In the fourth step, it is sufficient that at least the second particle group is present in the separation channel 10. In the fourth step, the first particle group may be present in the separation channel 10, but it is preferable that the first particle group has flowed out of the outlet port 15 and is not present in the separation channel 10. The third particle group described in the fourth operation may be present in the separation channel 10 in the fourth step.

[0083] In the fifth step, the first particle group and the second particle group are caused to flow out of the separation channel 10 from the outlet port 15 in this order (elution). In the fifth step, for example, the fifth operation of the FFF apparatus 1 can be executed, and each part of the FFF apparatus 1 may be controlled as described in the fifth operation.

[0084] The fifth step may be performed in parallel with or subsequent to the third step, and may also be performed subsequent to the fourth step. In this case, the first particle group may be discharged during or after the third step, and the second particle group may be discharged after the fourth step. Alternatively, the fifth step may be performed in parallel with or subsequent to the fourth step. In this case, the first particle group may be discharged during or after the fourth step, and the second particle group may be discharged after the fourth operation. In the fifth step, the third particle group may be discharged from the outlet port 15 to the outside of the separation channel 10, following the first and second particle groups.

[0085] In the sixth step, the second particle group that has flowed out from the outlet port 15 in the fifth step is detected by the detector 42. In the sixth step, for example, a sixth operation of the FFF apparatus 1 can be executed, and each part of the FFF apparatus 1 may be controlled as described in the sixth operation. The sixth step is preferably executed in parallel with the fifth step.

[0086] In the sixth step, at least one of the first particle group and the third particle group that are caused to flow out from the outlet port in the fifth step may be detected by the detector 42. In the sixth step, the detector 42 may detect both the first particle group and the third particle group.

[0087] In the seventh step, the liquid in the separation channel 10 is caused to flow out from the outlet port 15 while forming a reverse crossflow. The seventh step is performed at least either before the first step or after the sixth step. In the sixth step, for example, the seventh operation of the FFF apparatus 1 can be performed, and each part of the FFF apparatus 1 can be controlled as described for the seventh operation. In the seventh step, the remaining components of the membrane 11 can be moved from the membrane 11 side in the second direction D2, i.e., toward the center of the separation channel 10, and can be caused to flow out from the outlet port 15. In this way, the seventh step allows the first to sixth steps to be performed using the membrane 11 from which the remaining components have been removed by the classification method, thereby improving the detection accuracy of the target particles by the detector 42.

[0088] The classification method involves performing the first, second, third, and fourth steps in this order. In the classification method, a crossflow F1 may be formed during the first to third steps, and the flow rate of the crossflow F1 in the first step may be gradually reduced to zero during the second and third steps. Then, in the fourth step, the flow rate of the reverse crossflow F2 may be gradually increased from zero to a preset flow rate. These steps may be performed by the operations described for the FFF apparatus 1. The gradual reduction and gradual increase of the flow rate are as described for the FFF apparatus 1. By performing the classification method while controlling the flow rates of the crossflow F1 and the reverse crossflow F2 in this manner, target particles in a sample can be efficiently classified.

[0089] <Modifications of FFF Apparatus> In the FFF apparatus 1 shown in FIG. 1 , the sample supply unit 31 is disposed between the pump 32 and the first inlet port 13, but this is not limiting. For example, instead of or in addition to disposing the sample supply unit 31 between the pump 32 and the first inlet port 13, a sample supply unit may be disposed between the pump 34 and the second inlet port 14. Alternatively, a sample supply port may be formed between the first inlet port 13 and the second inlet port 14 of the separation channel 10, and a sample may be supplied into the separation channel 10 from a sample supply unit having this sample supply port. The sample supply unit disposed between the pump 34 and the second inlet port 14 may be disposed between the switching valve 33 and the second inlet port 14, or may be disposed between the pump 34 and the switching valve 33.

[0090] The FFF apparatus 1 may have a flow meter in the pipe p32 connecting the sample supply unit 31 and the pump 32, for measuring the flow rate of the liquid supplied from the pump 32. The FFF apparatus 1 may have a flow meter in the pipe p36 connecting the pump 34 and the switching valve 33, for measuring the flow rate of the liquid supplied from the pump 34. When the FFF apparatus 1 has a flow meter, the control unit 5 may control the flow meter. The flow meter can be an example of the first liquid supply unit or the second liquid supply unit.

[0091] 1, the suction side pipe p37 of the pump 34 is disposed in the container 35 in which the suction side pipe p33 of the pump 32 is disposed, but it may be disposed in a container different from the container 35. The liquid contained in the container different from the container 35 may be different from the liquid contained in the container 35, but it is preferable that they are the same.

[0092] 1 , the switching valve 33 is disposed between the pump 34 and the second inlet port 14, but it may be disposed between the pump 32 and the first inlet port 13. The switching valve 33 disposed between the pump 32 and the first inlet port 13 may be disposed between the pump 32 and the sample supply unit 31, or may be disposed between the sample supply unit 31 and the first inlet port 13.

[0093] Fig. 9 is a schematic diagram showing the configuration of an FFF apparatus according to another embodiment of the present invention. Hereinafter, the same reference numerals are used to designate components described in the FFF apparatus 1 shown in Fig. 1 , and detailed description thereof will be omitted. The FFF apparatus 2 shown in Fig. 9 differs from the FFF apparatus 1 in that it has a switching valve 38 and a pipe p38 instead of the switching valve 33 and the pipe p35 of the FFF apparatus 1, and does not have the introduction port 23 of the FFF apparatus 1.

[0094] The second inlet port 14 of the FFF apparatus 2 is connected to a switching valve 38 via, for example, a pipe p34. The switching valve 38 is connected to the discharge side of the pump 34 via a pipe p36. The suction side of the pump 34 is connected to a pipe p37 disposed in a container 35 containing a liquid (eluent). The pump 34 sucks the liquid contained in the container 35 through the pipe p37 and supplies it to the switching valve 38 via the pipe p36. The switching valve 38 selectively switches between a path (1) that supplies the liquid to the separation channel 10 without passing through the discharge chamber 20 and a path (2) that supplies the liquid to the discharge chamber 20 without passing through the separation channel 10. In the FFF apparatus 2, the path (1) is connected to the second inlet port 14, and the path (2) is connected from the switching valve 38 via the pipe p38 between the discharge chamber 20 and the flow rate regulator 43. The pipe p38 can be connected to the middle of the pipe p43 that connects the exhaust chamber 20 and the flow rate adjuster 43, for example, as shown in FIG.

[0095] When the switching valve 38 is switched to a state in which liquid is supplied to the separation channel 10 without passing through the discharge chamber 20, the liquid sucked by the pump 34 is supplied into the separation channel 10 through the pipe p36, the switching valve 38, the pipe p34, and the second inlet port 14. When the switching valve 38 is switched to a state in which liquid is supplied to the discharge chamber 20 without passing through the separation channel 10, the liquid sucked by the pump 34 can be supplied into the discharge chamber 20 through the pipe p36, the switching valve 38, the pipe p38, and the pipe p43.

[0096] In the FFF apparatus 2 shown in FIG. 9 , the path for supplying liquid to the discharge chamber 20 in the fourth operation differs from that of the FFF apparatus 1 shown in FIG. 1 . In the fourth operation performed by the FFF apparatus 2, the control unit 5 switches the switching valve 38 so that liquid is supplied to the discharge chamber 20 without passing through the separation channel 10. When the control unit 5 operates the pump 34 in this state, the liquid contained in the container 35 is sucked through the pipe p37 and supplied to the pipe p36, the switching valve 38, the pipe p38, and the pipe p43. At this time, the control unit 5 adjusts the flow rate of the flow rate adjuster 43 to be smaller than the flow rate of the liquid supplied from the second liquid supply unit. The flow rate of the flow rate adjuster 43 may be adjusted to zero. This allows liquid to be supplied into the discharge chamber 20 from the pipe p38 via the pipe p43.

[0097] The FFF apparatus 2 shown in Fig. 9 can execute the first to seventh operations described for the FFF apparatus 1 shown in Fig. 1, except for the operation of the fourth operation described above. The first to seventh steps of the classification method described above may be executed using the FFF apparatus 2.

[0098] The present invention will be described in more detail below with reference to a comparative example and a reference example. Fig. 10 is a graph showing the changes in crossflow and reverse crossflow flow rates in the comparative example and the reference example. Fig. 11 is a chromatogram obtained in the comparative example and the reference example. In the graph shown in Fig. 10, the crossflow flow rate is represented by a positive number, and the reverse crossflow flow rate is represented by a negative number. In Fig. 10 and Fig. 11, the horizontal axis represents time.

[0099] Comparative Example: An FFF device as shown in Figure 1 or Figure 9 was prepared. A polyethersulfone membrane was used as the membrane. A fluorescence detector was used as the detector, and analysis was performed at an excitation wavelength of 650 nm and a fluorescence wavelength of 650 nm. D-PBS was used as the liquid (eluent) flowing through the separation channel. Liposomes (product name: Control liposomes for Doxoves) were used as the target particles.

[0100] Liquid and target particles were supplied from the first liquid supply unit into the separation channel to form a crossflow (profile shown by the dashed line between 0 and 5 minutes in Figure 10 ), while the target particles were focused in the separation channel for 5 minutes (first step, first operation). The crossflow flow rate was kept constant during the target particle focusing. The crossflow flow rate was then decreased (profile shown by the dashed line at 5 minutes in Figure 10 ), and then linearly decreased over the next 10 minutes until the crossflow flow rate reached 0 (profile shown by the dashed line between 5 and 15 minutes in Figure 10 ). The crossflow flow rate was then maintained at 0 (profile shown by the dashed line after 15 minutes in Figure 10 ). After focusing (after 5 minutes in the profile in Figure 10 ), the target particle relaxation step (second step, second operation) was performed, the target particles were distributed in the longitudinal direction of the separation channel (third step, third operation), the target particles were released (corresponding to the fifth step and fifth operation), and the target particles were detected (sixth step, sixth operation).

[0101] The target particles were allowed to flow out of the outlet port of the separation channel and detected by a detector. The results are shown in Figure 11. As shown in Figure 11, a peak was observed around 17.5 minutes in the chromatograph of the comparative example.

[0102] [Reference Example] Using the same procedure as in the comparative example, target particles were focused and the crossflow flow rate was reduced to 0 (zero) (profile shown by the solid line between 0 and 15 minutes in FIG. 10). After the crossflow flow rate reached 0, the reverse crossflow flow rate was increased linearly (profile shown by the solid line between 15 and 15.4 minutes in FIG. 10) (fourth step, fourth operation). The reverse crossflow flow rate was then maintained constant at 15.4 minutes (profile shown by the solid line after 15.4 minutes in FIG. 10). After focusing (after 5 minutes in the profile in FIG. 10), the following steps were performed: relaxation of the target particles (second step, second operation), distribution of the target particles in the longitudinal direction of the separation channel (third step, third operation), application of the reverse crossflow (fourth step, fourth operation), release of the target particles (corresponding to fifth step and fifth operation), and detection of the target particles (sixth step, sixth operation).

[0103] The target particles flowed out of the outlet port of the separation channel and were detected by a detector. The results are shown in Figure 11. As shown in Figure 11, a peak was observed around 16.5 minutes in the chromatograph of the Reference Example. Comparing the Comparative Example and the Reference Example, it can be seen that the target particles are eluted earlier by the step of applying reverse crossflow to the target particles.

[0104] In the comparative example, the target particles flowed out after the crossflow flow rate became zero (15 minutes from Figure 10), which indicates that the membrane used in the comparative example and reference example has a strong interaction with the target particles, and the target particles are particles that require a long time to classify. From the above results, it can be expected that the classification method including the step of applying a reverse crossflow (step 4) and the FFF device controlled to include the operation of forming a reverse crossflow (operation 4) can efficiently classify target particles in a sample, even if the sample contains target particles that have a strong interaction with the membrane.

[0105] Aspects It will be understood by those skilled in the art that the exemplary embodiments and examples described above are examples of the following aspects.

[0106] (Item 1) The classification method according to one aspect is a classification method for classifying target particles in a sample using a field flow fractionation device, wherein the field flow fractionation device comprises: a separation channel having a longitudinal direction and an outlet port; a membrane arranged along the longitudinal direction and constituting a part of the wall surface of the separation channel; a discharge chamber arranged along the longitudinal direction so as to face the separation channel and having the membrane constituting a part of the wall surface; and a detector for detecting the target particles flowing out of the outlet port, wherein the target particles include a first particle group and a second particle group that differ from each other in at least one property selected from the group consisting of particle diameter, mass, and strength of interaction with the membrane, and the classification method comprises: a first step of supplying a liquid and the sample into the separation channel and focusing the target particles in the separation channel; and a second step of applying a cross flow that flows in a first direction from the separation channel side toward the discharge chamber via the membrane to the target particles after focusing, and then distributing the target particles in the first direction in the separation channel according to the property. a third step of distributing the target particles in the longitudinal direction within the separation channel after the second step, by supplying a liquid into the separation channel to form a parabolic flow that flows in the longitudinal direction and has a parabolic flow velocity distribution in the first direction, thereby allowing the first particle group and the second particle group to flow out of the outlet port in this order; a fourth step of applying a reverse cross flow that flows in a second direction opposite to the first direction to the second particle group after the third step, thereby moving the second particle group from the membrane side to the second direction within the separation channel; a fifth step of causing the first particle group and the second particle group to flow out of the separation channel from the outlet port in this order; and a sixth step of detecting the second particle group that flowed out of the outlet port in the fifth step with the detector.

[0107] According to the classification method described in paragraph 1, target particles in a sample can be efficiently classified using a field-flow fractionation device.

[0108] (Item 2) In the classification method described in item 1, the classification method includes performing the first step, the second step, the third step, and the fourth step in this order, the cross flow is formed during the period from the first step to the third step, the flow rate of the cross flow in the first step is gradually reduced to zero during the period from the second step to the third step, and the flow rate of the reverse cross flow is gradually increased from zero to a preset flow rate in the fourth step.

[0109] (Item 3) In the classification method described in item 1 or 2, the sixth step further comprises detecting the first particle group discharged from the outlet port in the fifth step with the detector.

[0110] (4) In the classification method described in any one of paragraphs 1 to 3, the classification method includes a seventh step of causing the liquid in the separation channel to flow out of the outlet port while forming the reverse crossflow, and the seventh step is carried out at least either before the first step or after the sixth step.

[0111] (Item 5) In the classification method described in any one of Items 1 to 4, the field-flow fractionation device further has a first liquid supply unit for supplying liquid to the separation channel without passing through the discharge chamber, and a flow rate adjustment unit for adjusting the flow rate of liquid discharged from the discharge chamber, and the cross flow is formed by supplying liquid from the first liquid supply unit into the separation channel while discharging liquid from the discharge chamber at a flow rate adjusted by the flow rate adjustment unit.

[0112] (Item 6) In the classification method described in any one of Items 1 to 5, the field-flow fractionation device further includes a flow rate regulator for regulating the flow rate of liquid discharged from the discharge chamber, and a second liquid supply unit for supplying liquid to the discharge chamber or between the discharge chamber and the flow rate regulator without passing through the separation channel, and the reverse crossflow is formed by supplying liquid from the second liquid supply unit at a flow rate greater than the flow rate of liquid discharged from the discharge chamber as adjusted by the flow rate regulator.

[0113] (Item 7) The field flow fractionation device according to one aspect is a field flow fractionation device for classifying target particles in a sample, comprising: a separation channel having a longitudinal direction and an outlet port; a membrane arranged along the longitudinal direction and constituting a part of the wall surface of the separation channel; a discharge chamber arranged facing the separation channel along the longitudinal direction and having the membrane constituting a part of the wall surface; a sample supply unit for supplying the sample into the separation channel; a first liquid supply unit for supplying liquid to the separation channel without passing through the discharge chamber; a flow rate regulator for regulating the flow rate of liquid discharged from the discharge chamber; a second liquid supply unit for supplying liquid to the discharge chamber or between the discharge chamber and the flow rate regulator for supplying liquid to the discharge chamber without passing through the separation channel; a detector for detecting the target particles flowing out from the outlet port; and a controller for controlling at least the first liquid supply unit, the flow rate regulator, the second liquid supply unit, and the detector. the target particles include a first particle group and a second particle group that differ from each other in at least one property selected from the group consisting of particle diameter, mass, and the magnitude of interaction with the membrane; and under the control of the control unit, in a first operation, the sample supplied from the sample supply unit and the liquid supplied from the first liquid supply unit are supplied into the separation channel so as to focus the target particles; and in a second operation, a cross flow that flows in a first direction from the separation channel side toward the discharge chamber through the membrane acts on the target particles after focusing, and then liquid is supplied from the first liquid supply unit into the separation channel so as to distribute the target particles in the first direction in the separation channel according to the property, and liquid is discharged from the discharge chamber at a flow rate adjusted by the flow rate adjuster to form the cross flow;a third operation, after the second operation, supplies liquid from the first liquid supply unit into the separation channel to distribute the target particles in the longitudinal direction within the separation channel and form a parabolic flow that flows in the longitudinal direction and has a parabolic flow velocity distribution in the first direction, so that the first particle group and the second particle group can flow out of the outlet port in this order; a fourth operation, supplies liquid from the second liquid supply unit into the discharge chamber to move the second particle group after the third operation from the membrane side within the separation channel in a second direction that is the opposite direction to the first direction, form a reverse cross flow that flows in the second direction; a fifth operation, supplies liquid from the first liquid supply unit into the separation channel so that the first particle group and the second particle group flow out of the separation channel from the outlet port in this order; and a sixth operation, detects the second particle group that has flowed out of the outlet port in the fifth operation with the detector.

[0114] According to the field flow fractionation device described in item 7, target particles in a sample can be efficiently classified.

[0115] (Item 8) In the field-flow fractionation device described in item 7, the first operation, the second operation, the third operation, and the fourth operation are performed in this order under the control of the control unit, the cross flow is formed during the first to third operations, the flow rate of the cross flow in the first operation is gradually reduced to zero during the second to third operations, and the flow rate of the reverse cross flow is gradually increased from zero to a preset flow rate in the fourth operation.

[0116] (Item 9) In the field flow fractionation device described in Item 7 or 8, under the control of the control unit, in the sixth operation, the first particle group that was caused to flow out of the outlet port in the fifth operation is further detected by the detector.

[0117] (Item 10) In the field flow fractionation device described in any one of items 7 to 9, under the control of the control unit, in a seventh operation, the liquid in the separation channel is caused to flow out from the outlet port while forming the reverse crossflow, and the seventh operation is performed at least either before the first operation or after the sixth operation.

[0118] (Item 11) In the field-flow fractionation apparatus according to any one of Items 7 to 10, the control unit further controls the sample supply unit.

[0119] (Item 12) In the field-flow fractionation device according to any one of Items 7 to 11, a porous support for supporting the membrane is disposed in the discharge chamber.

[0120] 1, 2 Field flow fractionation apparatus (FFF apparatus), 5 Control unit, 10 Separation channel, 11 Membrane, 13 First inlet port, 14 Second inlet port, 15 Outlet port, 20 Discharge chamber, 21 Porous support, 22 Discharge port, 23 Introduction port, 31 Sample supply unit, 32 Pump, 33, 38 Switching valve, 34 Pump, 35 Container, 41 Opening / closing valve, 42 Detector, 43 Flow rate control unit, p31, p32, p33, p34, p35, p36, p37, p38, p41, p42, p43 Piping, Fc Carrier flow, Ff Focused flow, Fp Parabolic flow (carrier flow), F1 Cross flow, F2 Reverse cross flow, L Longitudinal direction, D1 First direction, D2 Second direction.

Claims

1. A classification method for classifying target particles in a sample using a field flow fractionation device, wherein the field flow fractionation device includes: - a separation channel having a longitudinal direction and an outlet port; - a membrane disposed along the longitudinal direction and forming a part of the wall surface of the separation channel; - a discharge chamber disposed to face the separation channel along the longitudinal direction and having the membrane as a part of its wall surface; - a detector for detecting the target particles flowing out from the outlet port; the target particles include a first particle group and a second particle group in which at least one characteristic selected from the group consisting of particle diameter, mass, and the magnitude of interaction with the membrane is different from each other; the classification method includes: - a first step of supplying a liquid and the sample into the separation channel and focusing the target particles in the separation channel; - a second step of applying a cross-flow flowing in a first direction from the separation channel side through the membrane toward the discharge chamber to the focused target particles, and then distributing the target particles in the first direction in the separation channel according to the characteristics; - a third step of supplying a liquid into the separation channel after the second step to form a parabolic flow flowing in the longitudinal direction and having a parabolic flow velocity distribution in the first direction, so that the first particle group and the second particle group can flow out from the outlet port in this order, and distributing the target particles in the longitudinal direction in the separation channel; - a fourth step of applying a reverse cross-flow flowing in a second direction opposite to the first direction to the second particle group after the third step, and moving the second particle group from the membrane side in the second direction in the separation channel; - a fifth step of flowing out the first particle group and the second particle group from the outlet port to the outside of the separation channel in this order; - a sixth step of detecting the second particle group flowing out from the outlet port in the fifth step with the detector.

2. The classification method performs the first step, the second step, the third step, and the fourth step in this order. During the period from the first step to the third step, the cross flow is formed. During the period from the second step to the third step, the flow rate of the cross flow in the first step is gradually decreased until it becomes zero. In the fourth step, the flow rate of the reverse cross flow is gradually increased from zero to a preset flow rate. The classification method according to claim 1.

3. The sixth step further includes detecting, by the detector, the first particle group flowing out from the outlet port in the fifth step. The classification method according to claim 1.

4. The classification method includes a seventh step of causing the liquid in the separation channel to flow out from the outlet port while forming the reverse cross flow, and the seventh step is executed in at least one of a stage before the first step and a stage after the sixth step. The classification method according to claim 1.

5. The field flow fractionation device further includes a first liquid supply unit for supplying liquid to the separation channel without passing through the discharge chamber, and a flow rate adjustment unit for adjusting the flow rate of the liquid discharged from the discharge chamber. The cross flow is formed by supplying liquid from the first liquid supply unit into the separation channel and discharging liquid from the discharge chamber at a flow rate adjusted by the flow rate adjustment unit. The classification method according to claim 1.

6. The field flow fractionation device further includes a flow rate adjustment unit for adjusting the flow rate of the liquid discharged from the discharge chamber, and a second liquid supply unit for supplying liquid to the discharge chamber without passing through the separation channel, provided between the discharge chamber or between the discharge chamber and the flow rate adjustment unit. The reverse cross flow is formed by supplying liquid from the second liquid supply unit so that the flow rate is greater than the flow rate of the liquid discharged from the discharge chamber adjusted by the flow rate adjustment unit. The classification method according to claim 1.

7. A field flow fractionation device for classifying target particles in a sample, comprising: a separation channel having a longitudinal direction and an outlet port; a membrane disposed along the longitudinal direction and forming a part of the wall surface of the separation channel; a discharge chamber disposed to face the separation channel along the longitudinal direction and having the membrane forming a part of the wall surface; a sample supply unit for supplying the sample into the separation channel; a first liquid supply unit for supplying liquid into the separation channel without passing through the discharge chamber; a flow rate adjustment unit for adjusting the flow rate of the liquid discharged from the discharge chamber; a second liquid supply unit for supplying liquid into the discharge chamber without passing through the separation channel, disposed in the discharge chamber or between the discharge chamber and the flow rate adjustment unit; a detector for detecting the target particles flowing out from the outlet port; and a control unit for controlling at least the first liquid supply unit, the flow rate adjustment unit, the second liquid supply unit, and the detector. The target particles include a first particle group and a second particle group having at least one characteristic selected from the group consisting of particle diameter, mass, and magnitude of interaction with the membrane, which are different from each other. Under the control of the control unit, in a first operation, the sample supplied from the sample supply unit and the liquid supplied from the first liquid supply unit are supplied into the separation channel so as to focus the target particles. In a second operation, after applying a cross-flow flowing in a first direction from the separation channel side through the membrane toward the discharge chamber to the focused target particles, according to the characteristics, while supplying liquid from the first liquid supply unit into the separation channel so as to be distributed in the first direction in the separation channel, the liquid is discharged from the discharge chamber at a flow rate adjusted by the flow rate adjustment unit to form the cross-flow. In a third operation, after the second operation, liquid is supplied from the first liquid supply unit into the separation channel in the longitudinal direction of the separation channel so as to distribute the target particles in the longitudinal direction and enable the first particle group and the second particle group to flow out from the outlet port in this order, forming a parabolic flow having a parabolic velocity distribution flowing in the longitudinal direction and in the first direction.In the fourth operation, the second liquid supply unit supplies liquid into the discharge chamber so as to move the second particle group after the third operation from the membrane side in the separation channel in a second direction opposite to the first direction, thereby forming a reverse cross flow flowing in the second direction. In the fifth operation, the first liquid supply unit supplies liquid into the separation channel so that the first particle group and the second particle group flow out of the separation channel from the outlet port in this order. In the sixth operation, a field flow fractionation device detects the second particle group that has flowed out of the outlet port in the fifth operation with the detector.

8. Under the control of the control unit, the first operation, the second operation, the third operation, and the fourth operation are executed in this order, a cross-flow is formed during the operations of the first to third operations, during the operations of the second to third operations, the flow rate of the cross-flow in the first operation is gradually reduced until it becomes zero, and in the fourth operation, the flow rate of the reverse cross-flow is gradually increased from zero to a preset flow rate. The field flow fractionation device according to claim 7.

9. Under the control of the control unit, in the sixth operation, further, the first particle group flowing out from the outlet port in the fifth operation is detected by the detector. The field flow fractionation device according to claim 7.

10. Under the control of the control unit, in the seventh operation, while forming the reverse cross-flow, the liquid in the separation channel is caused to flow out from the outlet port, and the seventh operation is executed in at least one of before the first operation and after the sixth operation. The field flow fractionation device according to claim 7.

11. The control unit further controls the sample supply unit. The field flow fractionation device according to claim 7.

12. A porous support for supporting the membrane is disposed in the discharge chamber. The field flow fractionation device according to claim 7.

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