JP6346126B2 - Molecular weight distribution measuring method and molecular weight distribution measuring apparatus - Google Patents

Description

  The present invention relates to a molecular weight distribution measuring method and a molecular weight distribution measuring apparatus.

  Measurement of molecular weight is fundamentally important in chemical analysis, organic synthesis, industrial compound production, and biochemical experimental procedures. In organic synthesis and industrial compound production, the molecular weight is an index for evaluating a synthesized product. In the biochemical experimental operation, basically, the change in the molecular weight accompanying the cleavage and ligation of the protein that is a linear polypeptide and the DNA molecule that is a linear polydeoxyribonucleic acid is confirmed. Molecular weight is also important to confirm success.

  In addition, chromatography is mainly used to isolate a target molecule from a product obtained by a chemical reaction. In chromatography, the speed at which molecules move through the column varies depending on the interaction between the size of the molecules and the network of column packings. As a result, the retention time (Retention) varies depending on the molecular weight, and the molecules can be separated based on the difference in time from injection to elution.

  In general, in chromatography, molecules are separated by the interaction between the molecules to be separated and the walls of the column (container) or packing material. For example, in gel chromatography, due to the interaction between the size of the gel (filler) network and the size of the molecules to be separated, the smaller the molecules, the larger the free zone volume, and the longer the residence time. As a result, since molecules having a higher molecular weight pass through the column earlier, molecules can be separated by molecular weight (see, for example, Non-Patent Document 1).

  In gel electrophoresis, molecules charged by an electric field migrate, and when molecules pass through a matrix of acrylamide polymer (filler), large molecules are hindered by the matrix and mobility becomes small. Increases mobility.

  When a sample of a mixture of multiple molecules with different molecular weights is run from a single point on the gel, the migration speed differs depending on the molecular weight of the molecules. The molecule stops. If the stopped molecule is detected by staining or fluorescent method, the molecular weight can be measured based on the movement distance of the molecule from the starting point of electrophoresis.

  In order to determine the molecular weight, a sample having a known molecular weight (hereinafter referred to as “molecular weight marker”) is simultaneously migrated, and the stop position is compared with the stop position of the molecule to be measured. In this case, it is necessary to migrate the molecular weight marker and the molecule to be measured under the same conditions and measure the stop position after stopping the migration.

  Thus, in the conventional separation of molecules by chromatography, a sample of a mixture containing molecules to be separated (hereinafter sometimes referred to as “separated molecules”) is upstream of a column or a column-shaped flow path. The molecules are separated using the difference in the moving speed due to the interaction between the molecules to be separated and the stationary phase (packing agent) fixed in the column.

  As long as this interaction continues, the difference in the moving speed of the molecule will continue, so if you move through the column for a long distance or for a long time, the difference in the moving distance of the molecule according to the molecular weight will increase accordingly. Even molecules with small molecular weight differences can be separated based on the difference in distance. On the other hand, as time elapses due to the effect of diffusion and movement speed for each molecule, the distribution of elution time spreads even with the same molecular weight.

  For this reason, in order to determine the molecular weight of a plurality of molecules by separating a sample containing a mixture of molecules by chromatography, the experimental conditions are determined in consideration of the effect of dispersion of column length and moving speed. There is a need to.

  In chromatography, the concentration distribution of molecules generated in a column is detected by a sensor installed at the outlet of the column. This sensor has a method for measuring absorbance, fluorescence intensity, chemiluminescence intensity, refractive index, electrochemical activity, and conductivity.

  Molecules are measured by the properties of the molecules to be measured and the label molecules previously bound to the molecules. When a labeled molecule is used, the concentration distribution of the molecule can be measured with high sensitivity. However, when the molecule to be measured is unknown or the efficiency of the labeling reaction is unknown, measurement using the label becomes difficult.

  In the method using absorption, it is necessary to select a wavelength at which the molecule to be measured has absorption, but since a suitable wavelength differs depending on the molecule to be measured, a broadband spectrophotometer is required. Since the extinction coefficient varies greatly depending on the molecule to be measured, the extinction coefficient of the target molecule needs to be known in advance in order to obtain the concentration from the absorbance.

  In order to increase sensitivity with absorbance, it is necessary to increase the optical path length. However, if the optical path length is increased, the volume is required by that amount, and the concentration is averaged by the increased optical path length. Although it is separated in the column, the signal at the detector at the outlet of the column is measured with a lower resolution.

  In the electrochemical detection method, a molecule having a high electrochemical activity can be detected with high sensitivity, but other molecules having a low electrochemical activity cannot be detected, so that the molecules to be measured are limited.

  In the refractive index measurement, since the refractive index is substantially proportional to the concentration of the solution, the concentration of the molecule can be measured without being limited to the type of molecule to be measured. For this reason, it is suitable for measuring the molecular weight distribution of a sample mixed with unknown molecules. Further, in the measurement using the total reflection optical system, the concentration can be measured for each reflection point, so that the molecular weight distribution can be measured without losing information on the molecules separated in the column.

  In chromatography, the separation of molecules proceeds in the middle of the column, but the process is not measured. The time-dependent change in the concentration of molecules eluted at one point at the outlet is measured, and the molecular weight distribution is determined from the waveform (chromatogram). taking measurement.

  If the sample is a single molecule, or if the separation results in sufficient separation from other molecules and the single molecule flows to the detector in order during elution, the measured molecule measured by the detector The time change (chromatogram) of the concentration of A becomes a peak. Electrophoresis is the same as in the chromatogram because a peak-like result is obtained by the electrophoresis pattern.

  When two molecules having similar molecular weights pass through the column without sufficiently being separated and reach the detector, a chromatogram in which peaks are superimposed on the concentration pattern recorded by the detector is obtained.

  Since it is difficult to determine the molecular weight of overlapping peaks, it is necessary to optimize the column packing material, flow rate, sample volume, etc., and perform a separation operation so that the peaks do not overlap. Even in the case of gel electrophoresis, an operation for improving the separation performance is required by changing the gel composition.

  In both chromatography and gel electrophoresis, molecules are separated while the sample flows through the space filled with the packing material, so it is necessary to increase the residence time in the column in order to increase the resolution. The time cannot be shortened.

  Therefore, in chromatography, the flow rate is increased by increasing the pressure for liquid feeding. For this purpose, a pump capable of generating high pressure, a column capable of withstanding the pressure, and a connection part for liquid feeding are required.

  In electrophoresis, the speed of electrophoresis can be increased by increasing the voltage, but at the same time, the current also increases, so that the temperature rises and the reaction at the electrode becomes intense, making stable electrophoresis difficult. Thus, in chromatography, electrophoresis, etc., it takes a lot of time to separate molecules of a mixture or an unknown sample by molecular weight or to measure the molecular weight of the molecules.

  In view of this, a method has been proposed in which molecules are separated by molecular weight in a flow-through flow channel that flows away from the flow channel without filling the inside of the capillary column with a filler (see Non-Patent Document 2).

  In this Non-Patent Document 2, a microchannel having a length of 1 [um], a width of 0.5 [mm] and a length of 69 [mm] is produced, and particles (molecules) having a diameter of 155 [nm] or less and benzoic acid are mixed. A sample of the mixed mixture is injected into the microchannel from the injector, and the elution pattern is measured with an ultraviolet absorption detector (UV detector) installed at the outlet of the microchannel. And benzoic acid is detected last.

  This is because particles (molecules) having a large diameter do not approach the wall surface and are eluted earlier because they flow through the central portion with a higher linear flow velocity. Such a flow-through method is called hydrodynamic chromatography (HDC).

  In Non-Patent Document 3, in order to divide particles (molecules) by HDC having a cross-sectional aspect ratio close to 1, the size of the particles (molecules) is the height of the channel or the diameter of the column of the HDC. Need to be greater than a few percent.

  On the other hand, in a channel with a large aspect ratio, diffusion in the channel height and radial direction works more dominantly than particle (molecule) size, and diffusion and flow without chemical interaction with the outside. Molecular separation occurs only by the effect of.

  As a result, molecular separation actually occurs according to a simple theory. For example, in a capillary column having an inner diameter of 150 [um] and a length of 0.3 [m], a 220 [nm] light absorption detector is used to detect iodine ions (molecular weight 127). ) And nitrate ions (molecular weight 62) can be separated in about 100 seconds. In addition, since HDC having a large aspect ratio does not use chemical interaction, time is not required for the reaction, the flow rate can be increased, and molecules can be separated in a short time and at a short distance.

  Thus, although high-speed molecular separation is expected using HDC, the separation performance is low compared to liquid chromatography using a packed column, and the high-speed and simplicity of HDC achieved compared to that is the separation. The disadvantage of low performance could not be covered.

  This HDC is a measurement method based on so-called Taylor dispersion. On the other hand, Japanese Patent Application No. 2014-090269 according to the present application discloses a technique capable of measuring the molecular weight (or diffusion coefficient) in a short time and in a short flow path by measuring the concentration on the wall surface of the flow path.

  In this technique, the conventional Taylor dispersion method for measuring the diffusion coefficient was measured using the spatial dispersion of the solute in the flow direction after the solute moved to the average flow velocity as an index. The diffusion coefficient can be measured from the moving speed of the solute near the road wall.

  Specifically, when a solution is injected into a channel filled with a solvent, the solute moves along the flow in the vicinity of the center of the channel, and thus moves at the highest speed in the channel cross section. Then, it decelerates by diffusion, and finally moves at a speed that is 2/3 of the centerline flow velocity.

  On the other hand, the solute does not move near the channel wall surface because the flow velocity is zero due to the zero slip condition. However, since the concentration changes due to diffusion from the preceding flow path center portion, the solute begins to advance. Therefore, the solute speed is accelerated from 0 on the wall surface, and finally accelerated to 2/3 of the same centerline flow velocity as the flow path center.

  Since this concentration change process depends on diffusion, the diffusion coefficient of the solute can be obtained from the acceleration when the solute moves on the wall surface. In addition, the solute reaches the channel outlet in a time depending on the diffusion coefficient.

  Here, the diffusion coefficient has a simple tendency to correlate with the molecular weight. When the molecular weight is small and the diffusion coefficient is large, the diffusion coefficient is quickly diffused, and when the molecular weight is large and the diffusion coefficient is small, the diffusion coefficient flows slowly through the flow path. Since the time is different, it can be said that the elution time of the sample differs depending on the molecular weight. Therefore, if the diffusion coefficient is obtained with this technique, there is a possibility that molecules can be separated by molecular weight based on the obtained diffusion coefficient while having performance exceeding the high speed and simplicity of HDC.

Giddings, J. C., Unified Separation Science. John Wiley and Sons: 1991. Blom, MT; Chmela, E .; Oosterbroek, RE; Tijssen, R .; van den Berg, A., On-Chip Hydrodynamic Chromatography Separation and Detection of Nanoparticles and Biomolecules. Analytical Chemistry 2003, 75 (24), 6761-6768 .Chmela, E .; Tijssen, R .; Blom, MT; Gardeniers, HJGE; van den Berg, A., A Chip System for Size Separation of Macromolecules and Particles by Hydrodynamic Chromatography. Analytical Chemistry 2002, 74 (14), 3470 -3475. Non-patent literature Okada, T., HYDRODYNAMIC CHROMATOGRAPHY IN NARROW AND WIDE-BORES; WHETHER RADIAL DIFFUSION IS ESSENTIAL OR NOT. Journal of Liquid Chromatography & Related Technologies 2010, 33 (9-12), 1116-1129. Taylor, G., Dispersion of Soluble Matter in Solvent Flowing Slowly through a Tube.Proceedings of the Royal Society of London.Series A. Mathematical and Physical Sciences 1953, 219 (1137), 186-203.)

  However, the technique according to Japanese Patent Application No. 2014-090269 measures the molecular weight (diffusion coefficient) of one kind of molecule constituting a pure sample, and cannot measure the molecular weight distribution of a plurality of molecules constituting a mixture. There wasn't.

  The present invention has been made to solve the above problems, and a molecular weight distribution measurement method and a molecular weight distribution measurement method for measuring the molecular weight distribution of a plurality of molecules constituting a sample (second liquid) in a short time. The object is to propose a device.

  In the molecular weight distribution measuring method according to claim 1 of the present invention, at least a first liquid and a second liquid in which a plurality of molecules (j) are mixed in a flow path (113) of a flow path forming member (101). A liquid-liquid step for feeding the first liquid and the second liquid in contact with each other, and the first liquid and the second liquid in contact with each other in the flow path (113) diffused to each other. A measuring step for measuring the total concentration distribution (Call) of the second liquid, and a molecular weight of a plurality of molecules (j) constituting the second liquid based on the total concentration distribution (Call) measured in the measuring step A molecular weight distribution calculating step for calculating a distribution (A (Q)).

  2. The molecular weight distribution measuring method according to claim 1, wherein the measuring step measures the total concentration distribution (Call) in the vicinity of a channel wall surface of the channel forming member (101).

  2. The molecular weight distribution measuring method according to claim 1, wherein the flow path forming member (101) has an aspect ratio of greater than 1 in a cross section perpendicular to the liquid feeding direction.

  The molecular weight distribution measuring apparatus according to claim 4 of the present invention includes a flow path forming member (101) in which a flow path (113) is formed, and at least a first liquid and a plurality of molecules (in the flow path (113)). In the state where the second liquid in which j) is mixed is in contact with the first liquid and the liquid feeding section (106) for feeding the second liquid, the first liquid in contact with the second liquid in the flow path (113). A measuring unit (102) that measures the total concentration distribution (Call) of the second liquid when the first liquid and the second liquid diffuse to each other, and the total concentration (Call) measured by the measuring unit (102) ) Based on the molecular weight distribution calculation unit (102) for calculating the molecular weight distribution (A (Q)) of the plurality of molecules (j) constituting the second liquid.

  According to the first and fourth aspects of the present invention, the molecular weight distribution (A (Q)) of a plurality of molecules constituting the second liquid can be obtained in a short procedure and in a short time using a small short flow path (113). Can be calculated.

  According to the invention of claim 2, since the total concentration (Call) in the vicinity of the channel wall surface is measured, it is possible to measure a stable concentration change in a portion that is least affected by the flow.

  According to the invention of claim 3, since the flow path (113) has an aspect ratio of the cross section of the flow path larger than 1, the second liquid is molecular only by the effect of diffusion and flow without interaction with the outside. Can be separated.

It is a perspective view which shows the structure of the HDC apparatus in embodiment of this invention. It is a perspective view which shows the structure of a microchannel chip | tip. It is a graph which shows the measurement result of the state which the inside of a flow path switches from the refractive index A to the refractive index B. It is a model figure which modeled the main channel of a micro channel. It is a model figure which shows the initial state when switching from a 1st liquid to a 2nd liquid. It is a model figure which shows density | concentration distribution when switching from the 1st liquid to the 2nd liquid after progress for predetermined time. Model showing spatio-temporal concentration distributions showing the state in which the first liquid (pure substance liquid) and the second liquid (mixed solution of a plurality of molecules) are in contact with each other and flow through the flow path with three types of mutual diffusion coefficients Q. FIG. It is a figure when time-spatial density distribution of FIG. 7 is time-differentiated. It is a figure when the space time density distribution of FIG. 7 is spatially differentiated. FIG. 6 is a diagram showing the results of spatial differentiation of spatiotemporal concentration distributions of six types of samples in which six individual molecules are dissolved, and a sample in which three molecules are mixed, and a diagram showing the molecular detection results of a sample in which three molecules are mixed. is there. It is a figure which shows the diffusion coefficient distribution and molecular weight distribution of 6 types of samples which melt | dissolved 6 individual molecules.

  Hereinafter, an outline of an HDC (Hydrodynamic chromatography) apparatus as a molecular weight distribution measuring apparatus according to an embodiment of the present invention will be described. In this embodiment, since “density” is measured by replacing “refractive index”, it is considered that “density” and “refractive index” have a certain proportional relationship. Further, “diffusion coefficient” and “molecular weight” are monotonous and simple relationships, and can be converted into each other based on a predetermined relational expression.

<Outline of HDC device>
The HDC apparatus is configured to contact the first liquid that has filled at least a part of the flow path formed in the flow path forming member with the second liquid in which two or more types of molecules are mixed, The total concentration of a plurality of molecules contained in the second liquid (hereinafter, referred to as “total concentration”) when one liquid and the second liquid diffuse to each other while being sent through the flow path is measured. The concentration distribution (molecular weight distribution) for each molecular weight of a plurality of molecules contained in the second liquid is calculated by a predetermined component concentration calculation method such as a non-negative least square method.

<Configuration of HDC device>
As shown in FIG. 1, an HDC device 100 that measures a diffusion coefficient includes a micro-channel chip 101 as a channel-forming member and a refractive index measuring device connected in a state where the micro-channel chip 101 is placed. 102, a liquid dispensing device 104 as an injection device connected to the liquid introduction hole 110 opened to the atmosphere of the microchannel chip 101 by an FEP (fluororesin) tube 103, and the microchannel chip 101 by the FEP tube 103. The liquid discharge hole 111 is connected to the liquid discharge hole 111 in an airtight state, and the pressure control pump 106 is connected to the gas / liquid separation apparatus 105 and the FEP tube 103 as a liquid feeding start device. .

  As shown in FIG. 2, the microchannel chip 101 has a liquid introduction hole 110 formed at one end of the main channel 113 and a liquid discharge hole 111 formed at the other end of the main channel 113. The liquid introduction hole 110, the main flow path 113, and the liquid discharge hole 111 constitute a very small flow path (hereinafter referred to as “micro flow path”) 112 as a whole.

  The channel shape of the main channel 113 is created by a casting method using PDMS (polydimethylsiloxane). Specifically, the main flow path 113 has a simple rectangular parallelepiped shape with an aspect ratio larger than 1 and about 100 [μm] × 1 [mm], and the flow path length is about 5 [mm]. However, the present invention is not limited to this. The microchannel chip 101 is formed by bonding a glass (BK7) substrate and a microchannel 112.

  The refractive index measurement device 102 is a total reflection optical system refractive index measurement device, and includes a camera unit 120 such as an image sensor below the main flow path 113 in the vertical direction. Therefore, the refractive index measuring device 102 measures the refractive index (concentration) near the wall surface along the center line of the main flow path 113 of the micro flow path 112 by the camera section 120 as a measurement section. Since the refractive index measuring device 102 measures the refractive index (concentration) near the wall surface of the main flow path 113, it can measure a stable concentration change in a portion that is least affected by the flow of the main flow path 113. .

  As the refractive index measuring device 102, a surface plasmon resonance (SPR) type or critical angle or Brewster angle measuring type device can be used. In the case of the SPR type, a metal film is required on the wall surface of the micro flow channel 112 on the refractive index measuring device 102 side.

  The gas-liquid separation device 105 is a device that separates gas and liquid. The liquid dispensing device 104 is a device that supplies liquid to the liquid introduction hole 110 at a predetermined timing.

  The refractive index measuring apparatus 102 is realized by installing a computer program (software) in a computer (hardware) including a CPU (Central Processing Unit), a memory, an interface, and the like, and the diffusion coefficient in the refractive index measuring apparatus 102 is The functional units such as the calculation unit are realized by the cooperation of various computer hardware resources and a computer program. Further, the computer program may be provided in a state of being stored in a computer-readable recording medium or a storage device, or may be provided via an electric communication line.

<Injection>
A method of bringing the first liquid and the second liquid into contact with each other in the HDC device 100 having such a configuration will be described. In the HDC device 100, when water that is 10 times the volume of the main channel 113 in the microchannel 112 is dropped into the liquid introduction hole 110 by the liquid dispensing device 104 and sucked at a constant pressure by the pressure control pump 106, The inside of 113 can be made into a predetermined pressure state, the liquid (water) flows through the main flow path 113 at a liquid speed according to the pressure state, fills the main flow path 113, and then reaches the gas-liquid separation device 105. . The liquid accumulates at the bottom of the gas-liquid separator 105, and the gas necessary for pressure control reaches the pressure control pump 106.

  In the HDC device 100, the gas-liquid separator 105 and the pressure control pump 106 are used in a separated state. However, the present invention is not limited to this, and a pressure control pump capable of controlling the liquid pressure is also used. It may be used as a starting device.

  In a state where the main channel 113 is filled with liquid (water), the liquid dispensing apparatus 104 drops the first liquid (solvent) having a refractive index A into the liquid introduction hole 110 in the microchannel 112. The first liquid is a pure substance liquid. However, it is not restricted to this, The 1st liquid may be a solution containing a mixture and a blocking agent.

  When the first liquid is dropped, it comes into contact with the liquid (water) that has been filled up to the inlet of the microchannel 112 previously, so that the surface tension is remarkably lowered, and liquid feeding starts due to the pressure of the pressure control pump 106.

  When the volume of the sample (first liquid) in the liquid introduction hole 110 is gradually reduced and the water surface falls to the entrance of the micro flow path 112 (the connection point between the liquid introduction hole 110 and the main flow path 113), the surface of the first liquid The tension and the pressure of the pressure control pump 106 are balanced, and the first liquid does not move beyond that.

  At this time, when the refractive index of the main channel 113 is measured by the camera unit 120 of the refractive index measuring device 102, the value corresponding to the refractive index A of the first liquid is uniformly along the measurement axis Z parallel to the main channel 113. Measured. This is because the main channel 113 is filled with the first liquid.

<Contact and liquid feed of first liquid and second liquid>
Next, in the refractive index measuring apparatus 102, the camera unit 120 is set so that the change in refractive index with time can be measured every 1/150 seconds. In this setting, when the second liquid having the refractive index B is dropped into the liquid introduction hole 110 by the liquid dispensing device 104, the liquid supply of the second liquid is started by the pressure of the pressure control pump 106 also at this time. That is, after the first liquid and the second liquid are brought into contact with each other, the first liquid and the second liquid are fed in the main channel 113. In the main flow path 113, while the first liquid is being fed, the second liquid is dropped into the main flow path 113 so that the first liquid and the second liquid are brought into contact with each other. It may be.

  Here, the second liquid needs to contain the first liquid at the same composition ratio. In this case, the second liquid is a solution having a refractive index B containing three molecules of sodium chloride (Nacl), aprotinin, and immunoglobulin G (IgG) in the first liquid.

  At this time, the refractive index measuring apparatus 102 can observe a state where the inside of the main channel 113 is switched from the refractive index A to the refractive index B as shown in FIG. In this case, with respect to the refractive indexes A and B, the refractive index B is higher than the refractive index A. In FIG. 3A, the time is displayed on the X axis, the position from the upstream to the downstream in the main flow path 113 is displayed on the Y axis, and the change in refractive index with time is displayed on the Z axis.

  In the refractive index measuring apparatus 102, as shown in FIG. 3B, the refractive index A of the first liquid is first observed at the center of the refractive index observation range (Y axis) of the main channel 113, and the refractive index B It is possible to observe a state of change in which the refractive index B is observed over time from the arrival of the second liquid flow. In FIG. 3B, the refractive index is described as SPR angle proportional to the refractive index.

<Relationship between diffusion coefficient and total concentration spatiotemporal distribution>
Here, the general relationship between the diffusion coefficient and the total concentration spatiotemporal distribution will be described. In this case, a flow channel model having the same shape as that of the HDC apparatus 100 as shown in FIG. 1 is constructed for the second liquid containing one kind of molecule, and an OpenFOAM Inter Mixing Foam (for example, non-patent) The total concentration distribution (spatiotemporal distribution) was calculated using reference 4).

  As shown in FIG. 4, a flow path 200 that models the main flow path 113 of the HDC device 100 is used, a constant pressure is applied to both ends of the flow path 200, and the mutual diffusion coefficient between the first liquid and the second liquid is obtained. And the spatiotemporal distribution of the solute concentration was calculated.

  As an initial state, the second liquid is injected after the flow path 200 is filled with the first liquid, and the calculation is started when the flow stopped in the flow path 200 is resumed. The state in which the liquid switched to the second liquid was calculated. Here, in order to simplify the shape to be calculated, it is assumed that the second liquid has entered the same shape of the flow path in advance, and the flow path inlet surface 201 is in the middle of a rectangular parallelepiped flow path having a single cross-sectional shape. It shall be.

  The calculation is performed in two dimensions for efficiency, and the flow path 200 is set to be an infinite parallel plate, the substance concentration gradient on the lower surface of the flow path 200 is “0”, and the wall surface slips. A non slip boundary condition was set. In addition, symmetry is set in the upper direction of the flow path 200, and calculation is performed with a half thickness (height) of the flow path 200. In the following figure, the calculation result of this half thickness is shown.

  As shown in FIG. 5A, in the initial state, the first liquid occupies most of the flow path 200, and the second liquid is disposed upstream of the flow path inlet surface 201. In this case, it can be seen that the second liquid and the first liquid are in contact with each other at a boundary surface perpendicular to the longitudinal direction of the flow path (liquid feeding direction). Further, FIG. 5B shows the fraction (concentration) of the second liquid on the lower surface of the flow path 200 that is the object of refractive index measurement.

  As shown in FIG. 6A, when time elapses, the first liquid and the second liquid flow in the downstream direction of the flow path 200 due to the pressure applied to both ends of the flow path 200. At that time, the first liquid of the solvent and the second liquid of the solution flow while being mixed by the mutual diffusion coefficient Dm. In this calculation, since there are only the first liquid and the second liquid, the mutual diffusion coefficient Dm1 from the first liquid to the second liquid coincides with the mutual diffusion coefficient Dm2 from the second liquid to the first liquid. And

  In this calculation, if the flow path 200 is short with a total length of 500 [μm], the flow path 200 will flow through from the start to 0.1 [s] in terms of time, and therefore the first liquid and the first liquid in the cross section of the flow path 200 It can be seen that the two liquids flow in the downstream direction without becoming uniform. Further, it can be seen that the boundary surface first moves in the downstream direction at the center of the flow path where the flow velocity is maximum (above FIG. 6A) due to mixing by diffusion and flow velocity distribution in the cross section.

  In this case, the lower surface of the flow path 200 assumes a boundary condition where the flow velocity on the wall surface is “0” and there is no slip (because zero slip is assumed), the lower flow in FIG. On the lower surface (wall surface) of the path 200, the flow velocity that is the moving speed of the liquid is “0”.

  However, the liquid-liquid boundary also moves in the downstream direction on the lower surface of the flow path 200. This is because the first liquid and the second liquid flow in a direction perpendicular to the flow direction of the flow path 200 while being mixed by diffusion. In the graph of FIG. 6B, it can be seen that the concentration distribution of the second liquid on the wall surface of the flow path 200 is spread over a wide range.

  In the flow channel model of the flow channel 200, the first liquid and the second liquid having the same viscosity coefficient are in contact with each other, mixed without changing their viscosity, and mixed while flowing in the downstream direction of the flow path 200 in a laminar flow. .

  In the calculation, for simplicity, the first and second liquids are both pure solvents and mixed with a single mutual diffusion coefficient Dm (first liquid diffusion coefficient Dm1 = second liquid diffusion coefficient Dm2). The mixing ratio is read as the concentration by calculation, and the mixing ratio (concentration) is obtained based on each position in the flow direction in the flow path 200 and the time.

  The flow rate in the laminar flow depends on the viscosity, but does not depend on the diffusion coefficient. Further, in this calculation, the viscosity is assumed to be unchanged, and a flow path shape that assumes laminar flow is assumed. Therefore, the flow velocity distribution is the same regardless of the setting of the mutual diffusion coefficient Dm.

  Viscosity becomes negligible by lowering the concentration of the solution. Moreover, if the component of the 1st liquid as a mixed solvent is adjusted and the solute of a 2nd liquid is dissolved in a 1st liquid, the difference in a viscosity can be made small. Further, when the viscosity is known, the concentration distribution in the case of the viscosity can be obtained by numerical calculation.

<Measurement of total concentration distribution of second liquid>
On the other hand, when the second liquid in which a plurality of molecules having the same concentration (f) are mixed is targeted, the total concentration distribution of the second liquid measured by the camera unit 120 of the refractive index measuring apparatus 102 will be described.

  Here, regarding the concentration distribution of the first liquid, if the mutual diffusion coefficient “Dm1” of the first liquid is used, the concentration distribution of the first liquid at each point (y, z) and each time (t) of the main flow path 113. Can be represented by “c (y, z, t, Dm1)”.

  Further, since the second liquid is a mixed solution of a plurality of molecules having the same concentration (f), the mutual diffusion coefficient of each molecule (j) constituting the second liquid with the first liquid is “Dm (j)”. Assuming that “Dm (j)” is independent, the total concentration distribution Call of the second liquid can be expressed by the following (formula 1). In each molecule (j), j = 1 to n and n is an integer of 2 or more.

Call = Σ _j f * c (y, z, t, Dm (j)) …………………………………………………………… (Formula 1)

  Here, “f” is the same concentration value (fixed value) independent of the molecule (j), “y” is a position in the flow direction of the main flow path 113, “z” is a position in the height direction of the main flow path 113, “T” is time. C (y, z, t, Dm (j)) is the value of each molecule (j) at each point (y, z) and each time (t) when the second liquid is normalized at the highest concentration. Concentration distribution. That is, the density value represented by the density distribution c (y, z, t, Dm (j)) is a value from 0 to 1.

  That is, the total concentration distribution Call of the second liquid is the concentration distribution c (y, z, t, of each molecule (j) of the second liquid calculated by the mutual diffusion coefficient Dm (j) of the single molecule (j). Dm (j)). Between solutes with different mutual diffusion coefficients Dm (j) of the first and second liquids, the diffusion coefficients affect each other, so the second liquid may not be established strictly at high concentrations, but in other cases In many cases, the lower the concentration, the better.

<Molecular weight separation>
Furthermore, when the second liquid is a mixture of a plurality of molecules (j) (where j = 1 to n) having different concentrations or a polymer having a molecular weight distribution of the plurality of molecules (j) Assuming that the concentration of each molecule (j) having a single molecular weight is A (j), the total concentration distribution Call of the second liquid can be expressed by the following (Equation 2). The concentration A (j) of the molecule (j) is a non-negative value. In this case, it is assumed that the concentration A (j) of the molecule (j) varies depending on the type (j) of the molecule.

Call = Σ _j A (j) * c (y, z, t, Dm (j)) ………………………………………………………… (Formula 2)
In this case, the measured value of the total concentration distribution Call of the second liquid acquired by the camera unit 120 of the refractive index measuring apparatus 102 and the concentration distribution c (y of the second liquid calculated by the calculating unit of the refractive index measuring apparatus 102 , z, t, Dm (j)), the concentration A (j) of the molecule (j) can be obtained by a component concentration calculation method such as a non-negative least square method.

  Here, when the concentration distribution c (y, z, t, Dm (j)) of the second liquid is obtained, the type and molecular weight of each molecule (j) contained in the second liquid are known. The molecule (j) can be represented by a spatiotemporal concentration distribution of the molecular weight and the concentration A (j). That is, the molecule (j) can be separated based on the molecular weight based on the concentration distribution c (y, z, t, Dm (j)) of the second liquid.

<Molecular weight distribution>
Next, a method for calculating the molecular weight distribution A (Q) of the second liquid will be described. The concentration A (j) of the molecule (j) may be considered to be continuously distributed, the type and molecular weight of each molecule (j) is known, and based on the molecular weight of each molecule (j) Since it can be expressed continuously, the total concentration distribution Call of the second liquid can be expressed by the following (formula 3).

Call = ∫A (Q) * c (y, z, t, Q) dQ ………………………………………………………… (Formula 3)
Here, A (Q) is the molecular weight distribution of the second liquid and is a non-negative value. “Q” is an integral variable representing the mutual diffusion coefficient of the molecule (j) of the second liquid.

  The integration according to (Equation 3) is performed using the mutual diffusion coefficient Q of the plurality of molecules (j) contained in the second liquid. The concentration distribution c (y, z, t, Q) of each molecule (j) in the second liquid in (Expression 3) is calculated by the calculation unit of the refractive index measuring device 102 based on the diffusion coefficient Q of any molecule (j). It is possible to obtain by numerical calculation according to.

  The total concentration distribution Call of the second liquid is a measurement value that can be measured by the camera unit 120 of the refractive index measurement device 102 of the total reflection optical system as described above. In (Equation 3), the concentration distribution c (y, z, t, Q) of each molecule (j) in the second liquid is the value of the channel wall surface of the main channel 113, so z = 0 and the concentration Distribution c (y, 0, t, Q).

  In this case, the total concentration distribution Call of the second liquid is measured as a two-dimensional array (image) by the camera unit 120 of the refractive index measuring device 102. At that time, each image measured at each point (y, z) is a concentration distribution c (y, z, t, Q) of each molecule (j) of the second liquid at that point.

  Therefore, in the refractive index measurement apparatus 102, the spatio-temporal concentration distribution (image) representing the total concentration distribution Call of the second liquid by adjusting the measurement time interval by the camera unit 120 and the length of the measured portion of the main channel 113. Can be obtained with high resolution.

  Since the concentration distribution c (y, 0, t, Q) of each molecule (j) in the second liquid and the total concentration distribution Call of the second liquid acquired by the refractive index measuring device 102 are known, the non-negative least square method It is possible to estimate the molecular weight distribution A (Q) of the second liquid by a predetermined component concentration calculation method (mathematical calculation method) using the above. In this way, the molecular weight distribution A (Q) of the second liquid can be calculated.

Specifically, as shown in FIGS. 7A to 7C, the first liquid and the second liquid come into contact with each other at a position 400 on the vertical axis y and then flow through the main flow path 113. The diffusion coefficient Q is shown for each of 1e-8 [m ² / s], 1e-9 [m ² / s], and 1e-10 [m ² / s].

  That is, in FIG. 7, the light and shade represent the difference in concentration. The larger the diffusion coefficient Q, the more the spatial concentration gradient of the total concentration distribution Call of the second liquid becomes gentle, and the smaller the diffusion coefficient Q, the total concentration of the second liquid. It can be seen that the spatial concentration gradient of the distribution Call becomes steep.

Thus, for each diffusion coefficient Q of 1e-8 [m ² / s], 1e-9 [m ² / s], 1e-10 [m ² / s], each molecule (j) of the second liquid The concentration distribution c (y, 0, t, Q) can be obtained by calculation. In this case, it is possible to calculate the concentration distribution c (y, 0, t, Q) as finely as possible by setting the diffusion coefficient Q in the entire diffusion coefficient range of each molecule (j) of the second liquid. The concentration distribution c (y, 0, t, Q) can be calculated as a continuous function with respect to the diffusion coefficient Q. Thereby, the molecular weight distribution A (Q) of the second liquid can be obtained with high resolution.

  As described above, before obtaining the molecular weight distribution A (Q) of the second liquid from (Equation 3), before (Equation 3) is set to the time variable t or the position of the main flow passage 113 in the flow direction. The variable y or both may be first-order differentiated or higher-order differentiated (time variable t is n (n is an integer of 2 or more), position variable y is n-th derivative, etc.). Thereby, the boundary surface which a 1st liquid and a 2nd liquid contact can be coordinated.

  FIGS. 8A to 8C are spatiotemporal concentration distributions of the second liquid obtained by temporally differentiating the concentration distribution c (y, 0, t, Q) of (Equation 3). Represents the result. 9A to 9C show the results of spatial differentiation of the concentration distribution c (y, 0, t, Q) in (Equation 3) with respect to the time variable t and the position variable y in the flow path direction of the main flow path 113. Is a spatiotemporal concentration distribution of the second liquid.

  As described above, when the concentration distribution c (y, 0, t, Q) is time-differentiated or spatially differentiated, the boundary surface where the first liquid and the second liquid are in contact with each other and the calculation processing amount is reduced. The molecular weight distribution A (Q) of the second liquid can be measured with high resolution.

<Estimation of molecular weight distribution for a mixture of three molecules>
Next, using the concentration distribution c (y, 0, t, Q) of each molecule (j) of the second liquid obtained experimentally rather than by calculation, each of the second liquid of the mixture of three molecules is used. The result of the experiment for calculating (estimating) the molecular weight distribution of the concentration A (j) of the molecule (j) will be described.

  In this case, for example, a block ace solution is used as a first solution, and six types of sodium glycerol, glucose, fructose, aprotinin, human albumin (HSA), and human IgG (hIgG) are individually dissolved in the block ace solution. Changes in the refractive index (concentration) of the second liquid when the solution was the second liquid were measured. Moreover, the change of the refractive index (concentration) of the sample which mixed three molecules, sodium chloride, aprotinin, and human IgG among said 6 molecules was measured similarly.

  FIG. 10A shows the result of spatial differentiation of the change in refractive index (concentration) in each of the six individual molecules and the result of spatial differentiation of the change in refractive index (concentration) in a sample in which three molecules are mixed. Show. In FIG. 10A, measurement data from the time point of 0.1 seconds before the contact between the first liquid and the second liquid to the time point of 0.67 seconds after the contact was used. That is, the time required for measuring the molecular weight distribution in this case is 0.77 seconds.

  The change in the refractive index (concentration) of the sample in which three molecules are mixed is the sum of the change in the refractive index (concentration) of the corresponding three individual molecules. Here, when the molecular weight distribution A (Q) of the second liquid that minimizes the square sum of the difference between the right side and the left side of (Equation 3) is calculated by the non-negative least square method, the result shown in FIG. Obtained.

  Actually, sodium chloride, aprotinin, and human IgG were detected as mixed with the sample, and unmixed glucose, sucrose, and human albumin (HSA) were not detected in the sample. In this case, the concentration ratio of the detected three molecules should be 1: 1: 1, but in this example, the noise contained in the measured value of the total concentration distribution Call of the second liquid and the measurement Since the pressure difference between both ends of the main channel 113 could not be kept constant, the concentration ratio could not be determined with high accuracy.

  However, it can be seen that the molecules (sodium chloride, aprotinin, human IgG) mixed in the sample can be accurately separated from the molecules (glucose, sucrose, human albumin (HSA)) not mixed in the sample.

  Since the diffusion coefficient Q of 6 molecules and the molecular weight of 6 molecules are already known, if this value is used to graph FIG. 10B corresponding to the diffusion coefficient and the molecular weight, FIG. 11A and FIG. As shown, the diffusion coefficient distribution and molecular weight distribution of the sample can be obtained. That is, it can be seen that chromatography can be performed by this method.

  Thus, in the refractive index measuring apparatus 102, for example, a mixture of inorganic salt, sugar, and protein having a molecular weight range (50 to 150,000) from sodium chloride (Nacl) to immunoglobulin G (IgG) can be reduced by one operation. Separation can be achieved in a short time of 0.77 seconds.

  In this case, the HDC device 100 does not require a special packed column, gel, or a special structure inside the flow path, and may simply have a main flow path 113 having a hollow length of about 5 mm, and a high pressure for liquid feeding. These pumps are unnecessary, and can be realized with only a very small pump of 100 [Pa] or less.

<Effect>
The HDC apparatus 100 can measure the molecular weight distribution of a plurality of molecules constituting the second liquid in a short time with high accuracy with a very simple apparatus and procedure including the minute main channel 113.

  The main flow path 113 realized in the HDC device 100 is a short flow path with low back pressure, and the pressure to feed the liquid there may be extremely low as it is realized with surface tension. And easy to seal. When injecting the sample into the main flow path 113, since the pressure for feeding the liquid is low, a special injector is not necessary, and the sample may be simply supplied from the connection point between the liquid introduction hole 110 and the main flow path 113.

  Further, in the HDC device 100, since the aspect ratio of the cross section of the micro flow channel 112 perpendicular to the liquid flow direction of the main flow channel 113 is larger than 1, the second liquid diffuses without interaction with the outside. It is possible to cause molecular separation only by the flow effect.

  Furthermore, the HDC device 100 has no substance that interacts with the solute in the main flow path 113 and is merely hollow, but separates within a range from a molecular weight of 50 or less to a molecular weight exceeding 160,000 in the sample of the mixture. be able to.

  In the HDC device 100, the refractive index measuring device 102 made of a total reflection optical system is used to measure the total concentration Call of the second liquid, and the reflected light from the light source is transmitted from below the microchannel 112 by the camera unit 120. Since it is the structure which images, the movable part which moves the camera part 120 is unnecessary, and it can implement | achieve at low cost while the whole structure can be simplified.

<Other embodiments>
In the above-described embodiment, the case where the molecular weight distribution A (Q) of the second liquid is obtained by the component concentration measurement method such as the non-negative least square method has been described. The supervised learning method by collating the spatiotemporal distribution of the total concentration Call of the second liquid measured by the rate measuring device 102 with the spatiotemporal distribution (teacher data) of the total concentration Call that has been obtained in the past. Thus, the molecular weight distribution of the second liquid may be obtained.

  In the above-described embodiment, the case where the molecular weight distribution A (Q) of the second liquid is obtained by the component concentration measurement method (calculation method) such as the non-negative least square method has been described. However, the molecular weight distribution A (Q) of the second liquid may be obtained using a component concentration measurement method (calculation method) such as other pseudo inverse matrix, spectral decomposition, and general harmonic analysis.

  Further, in the above-described embodiment, the case where the molecular weight distribution A (Q) of the second liquid is obtained on the assumption that the diffusion coefficient Q of the molecule is constant has been described. However, the present invention is not limited to this. However, the present invention can be applied even when the molecular diffusion coefficient Q changes in the mixing process of the first liquid and the second liquid. In this case, in the HDC device 100, since it flows out in a short time in the very short main channel 113, the molecular weight distribution A (Q) of the second liquid can be obtained before the molecular diffusion coefficient Q changes.

  Furthermore, in the above-described embodiment, the case where the pressure control pump 106 having a constant pressure is used for feeding the second liquid has been described. However, the present invention is not limited to this, and a filter paper or a pillar structure is used. The second liquid may be fed by the surface tension.

  The molecular weight distribution measuring method and the molecular weight distribution measuring apparatus of the present application can be easily automated, and are suitable for continuous measurement using a short measurement time. In particular, since it has a feature that an initial process of a chemical reaction with a large diffusion coefficient can be measured, it is suitable for shortening the measurement time of a long chemical reaction. Therefore, the utility value in such biochemical, medical, food and chemical engineering fields is high.

  DESCRIPTION OF SYMBOLS 100 ... HDC measuring apparatus (molecular weight distribution measuring apparatus), 101 ... Microchannel chip, 102 ... Refractive index measuring apparatus (calculation part), 103 ... FEP tube, 104 ... Liquid dispensing apparatus (injection apparatus), 105 ... Gas-liquid Separator, 106 ... Pressure control pump (liquid feeding part), 110 ... Liquid introduction hole, 111 ... Liquid discharge hole, 112 ... Micro-channel chip, 113 ... Main channel (channel), 120 ... Camera part, 200 ... A flow path, 201: a flow path inlet surface, 202: a flow path outlet.

Claims (4)

A liquid feeding step for feeding the first liquid and the second liquid in a state where at least the first liquid and the second liquid in which a plurality of molecules are mixed are in contact with each other in the flow path of the flow path forming member;
A measurement step of measuring a total concentration distribution of the second liquid when the first liquid and the second liquid contacted in the flow path diffuse to each other;
A molecular weight distribution measuring method comprising: calculating a molecular weight distribution of the plurality of molecules constituting the second liquid based on the total concentration distribution measured in the measuring step. The molecular weight distribution measurement method according to claim 1,
The measurement step includes measuring the total concentration distribution in the vicinity of the flow path wall surface of the flow path forming member. The molecular weight distribution measurement method according to claim 1,
The flow path forming member has an aspect ratio of a cross section perpendicular to the liquid feeding direction of greater than 1. A molecular weight distribution measurement method, wherein: A flow path forming member in which a flow path is formed;
A liquid feeding section for feeding the first liquid and the second liquid in a state where at least the first liquid and the second liquid in which a plurality of molecules are mixed are in contact with each other in the flow path;
A measuring unit for measuring a total concentration distribution of the second liquid when the first liquid and the second liquid contacted in the flow path diffuse to each other;
A molecular weight distribution measuring device comprising: a molecular weight distribution calculating unit that calculates a molecular weight distribution of the plurality of molecules constituting the second liquid based on the total concentration distribution measured by the measuring unit.