WO2025104842A1 - Capillary electrophoresis device and capillary electrophoresis analysis method - Google Patents

Abstract

According to an embodiment, this capillary electrophoresis device is equipped with an air-type pressurizing mechanism on a sample injection end side and with a plunger-type pressing mechanism and an air-type pressurizing mechanism on a sample elution end side, as a result of which polymer solution filling can be performed within a practical time using a polymer solution of high viscosity for DNA sequencing and DNA fragment analysis, and sample pressure injection as well as application of pressure to both ends during electrophoresis are also made possible (see fig. 8).

Description

CAPILLARY ELECTROPHORESIS APPARATUS AND CAPILLARY ELECTROPHORESIS ANALYSIS METHOD

The present invention relates to an apparatus and method for analyzing components contained in a sample solution using capillary electrophoresis.

In this specification, pressure is defined as the pressure added to atmospheric pressure (approximately 1 atmosphere). In other words, atmospheric pressure is 0 atmospheres. For convenience, pressures between 0 and 7 atmospheres are called low pressure, and pressures above 7 atmospheres are called high pressure. 1 atmosphere is approximately 0.1 MPa.

A capillary electrophoresis device is an automated device that separates various types of components contained in a sample solution by electrophoresis and analyzes them according to charge and size. For example, the Applied Biosystems ^™ SeqStudio ^™ Flex Series Genetic Analyzers (hereinafter referred to as Device A) sold by Thermo Fisher Scientific can perform DNA sequencing and DNA fragment analysis, as described in Non-Patent Document 1. Device A can perform parallel analysis using 8 or 24 capillaries, but this specification mainly describes one capillary (similar explanations apply to other capillaries). Since DNA fragments are negatively charged, the sample injection end of the capillary is the cathode side, and the sample elution end is the anode side, and electrophoresis is performed from the cathode to the anode. Device A can apply high pressure to the sample elution end of the capillary by a plunger-type pressure mechanism. The sample elution end is connected to a flow path containing a polymer solution, and a syringe containing the polymer solution is further connected to the flow path. In addition, an anode buffer solution tank containing an anode buffer solution is also connected to the flow path, and the anode buffer solution in the anode buffer solution tank is connected to the polymer solution in the flow path, with a valve installed at the boundary between them. With the valve closed, the plunger-type pressure mechanism mechanically pushes in the plunger of the syringe, increasing the pressure of the polymer solution inside to a maximum of approximately 70 atmospheres, with a practical value of 35 atmospheres. At this time, the sample injection end is open to atmospheric pressure, so the highly viscous polymer solution can be filled into the capillary.

On the other hand, when the valve is opened, the inside of the anode buffer solution tank and the sample elution end are exposed to atmospheric pressure. The negative electrode is shaped like a pipe, and the sample injection end of the capillary is inserted into the negative electrode to integrate the two. The positive electrode is not integrated with the sample elution end of the capillary, but is inserted into the anode buffer solution tank and immersed in the anode buffer solution. When both ends of the capillary are open to atmospheric pressure, the sample injection end is immersed in the sample solution and a voltage is applied between the two electrodes, i.e., to both ends of the capillary, and the sample can be injected into the capillary by electric field. Also, when both ends of the capillary are open to atmospheric pressure, the sample injection end is immersed in the cathode buffer solution and a voltage is applied to both ends of the capillary, and electrophoresis (called atmospheric pressure electrophoresis) can be performed.

The PA 800 Plus Pharmaceutical Analysis System (hereinafter referred to as Instrument B) sold by Sciex is a different type of capillary electrophoresis instrument from Instrument A, and as described in Non-Patent Document 2, it is capable of analyzing proteins such as antibodies. Instrument B can analyze both negatively and positively charged components. When analyzing negatively charged components, the sample injection end of the capillary is the cathode side, and the sample elution end is the anode side, and electrophoresis is performed from the cathode to the anode.

On the other hand, when analyzing positively charged components, the sample injection end of the capillary is the anode side, and the sample elution end is the cathode side, and electrophoresis is performed from the anode to the cathode. For simplicity, the following describes the case of analyzing a sample containing a negatively charged component. In device B, low pressure can be applied independently to both ends of the capillary, the sample injection end and the sample elution end, by the air pressure mechanism. In addition, both ends can be independently opened to atmospheric pressure. The negative electrode is pipe-shaped, and the sample injection end of the capillary is penetrated into the negative electrode to integrate the two. The positive electrode is also pipe-shaped, and the sample elution end of the capillary is penetrated into the positive electrode to integrate the two. The air pressure mechanism inserts one of the capillary ends into a container that contains the solution and immerses it in the solution, and introduces compressed air into the container with the container sealed, thereby increasing the pressure inside the container to a maximum of about 7 atmospheres. The capillary can be filled with the polymer solution from the sample elution end toward the sample injection end by immersing the sample elution end of the capillary in the polymer solution, applying a low pressure of 5 atmospheres by the air pressure mechanism, and releasing the sample injection end to atmospheric pressure (or, the capillary can be filled with the polymer solution from the sample injection end toward the sample elution end by immersing the sample injection end of the capillary in the polymer solution, applying a low pressure of 5 atmospheres by the air pressure mechanism, and releasing the sample elution end to atmospheric pressure). Also, the sample can be pressure-injected into the capillary by immersing the sample injection end of the capillary in the sample solution, applying a low pressure of 1 atmosphere by the air pressure mechanism, and releasing the sample elution end to atmospheric pressure. Alternatively, the sample can be electric-field-injected into the capillary by immersing the sample elution end of the capillary in the anode buffer solution, immersing the sample injection end in the sample solution, and applying a voltage to both ends of the capillary. Atmospheric pressure electrophoresis can be performed by immersing the sample elution end of the capillary in the anode buffer solution, immersing the sample injection end in the cathode buffer solution, and applying a voltage to both ends of the capillary. Furthermore, by applying an equal low pressure of 1 atmosphere to both ends of the capillary using an air pressure mechanism during electrophoresis (double-end pressure mechanism), it is possible to suppress the generation of bubbles in the polymer solution inside the capillary and stabilize the electrophoretic analysis.

The device shown in Fig. 10 of Patent Document 1 (hereinafter referred to as Device C) can perform DNA sequencing and DNA fragment analysis using a capillary with a short effective length. In Device C, the entire capillary is vertically erected with the sample injection end facing vertically downward and the sample elution end facing vertically upward. The sample elution end is connected to an anode buffer tank containing an anode buffer solution via a flow path containing a polymer solution. Here, the cathode buffer tank has a sealed structure to prevent the polymer solution in the capillary from falling due to gravity. Also, as with Device A, a high pressure of 35 atmospheres can be applied to the sample elution end of the capillary by a plunger-type pressure mechanism to fill the capillary with a high-viscosity polymer solution. Also, a low pressure of 1 atmosphere can be applied to the sample injection end by an air-type pressure mechanism. Furthermore, since the cathode buffer tank has a sealed structure, it is possible to more effectively prevent the polymer solution from falling due to gravity. In this state, the sample injection end is immersed in the sample solution and a voltage is applied to both ends of the capillary, allowing the sample to be electric-field injected into the capillary.

　The plunger type pressurizing mechanism compresses the liquid stored in a sealed container by moving a solid that is in direct contact with the liquid, thereby increasing the pressure. Compared to gas, the volumetric change in response to pressure of solids and liquids is small, so high pressure can be obtained by small movements of solids. In other words, the plunger type pressurizing mechanism is a suitable method for applying high pressure to liquids stored in a sealed container. Even if a small amount of gas is contained in the sealed container, if the volume of the gas is reduced sufficiently by pressurization, the pressure increase due to the subsequent movement of solids will be large. Similarly, high pressure can be obtained by the plunger type pressurizing mechanism even when a small amount of gas is sandwiched between the liquid and the solid and a solid that is in indirect contact with the liquid stored in a sealed container is moved.

In contrast, the air-operated pressurizing mechanism compresses the gas that is in direct contact with the liquid stored in the sealed container (the density of the gas increases) to increase the pressure, thereby compressing the liquid and increasing the pressure. Since the volume change in response to the pressure of the gas is large, a large device is required to generate compressed air. Therefore, the sealed container and the large device must be placed at a distance from each other, and the two must be connected using air tubes, connectors, valves, etc. As a result, the volume and surface area of the compressed air handled also become large, so a small pressure generates a large force, making it easier to cause leaks and explosions. For the above reasons, it is generally difficult to increase the pressure of compressed air, and low-pressure compressed air of 5 atmospheres or less is used in practice. In other words, the air-operated pressurizing mechanism is a suitable method for applying low pressure to the liquid stored in a sealed container. In addition, even when a small amount of solid is sandwiched between the liquid and the gas and gas that is indirectly in contact with the liquid stored in the sealed container is compressed, low pressure can be obtained by the air-operated pressurizing mechanism in the same way.

JP 2001-124736 A

SeqStudioTM Flex Series Genetic Analyzer with Instrument Software v1.1, USER GUIDEhttps://assets.thermofisher.com/TFS-Assets/LSG/manuals/100104689_SeqStudioFlex_v1_RUO_UG.pdf PA 800 Plus Pharmaceutical Analysis System, Methods Development Guidehttps://sciex.com/content/dam/SCIEX/pdf/customer-docs/user-guide/pa-800-plus-methods-development-guide.pdf

In capillary electrophoresis, the amount of sample injected by electric field injection is proportional to the sample concentration when the sample concentration is low, but is saturated with respect to the sample concentration when the sample concentration is high. On the other hand, the amount of sample injected by pressure injection is proportional to the sample concentration regardless of the sample concentration. Therefore, pressure injection may be superior to electric field injection in order to prepare a high-concentration sample and obtain a larger injection amount. In addition, compared to electric field injection, pressure injection can obtain a linear relationship between the sample concentration and peak intensity over a wide range of sample concentrations. From the above, pressure injection is expected to be superior in terms of improving sensitivity and quantitativeness compared to electric field injection. In addition, the double-end pressurization function during electrophoresis contributes to suppressing the generation of bubbles by increasing the pressure of the polymer solution inside the capillary, thereby contributing to stably obtaining high separation performance. In particular, improving the decrease in separation ability due to the generation of bubbles is important when injecting a large amount of high-concentration samples.

The above sample pressure injection and both-end pressurization during electrophoresis are expected to have the same effect when performing DNA sequencing and DNA fragment analysis. DNA sequencing and DNA fragment analysis were performed with System A using POP-7 ^TM Polymer, for 3500/SeqStudio ^TM Flex (Thermo Fisher Scientific, hereafter POP-7), a high-viscosity polymer solution for DNA sequencing and DNA fragment analysis. The polymer solution was filled by applying a high pressure of 35 atmospheres using a plunger-type pressurization mechanism, so the polymer solution could be filled within a practical time of about 5 minutes, despite the high viscosity of the polymer solution. However, because System A cannot use an air-type pressurization mechanism, it was not possible to perform sample pressure injection or both-end pressurization during electrophoresis (both-end pressurized electrophoresis).

We therefore attempted DNA sequencing and DNA fragment analysis using Apparatus B, which is equipped with air pressure mechanisms on both ends of the capillary. The sample elution end of the capillary was immersed in the highly viscous polymer solution POP-7, and a low pressure of 5 atmospheres was applied using the air pressure mechanism, and the sample elution end was released to atmospheric pressure. However, the viscosity of POP-7 was higher than that of the polymer solutions for protein analysis typically used in Apparatus B (e.g., Sciex's SDS-MW Gel Buffer), and the filling speed of the polymer solution was significantly reduced. As a result, it was not possible to fill the polymer solution within a practical time frame. This made it difficult to perform DNA sequencing and DNA fragment analysis using Apparatus B.

Next, DNA sequencing and DNA fragment analysis were performed using Apparatus C. As with Apparatus A, the high viscosity polymer solution POP-7 could be filled into the capillary within a practical time by applying a high pressure of 35 atmospheres using the plunger-type pressure mechanism at the sample elution end. On the other hand, it was found that the sample could not be pressure-injected even though a low pressure of 1 atmosphere could be applied to the sample injection end using the air-type pressure mechanism at the sample injection end. This was because the anode buffer tank to which the sample elution end is connected has a sealed structure and cannot be released to atmospheric pressure, so the pressure applied to the sample injection end is transmitted to the entire polymer solution inside the capillary, and no pressure difference occurs at both ends of the capillary.

　In light of the above, the present invention proposes a capillary electrophoresis technology that achieves either (1) "enabling filling of the polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis" and (2) "enabling pressure injection of a sample" (first objective), (1) "enabling filling of the polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis" and (3) "enabling both ends to be pressurized during electrophoresis" (second objective), or (1) "enabling filling of the polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis" and (2) "enabling pressure injection of a sample" and (3) "enabling both ends to be pressurized during electrophoresis" (third objective).

In order to solve the above problems, the present invention proposes a capillary electrophoresis device comprising a first liquid system with which a first capillary end of a capillary is in contact, a second liquid system with which a second capillary end of a capillary is in contact, a power source for applying a voltage between the first liquid system and the second liquid system, a first pressurization mechanism for increasing a first pressure of the first liquid system by compressing a first gas that is in direct or indirect contact with a first interface of the first liquid system, a first pressure reduction mechanism for reducing the first pressure of the first liquid system, a second pressurization mechanism for increasing a second pressure of the second liquid system by moving a solid that is in direct or indirect contact with a second interface of the second liquid system, and a second pressure reduction mechanism for reducing the second pressure of the second liquid system, wherein the second liquid system has a second gas that is in direct or indirect contact with a third interface different from the second interface, which is an interface within a vessel that holds the liquid when the liquid is filled into the capillary.

Further features related to the present disclosure will become apparent from the description of the present specification and the accompanying drawings. Also, aspects of the present disclosure may be realized and realized by the elements and combinations of various elements and aspects set forth in the following detailed description and the appended claims.
The descriptions in this specification are merely exemplary and illustrative and are not intended to limit the scope or application of the present disclosure in any way.

In one embodiment of the present invention (configuration of Apparatus F: Apparatus F is described below), a high-viscosity polymer solution for DNA sequence and DNA fragment analysis can be used to fill the polymer solution within a practical time, and pressure injection of the sample is also possible. As a result, in DNA sequence and DNA fragment analysis, a larger sample injection volume than before can be used to improve sensitivity, while at the same time enabling highly quantitative analysis. Furthermore, in another embodiment of the present invention (configuration of Apparatus I: Apparatus I is described below), in addition to the effects of Apparatus F described above, both-end pressurized electrophoresis is possible. This makes it possible to suppress the generation of air bubbles and stably obtain high separation performance.

FIG. 1 is a diagram showing an example of the configuration of a conventional apparatus 1 for performing atmospheric pressure electrophoresis. FIG. 1 is a diagram showing a configuration example of a conventional device 1 for carrying out high-pressure polymer solution filling. FIG. 1 is a diagram showing a configuration example of a conventional apparatus 1 for performing sample field injection. FIG. 1 is a diagram showing a configuration example of a conventional device 2 performing double-ended pressure electrophoresis. FIG. 1 is a diagram showing a configuration example of a conventional device 2 for carrying out low-pressure polymer solution filling. FIG. 1 is a diagram showing a configuration example of a conventional device 2 for performing sample field injection. FIG. 1 is a diagram showing an example of the configuration of a capillary electrophoresis apparatus according to Improvement Example 1 performing atmospheric pressure electrophoresis. FIG. 1 is a diagram showing an example of the configuration of a capillary electrophoresis apparatus (Example 1) according to Improvement Example 2 in which atmospheric pressure electrophoresis is performed. FIG. 13 is a diagram showing a configuration example of a capillary electrophoresis apparatus (embodiment 1) according to improvement example 2 in which high-pressure polymer solution filling is performed. FIG. 13 is a diagram showing a configuration example of a capillary electrophoresis apparatus (embodiment 1) according to improvement example 2 in which sample pressure injection is performed. FIG. 13 is a diagram showing a configuration example of a capillary electrophoresis apparatus (Example 2) according to Improvement Example 3 in which atmospheric pressure electrophoresis is performed. FIG. 13 is a diagram showing a configuration example of a capillary electrophoresis apparatus (embodiment 2) according to improvement example 3 in which high-pressure polymer solution filling is performed. FIG. 13 is a diagram showing a configuration example of a capillary electrophoresis apparatus (embodiment 2) according to improvement example 3 in which sample pressure injection is performed. FIG. 13 is a diagram showing a configuration example of a capillary electrophoresis apparatus (Example 2) according to Improvement Example 3 in which both-end pressure electrophoresis is performed. FIG. 13 is a diagram showing a configuration example of a capillary electrophoresis apparatus (embodiment 3) according to improvement example 4 in which high-pressure polymer solution filling is performed. FIG. 13 is a diagram showing a configuration example of a capillary electrophoresis apparatus (embodiment 3) according to improvement example 4 in which sample pressure injection is performed. FIG. 13 is a diagram showing a configuration example of a capillary electrophoresis apparatus (Example 4) according to Improvement Example 5 in which both-end pressure electrophoresis is performed. FIG. 13 is a diagram showing a configuration example of a capillary electrophoresis apparatus (embodiment 4) according to improvement example 5 in which high-pressure polymer solution filling is performed. FIG. 13 is a diagram showing a configuration example of a capillary electrophoresis apparatus (Example 4) according to Improvement Example 5 in which sample pressure injection is performed. FIG. 13 is a diagram showing a configuration example of a capillary electrophoresis apparatus (Example 5) according to Improvement Example 6 in which both-end pressure electrophoresis is performed. FIG. 13 is a diagram showing a configuration example of a capillary electrophoresis apparatus (Example 6) according to Improvement Example 7 in which both-end pressure electrophoresis is performed. FIG. 13 is a diagram showing a configuration example of a capillary electrophoresis apparatus (Example 7) according to Improvement Example 8 in which both-end pressure electrophoresis is performed. FIG. 13 is a diagram showing a configuration example of a capillary electrophoresis apparatus (Example 7) according to Improvement Example 8 in which high-pressure polymer solution filling is performed. FIG. 13 is a diagram showing a configuration example of a capillary electrophoresis apparatus (Example 7) according to Improvement Example 8 in which sample pressure injection is performed. FIG. 13 is a diagram showing a configuration example of a capillary electrophoresis apparatus (Example 8) according to Improvement Example 9 in which both-end pressure electrophoresis is performed. FIG. 13 is a diagram showing a configuration example of a capillary electrophoresis apparatus (embodiment 8) according to improvement example 9 in which high-pressure polymer solution filling is performed. FIG. 13 is a diagram showing a configuration example of a capillary electrophoresis apparatus (Example 8) according to Improvement Example 9 in which sample pressure injection is performed. FIG. 13 is a diagram showing a configuration example of a capillary electrophoresis apparatus (Example 9) according to Improvement Example 10 in which both-end pressure electrophoresis is performed. 13 is an electropherogram obtained by electric field injection using the capillary electrophoresis apparatus of Improvement Example 3 (Example 2). FIG. 30 is a diagram showing the relationship between the electric field injection time and the peak area in FIG. 29. 1 is an electropherogram obtained by pressure injection using Improved Example 3 (Example 2). FIG. 32 is a graph showing the relationship between pressure injection time and peak area in FIG. 31. FIG. 1 is a diagram showing the process of capillary electrophoresis analysis using electric field injection. FIG. 1 shows the steps of capillary electrophoresis analysis using pressure injection. FIG. 1 shows the steps of capillary electrophoresis analysis using pressure injection and double-ended pressure electrophoresis.

The configuration of the capillary electrophoresis device of the present invention (improved example) and the features of the analysis method will be explained in comparison with the conventional method. In the following, for simplicity of explanation, a capillary electrophoresis device using one capillary will be mainly described, but this method may also be applied to a multi-capillary electrophoresis device using multiple capillaries.
In the following embodiments, the devices having different configurations are named Device A, Device B, etc., and each will be described (however, Device A, B, and C are also described in the Background Art above). In the following description, in order to handle a plurality of different devices, the devices having different configurations will be described in detail with reference to the drawings, with the conventional method (conventional example) referred to as Conventional Example 1, Conventional Example 2, etc., and the new method (improved example) referred to as Improved Example 1, Improved Example 2, etc. However, the mutual correspondence will be described in each case.

Each instrument is equipped with a temperature control device that maintains a constant temperature of the capillary, but this is not described in the explanation of each figure. For example, when performing DNA sequencing and DNA fragment analysis, it is best to control the temperature of the capillary at 60°C.

As shown in the upper left corner of each figure in this specification, a right-handed XYZ Cartesian coordinate system is defined for each figure. That is, in each figure, the X axis is taken horizontally, with the rightward direction being the positive X axis, the Z axis is taken vertically, with the upward direction being the positive Z axis, and the Y axis is taken perpendicular to the paper surface, with the direction into the paper being the positive Y axis. In each figure, parts with similar structures and similar functions are given the same reference numerals.

In this specification, the liquid system is defined as the entire liquid (buffer solution, polymer solution, sample solution, etc.) in which the capillary end (sample injection end or sample elution end) is immersed. A liquid system is a continuous string of liquid (it does not have to be contained in a single container) and can change over time (for example, if a liquid system is divided by a valve, the liquid system becomes smaller). The composition of a liquid system does not have to be uniform (for example, if a buffer solution and a polymer solution are in contact without mixing, they may be collectively referred to as a single liquid system).

<Conventional Example 1>
Figures 1 to 3 show an example of the configuration of a capillary electrophoresis device (corresponding to device A) according to conventional example 1 and an analysis method using the device. The sample injection end 2 of one capillary 1 is placed on the right side, and the sample elution end 3 is placed on the left side, facing vertically downward (in the -Z-axis direction). A plunger-type pressure mechanism is provided on the anode side (sample elution end side). Figure 1 shows the state in which electrophoresis (atmospheric pressure electrophoresis) is performed in conventional example 1. A sample is injected from the sample injection end 2 into capillary 1 (outer diameter 360 μm, inner diameter 50 μm) filled with a polymer solution 18 (POP-7), and electrophoresed through the capillary toward the sample elution end 3, and multiple different components contained in the sample are electrophoretically separated based on charge and size. The sample injection end 2 is inserted into the pipe-shaped negative electrode 4 to integrate the two. The cathode buffer solution tank 8, which contains the cathode buffer solution 6, and the sample solution tank 11, which contains the sample solution 10, are fixed on the cathode stage 12, and the cathode stage 12 is further connected to an XYZ axis drive mechanism (not shown). Of course, other containers, such as a plurality of sample solution tanks containing different samples, are also fixed on the cathode stage 12, but for simplicity, they are not shown. The sample injection end 2 equipped with the cathode 4 is inserted into the cathode buffer solution tank 8 and immersed in the cathode buffer solution 6. The sample elution end 3 is connected to an acrylic T-block 15 filled with a polymer solution 18 using a connector 14, and immersed in the polymer solution 18. An inverted T-shaped flow path is formed inside the T-block 15, and the flow path is filled with the polymer solution 18. A pressure-resistant syringe 16 and a polymer solution tube 19 are also connected to the T-block 15, and both are filled with the polymer solution 18 (they are connected using connectors, but not shown). Each connection is sealed so that the contents do not leak out even if the pressure of the polymer solution inside increases (it has pressure resistance). The anode buffer solution tank 9, in which the anode buffer solution 7 is stored, is fixed on the anode stage 13. The end of the polymer solution tube 19 opposite the T-block 15 is inserted into the anode buffer solution tank 9 and immersed in the anode buffer solution 7. A polymer solution valve 20 is installed at the end of the polymer solution tube 19, at the boundary between the polymer solution 18 and the anode buffer solution 7. The polymer solution valve 20 has a plunger part that moves up and down (Z-axis direction), and when the plunger part moves up (+Z-axis direction), the polymer solution valve 20 is open, and when the plunger part moves down (-Z-axis direction), the polymer solution valve 20 is closed.

In Fig. 1, only the plunger part of the polymer solution valve 20 is drawn, but in reality, the polymer solution valve 20 includes a solenoid mechanism (not shown) for moving the plunger part up and down, and a connection mechanism (not shown) for linking the solenoid mechanism and the plunger part. The cylindrical anode 5 is inserted into the anode buffer solution tank 9 and immersed in the anode buffer solution 7. The anode 5 and cathode 4 are connected to a DC power source 21 by an electric wire 22. A voltage is applied between the anode 5 and cathode 4, that is, between the sample elution end 3 and the sample injection end 2 of the capillary 1, to perform atmospheric pressure electrophoresis. The vertical height (Z coordinate) of the interface 101 between the cathode buffer solution 6 and the air (i.e., the liquid level of the cathode buffer solution 6) and the interface 102 between the anode buffer solution 7 and the air (i.e., the liquid level of the anode buffer solution 7) are aligned. For example, it is recommended that the difference in height between the two liquid levels be 1 mm or less. This prevents the polymer solution 18 inside the capillary 1 from moving due to gravity during electrophoresis. A position electrophoresed a certain distance from the sample injection end 2 on the capillary 1 is set as the detection position, and the polyimide coating of the capillary near the detection position is removed in advance. A laser beam 24 emitted from a laser light source 23 is irradiated onto the detection position, and the fluorescence emitted from the detection position is detected by a fluorescence detection device (not shown).

FIG. 2 shows the state in which the capillary 1 is filled with the polymer solution 18 at high pressure in the capillary electrophoresis device according to the conventional example 1. This filling can also replace the polymer solution 18 already filled in the capillary 1 with the newly filled polymer solution 18. The polymer solution valve 20 is closed, and the plunger 17 of the pressure-resistant syringe 16 is mechanically pushed in by a motor to apply pressure to the polymer solution 18 inside. The polymer solution 18 in the polymer solution tube 19, the polymer solution 18 in the T-block 15, and the polymer solution 18 in the pressure-resistant syringe 16 form a continuous liquid system. This liquid system fills an enclosed space, and this enclosed space contains almost no air. The sample elution end 3 is immersed in this liquid system. In this state, the plunger 17, which is a solid, directly pressurizes the liquid polymer solution 18 at the interface 104 with the polymer solution 18, so that a high pressure of up to about 70 atmospheres can be obtained. Even if a gas such as air is inserted between the plunger 17 and the polymer solution 18, if the volume of the gas is sufficiently small, the plunger 17 can indirectly pressurize the polymer solution 18, and a similar high pressure can be obtained. The high pressure is transmitted to the entire liquid system, including the sample elution end 3.

In each figure in this specification, the polymer solution under atmospheric pressure is shown with a vertical dotted line pattern, and the polymer solution under high pressure is shown with a checkered pattern. Therefore, in Fig. 1, the liquid system under atmospheric pressure is shown with a vertical dotted line pattern, and in Fig. 2, the liquid system under high pressure is shown with a checkered pattern. Since the sample injection end 2 is under atmospheric pressure, a high pressure difference is generated between the sample elution end 3 and the sample injection end 2, and the polymer solution 18 is filled into the capillary 1 from the sample elution end 3 toward the sample injection end 2. The pressure applied to the liquid system can be controlled by controlling the force of the motor that pushes the plunger 17. In practice, a high pressure of 35 atmospheres is applied to the liquid system to fill the capillary 1 with the polymer solution 18 at high pressure. After filling the polymer solution, the mechanical pushing of the plunger 17 is stopped, the plunger 17 and the mechanical pushing mechanism are separated, and the pressure applied to the liquid system is set to almost zero. However, in practice, some pressure may remain. Therefore, the polymer solution valve 20 is opened to expose the sample elution end 3 and the above liquid system to atmospheric pressure.

Figure 3 shows the state in which a sample is electric-field injected into the capillary 1 in the conventional example 1. The cathode stage 12 is moved by the XYZ-axis drive mechanism to insert the sample injection end 2 into the sample solution tank 11 and immerse it in the sample solution 10. In this state, a voltage is applied between the sample elution end 3 and the sample injection end 2 of the capillary 1 for a certain period of time, so that the sample is electric-field injected into the capillary 1 from the sample injection end 2. At this time, it is not necessary to align the vertical heights (Z coordinates) of the interface 105 between the sample solution 10 and air (i.e., the liquid surface of the sample solution 10) and the interface 102 (i.e., the liquid surface of the anode buffer solution 7). If these heights are misaligned, the polymer solution 18 inside the capillary 1 may move due to gravity, but if the electric field injection time is short, the effect is small.

After this, the XYZ-axis drive mechanism of the cathode stage 12 is used to move the sample injection end 2 into the cathode buffer solution tank 8 and immerse it in the cathode buffer solution 6. Returning to the state shown in FIG. 1, a voltage is applied between the sample elution end 3 and the sample injection end 2, and both ends of the capillary 1 are placed under atmospheric pressure to perform electrophoresis (atmospheric pressure electrophoresis). The vertical heights (Z coordinates) of the interface 101 between the cathode buffer solution 6 and the air (i.e., the liquid level of the cathode buffer solution 6) and the interface 102 between the anode buffer solution 7 and the air (i.e., the liquid level of the anode buffer solution 7) are aligned. This makes it possible to prevent the polymer solution 18 inside the capillary 1 from moving due to gravity during electrophoresis.

As described above, the capillary electrophoresis device of conventional example 1 does not have an air pressure device, so it is not possible to perform pressure injection of the sample, and it is not possible to apply pressure to both ends during electrophoresis.

<Conventional Example 2>
4 to 6 show the configuration of a capillary electrophoresis apparatus (corresponding to apparatus B) according to conventional example 2 and an analysis method using the same. In the following, some of the explanations that overlap with conventional example 1 will be omitted, and the explanation will focus on the differences from conventional example 1. An air-type pressurizing mechanism is provided on the cathode side (sample injection end), and an air-type pressurizing mechanism is also provided on the anode side (sample elution end).

Figure 4 shows the state in which electrophoresis (double-ended pressurized electrophoresis) is being performed in the capillary electrophoresis device of conventional example 2. The sample injection end 2 is inserted into the pipe-shaped cathode 4, and the two are integrated. A cathode buffer solution tank 8 containing a cathode buffer solution 6 and a sample solution tank 11 containing a sample solution 10 are fixed onto the cathode stage 12, which is further connected to an XYZ-axis drive mechanism (not shown). Of course, other containers such as multiple sample solution tanks containing different samples, a polymer solution tank containing a polymer solution, and a waste liquid tank containing waste liquid can also be fixed onto the cathode stage 12, but for simplicity, these are not shown.

　An O-ring 33 is placed on the edge of the upper end of each of the cathode buffer solution chamber 8 and the sample solution chamber 11. The fixed block 31 has a flat surface that is parallel to the XY plane and faces the -Z direction. By moving the cathode stage 12 using the XYZ axis drive mechanism, the sample injection end 2 is inserted into the cathode buffer solution chamber 8 together with the cathode 4 and immersed in the cathode buffer solution 6. At the same time, by moving the cathode stage 12 in the +Z direction, the O-ring 33 is pressed against the flat surface of the fixed block 31 and compressed, sealing the cathode buffer solution chamber 8.

The gap between the capillary 1 and the cathode 4 and the fixed block 31 is sealed. At this time, the cathode buffer solution tank 8 contains the cathode buffer solution 6 and air 34 in contact with the cathode buffer solution 6. The air 34 is connected to the compressed air source 25 or the atmosphere via the cathode pressure valve 28 and the cathode release valve 29 via the air tube 30. The pressure of the compressed air discharged from the compressed air source 25 can be adjusted and controlled to a constant low pressure of 0 to 7 atmospheres.

As shown in FIG. 4, when the cathode pressure valve 28 is opened and the cathode release valve 29 is closed, the air 34 is connected to the compressed air source 25, the compressed air flows into the cathode buffer solution tank 8 via the air tube 30, and the pressure of the air 34 rises until it becomes equal to the pressure of the compressed air discharged from the compressed air source 25. At the same time, the pressure of the cathode buffer solution 6 contained in the cathode buffer solution tank 8 similarly rises until it becomes equal to the pressure of the compressed air discharged from the compressed air source 25. In each figure in this specification, air under atmospheric pressure is shown with a blank pattern, and air under low pressure is shown with a dotted pattern. In addition, the buffer solution under atmospheric pressure is shown with a horizontal dotted line pattern, and the buffer solution under low pressure is shown with a horizontal solid line pattern. For this reason, the air 34 under low pressure is shown with a dotted pattern in FIG. 4, and the air 34 under atmospheric pressure is shown with a blank pattern in FIG. 5. In addition, in Figure 4, the cathode buffer solution 6 under low pressure is shown with a horizontal solid line pattern, and in Figure 5, the cathode buffer solution 6 under atmospheric pressure is shown with a horizontal dotted line pattern. These patterns are also used in other figures.

The anode side has the same device configuration as the cathode side. The sample elution end 3 is inserted into the pipe-shaped anode 5, and the two are integrated. The anode buffer solution tank 9, which contains the anode buffer solution 7, and the polymer solution tank 32, which contains the polymer solution 18, are fixed onto the anode stage 13, which is further connected to an XYZ-axis drive mechanism (not shown). Of course, other containers such as multiple sample solution tanks containing different samples and a waste liquid tank containing waste liquid can also be fixed onto the anode stage 13, but for simplicity, these are not shown.

　O-rings 33 are placed on the edges of the upper ends of the anode buffer solution tank 9 and the polymer solution tank 32. The fixed block 31 has a plane parallel to the XY plane and facing the -Z direction. The sample elution end 3 is inserted into the anode buffer solution tank 9 together with the anode 5 by the movement of the cathode stage 13 by the XYZ axis drive mechanism, and is immersed in the anode buffer solution 7. At the same time, the anode stage 13 is moved in the +Z direction, and the O-ring 33 is pressed against the flat part of the fixed block 31 and compressed, sealing the anode buffer solution tank 9. The gap between the capillary 1 and the anode 5 and the fixed block 31 is sealed. At this time, the anode buffer solution tank 9 contains the anode buffer solution 7 and air 35 in contact with the anode buffer solution 7. The air 35 is connected to the compressed air source 25 or the atmosphere via the air tube 30 and the anode pressure valve 26 and the anode release valve 27.

As shown in Figure 4, when the anode pressure valve 26 is opened and the anode release valve 27 is closed, the air 35 is connected to the compressed air source 25, the compressed air flows into the anode buffer solution tank 9 via the air tube 30, and the pressure of the air 35 rises until it becomes equal to the pressure of the compressed air discharged from the compressed air source 25. At the same time, the pressure of the anode buffer solution 7 contained in the anode buffer solution tank 9 similarly rises until it becomes equal to the pressure of the compressed air discharged from the compressed air source 25. As with the cathode side, the air 35 under low pressure is shown with a dot pattern in Figure 4, and the air 35 under atmospheric pressure is shown with a blank pattern in Figure 5. Also, the anode buffer solution 7 under low pressure is shown with a horizontal solid line pattern in Figure 4, and the anode buffer solution 7 under atmospheric pressure is shown with a horizontal dotted line pattern in Figure 5.

As described above, when the pressure of the compressed air discharged from the compressed air source 25 is set to 1 atmosphere, a low pressure of 1 atmosphere is applied to both the cathode buffer solution 6 and the anode buffer solution 7, and a low pressure of 1 atmosphere is applied to both ends of the sample injection end 2 and the sample elution end 3 of the capillary 1. In this state, a voltage is applied between the positive electrode 5 and the negative electrode 4, and a voltage is applied to both ends of the sample injection end 2 and the sample elution end 3 of the capillary 1, performing double-end pressure electrophoresis. This increases the pressure of the polymer solution 18 inside the capillary 1 to 1 atmosphere, suppresses the generation of bubbles inside the capillary 1 during electrophoresis, and enables stable high separation performance to be obtained. In addition, the vertical height (Z coordinate) of the interface 101 between the cathode buffer solution 6 and the air (i.e., the liquid level of the cathode buffer solution 6) and the interface 102 between the anode buffer solution 7 and the air (i.e., the liquid level of the anode buffer solution 7) are aligned. For example, it is advisable to set the difference in height between the two liquid levels to 1 mm or less. This will prevent the polymer solution 18 inside the capillary 1 from moving due to gravity during electrophoresis.

Figure 5 shows the state in which the capillary 1 is filled with the polymer solution 18 at low pressure in the capillary electrophoresis device of conventional example 2. The sample elution end 3 is inserted into the polymer solution tank 32 together with the anode 5 by moving the anode stage 13 by the XYZ axis drive mechanism, and is immersed in the polymer solution 18. At the same time, the O-ring 33 is pressed against the fixed block 31 and compressed, sealing the polymer solution tank 32. The pressure of the compressed air discharged from the compressed air source 25 is set to 5 atmospheres. The anode pressure valve 26 is opened, and the anode release valve 27 is closed to allow compressed air to flow into the polymer solution tank 32 via the air tube 30, and the pressure of the polymer solution 18 inside the polymer solution tank 32 and the air 36 in contact with it is increased to 5 atmospheres.

In Figure 5, air 36 under a low pressure of 5 atmospheres is shown with a dotted pattern. In each figure in this specification, polymer solutions under atmospheric pressure are shown with a vertical dotted line pattern, and polymer solutions under low pressure are shown with a vertical solid line pattern. For this reason, in Figure 5, polymer solution 18 under low pressure is shown with a vertical solid line pattern. These patterns are commonly used in other figures.

Meanwhile, the cathode pressure valve 28 is closed and the cathode release valve 29 is opened to connect the air 34 inside the cathode buffer solution tank 8 to the atmosphere, and the pressure of the air 34 is set to atmospheric pressure (0 atm). The air 34 is blank and the cathode buffer solution 6 is shown with a horizontal dotted line pattern to indicate the atmospheric pressure state. As a result, a low pressure difference of 5 atm is created between the sample elution end 3 and the sample injection end 2, and the polymer solution 18 is filled at low pressure in the capillary 1 from the sample elution end 3 toward the sample injection end 2. After the polymer solution is filled, the anode pressure valve 26 is closed and the anode release valve 27 is opened to exhaust part of the air 36 inside the anode buffer solution tank 9 to the atmosphere via the air tube 30, and the pressure of the polymer solution 18 and the air 36 is set to atmospheric pressure.

FIG. 6 shows the state in which a sample is pressure-injected into the capillary 1 in the capillary electrophoresis apparatus of the conventional example 2. The sample elution end 3 is inserted into the anode buffer solution tank 9 and immersed in the anode buffer solution 7 by the movement of the anode stage 13 by the XYZ-axis drive mechanism. The pressure of the anode buffer solution 7 and the air 32 inside the anode buffer solution tank 9 is atmospheric pressure. Meanwhile, the sample injection end 2 is inserted into the sample solution tank 11 by the movement of the cathode stage 12 by the XYZ-axis drive mechanism and immersed in the sample solution 10. At the same time, the O-ring 33 is pressed against the fixed block 31 and compressed, sealing the sample solution tank 11. The pressure of the compressed air discharged from the compressed air source 25 is set to 1 atmosphere. When the cathode pressure valve 28 is opened and the cathode release valve 29 is closed, the pressure of the sample solution 10 inside the sample solution tank 11 and the air 37 in contact with it rises to 1 atmosphere.

In each figure in this specification, sample solutions under atmospheric pressure are shown with horizontal dotted lines, and sample solutions under low pressure are shown with horizontal solid lines. Therefore, in Figure 6, sample solution 10 under low pressure is shown with horizontal solid lines. These patterns are also used in other figures. Air 37 under similarly low pressure is shown with dots. As a result, a low pressure difference of 1 atmosphere is created between the sample injection end 2 and the sample elution end 3, and sample solution 10 is injected from the sample injection end 2 into the capillary 1. After a predetermined time has passed, the cathode pressure valve 28 is closed and the cathode release valve 29 is opened to release the pressure of the sample solution 10 and air 37 inside the cathode sample solution tank 11 to atmospheric pressure. In this state, it is also possible to apply a voltage between the sample elution end 3 and the sample injection end 2 to perform electric field injection of the sample.

After this, the XYZ-axis drive mechanism of the cathode stage 12 moves the sample injection end 2 into the cathode buffer solution tank 8, immersing it in the anode buffer solution 6 and sealing the cathode buffer solution tank 8. When the anode pressure valve 26 is opened, the anode release valve 27 is closed, the cathode pressure valve 28 is opened, and the cathode release valve 29 is closed, the cathode buffer solution 7 and air 35 inside the anode buffer solution tank 9, and the anode buffer solution 6 and air 34 inside the cathode buffer solution tank 8 are all pressurized to 1 atmosphere, returning to the state shown in Figure 4. Since the compressed air introduced into the anode buffer solution tank 9 and the cathode buffer solution tank 8 comes from the same compressed air source 25, the pressure inside the anode buffer solution tank 9 and the cathode buffer solution tank 8 becomes strictly equal. In this state, a voltage is applied between the sample elution end 3 and the sample injection end 2, and double-end pressurized electrophoresis is performed. This increases the pressure of the polymer solution 18 inside the capillary 1 to 1 atmosphere, suppressing the generation of bubbles inside the capillary 1 during electrophoresis, and enabling stable high separation performance to be obtained. In addition, the vertical heights (Z coordinates) of the interface 101 between the cathode buffer solution 6 and the air (i.e., the liquid level of the cathode buffer solution 6) and the interface 102 between the anode buffer solution 7 and the air (i.e., the liquid level of the anode buffer solution 7) are aligned. This makes it possible to prevent the polymer solution 18 inside the capillary 1 from moving due to gravity during electrophoresis.

<Improvement Example 1>
7 shows an example of the configuration of a capillary electrophoresis device (corresponding to device D) according to Improved Example 1. Improved Example 1 is configured by making modifications to Conventional Example 2. However, as a result of investigation, it has become clear that Improved Example 1, like device C of Patent Document 1, is unable to achieve any of the objects of the present invention.

Improved Example 1 is equipped with an air-type pressure mechanism on the cathode side (sample injection end) and a plunger-type pressure mechanism on the anode side (sample elution end). Figure 7 shows the state in which atmospheric pressure electrophoresis is being performed in the capillary electrophoresis device of improved example 1. The configuration of the cathode side is the same as that of conventional device 2. However, unlike Figure 4, the cathode pressure valve 28 is closed and the cathode release valve 29 is opened to open the inside of the cathode buffer solution tank 8 to atmospheric pressure. As in conventional example 2, the sample elution end 3 is inserted into the pipe-shaped anode 5 and the two are integrated. The pressure-resistant syringe 16 containing the polymer solution 18 and the polymer solution tank 32 storing the polymer solution 18 are combined and fixed on the anode stage 13. The sample elution end 3 is inserted into the polymer solution tank 32 together with the anode 5, and is immersed in the polymer solution 18 while the polymer solution tank 32 is sealed. However, unlike the conventional device 2, the sealed structure of the polymer solution tank 32 cannot be easily eliminated.

The polymer solution tank 32 and the pressure-resistant syringe 16 have a single internal space, which is filled with the polymer solution 18 to form a single liquid system. This internal space contains almost no air. In this state, electrophoresis can be performed by applying a voltage between the sample elution end 3 and the sample injection end 2. In addition, by releasing the cathode buffer solution 6 and air 34 inside the cathode buffer solution tank 8 to atmospheric pressure while mechanically pushing in the plunger 17 of the pressure-resistant syringe 16, the pressure of the polymer solution inside the polymer solution tank 32 and the syringe 16 can be increased to 35 atmospheres, and the polymer solution 18 can be filled into the capillary 1 at high pressure (not shown).

However, it was found that even if the sample injection end 2 is inserted into the sample solution tank 11, the sample solution tank 11 is sealed, and the internal pressure is increased to 1 atmosphere as in Conventional Example 2 after the mechanical pushing of the plunger 17 is stopped, pressure injection of the sample is not performed. This is because the sealed structure of the polymer solution tank 32 is maintained, so the pressure at the sample injection end and the sample elution end are equal (1 atmosphere), and no pressure difference is obtained at both ends. This structural problem is the same as the problem with Device C in Patent Document 1. Therefore, Improved Example 1 cannot achieve any of the first to third objects of the present invention.

<Improvement Example 2: Example 1>
8 to 10 show an example of the configuration of a capillary electrophoresis device according to Improved Example 2 (Example 1) and an analysis method using the same. Improved Example 2 (Example 1) is configured by adding modifications to Conventional Example 2, and has a configuration that solves the problems of Improved Example 1.

The capillary electrophoresis device according to the improved example 2 (embodiment 1) is equipped with an air-type pressure mechanism on the cathode side (sample injection end side) and a plunger-type pressure mechanism on the anode side (sample elution end side). Figure 8 shows the state in which atmospheric pressure electrophoresis is being performed in the improved example 2 (embodiment 1). The configuration on the cathode side is the same as that of the conventional example 2. However, unlike Figure 4, the cathode pressure valve 28 is closed and the cathode release valve 29 is opened to open the inside of the cathode buffer solution tank 8 to atmospheric pressure. As in the conventional example 2, the sample elution end 3 is inserted into the pipe-shaped anode 5, and the two are integrated. The anode buffer solution tank 9 containing the anode buffer solution 7 and the polymer solution tank 32, which is connected to the pressure-resistant syringe 16 containing the polymer solution 18, are fixed on the anode stage 13, and the anode stage 13 is further connected to the XYZ axis drive mechanism (not shown).

No O-ring 33 is installed on the edge of the upper end of the anode buffer solution tank 9, but an O-ring 33 is installed on the upper end of the polymer solution tank 32. The sample elution end 3 is inserted into the anode buffer solution tank 9 and immersed in the anode buffer solution 7 by moving the cathode stage 13 using the XYZ axis drive mechanism. Unlike the conventional example 2 and the improved example 1, the anode buffer solution tank 9 is not sealed, and the anode buffer solution 7 inside is opened to atmospheric pressure. A voltage is applied between the sample elution end 3 and the sample injection end 2 of the capillary 1 to perform atmospheric pressure electrophoresis. The vertical heights (Z coordinate) of the interface 101 between the cathode buffer solution 6 and the air (i.e., the liquid level of the cathode buffer solution 6) and the interface 102 between the anode buffer solution 7 and the air (i.e., the liquid level of the anode buffer solution 7) are aligned. For example, it is recommended that the difference in height between the two liquid levels be 1 mm or less. This prevents the polymer solution 18 inside the capillary 1 from moving due to gravity during electrophoresis.

FIG. 9 shows the state in which the capillary 1 is filled with the polymer solution 18 at high pressure in the capillary electrophoresis apparatus according to the improved example 2 (embodiment 1). The sample elution end 3 is inserted into the polymer solution tank 32 by the movement of the anode stage 13 by the XYZ axis drive mechanism, and is immersed in the polymer solution 18. At the same time, the O-ring 33 is pressed against the fixed block 31 and compressed, sealing the polymer solution tank 32. The polymer solution tank 32 and the pressure-resistant syringe 16 are integrated and have one internal space, which is filled with the polymer solution 18 and contains almost no air. The cathode buffer solution 6 and air 34 inside the cathode buffer solution tank 8 are released to atmospheric pressure, while the plunger 17 of the pressure-resistant syringe 16 is mechanically pressed in, increasing the pressure of the polymer solution 18 inside the polymer solution tank 32 and the syringe 16 to 35 atmospheres, and the polymer solution 18 is filled into the capillary 1 at high pressure. After the polymer solution is filled, the mechanical pushing of the plunger 17 is stopped, the plunger 17 and the mechanical pushing mechanism are separated, and the pressure applied to the polymer solution 18 is reduced to almost zero. However, in reality, some pressure may remain. Therefore, the anode stage 13 is moved in the -Z axis direction by the XYZ axis drive mechanism, releasing the compression of the O-ring and releasing the polymer solution 18 to atmospheric pressure.

Figure 10 shows the state in which a sample is pressure-injected into the capillary 1 in the capillary electrophoresis device according to Improved Example 2 (Example 1). The sample elution end 3 is inserted into the anode buffer solution tank 9 and immersed in the anode buffer solution 7 by moving the anode stage 13 using the XYZ-axis drive mechanism. The pressure of the anode buffer solution 7 inside the anode buffer solution tank 9 is atmospheric pressure.

Meanwhile, the XYZ-axis drive mechanism of the cathode stage 12 moves the sample injection end 2 into the sample solution tank 11, immersing it in the sample solution 10, while at the same time pressing the O-ring 33 against the fixed block 31 to compress it, sealing the sample solution tank 11. The pressure of the compressed air discharged from the compressed air source 25 is set to 1 atmosphere. When the cathode pressure valve 28 is opened and the cathode release valve 29 is closed, the pressure of the sample solution 10 inside the sample solution tank 11 and the air 37 in contact with it rises to 1 atmosphere. As a result, a low pressure difference of 1 atmosphere is created between the sample injection end 2 and the sample elution end 3, and the sample solution 10 is injected from the sample injection end 2 into the capillary 1. After a predetermined time has passed, the cathode pressure valve 28 is closed and the cathode release valve 29 is opened to release the pressure of the sample solution 10 and air 37 inside the cathode sample solution tank 11 to atmospheric pressure.

After this, the XYZ-axis drive mechanism of the cathode stage 12 moves the sample injection end 2 into the cathode buffer solution tank 8, immersing it in the anode buffer solution 6 while simultaneously sealing the cathode buffer solution tank 8. However, by closing the cathode pressure valve 28 and opening the cathode release valve 29 to release the inside of the cathode buffer solution tank 8 to atmospheric pressure, the state of FIG. 8 is restored. In this state, a voltage is applied between the sample elution end 3 and the sample injection end 2, and atmospheric pressure pressurized electrophoresis is performed.

Therefore, Improved Example 2 (Example 1) achieves the first objective of the present invention, which is to provide a capillary electrophoresis device that (1) "enables filling of a polymer solution within a practical time using a high-viscosity polymer solution for DNA sequencing and DNA fragment analysis" and (2) "enables pressure injection of a sample." However, Improved Example 2 (Example 1) does not achieve the second and third objectives of the present invention.

In improved example 2 (embodiment 1), not only pressure injection of the sample is possible, but also electric field injection. In FIG. 10, the cathode pressure valve 28 is closed and the cathode release valve 29 is opened to release the sample solution 10 inside the sample solution tank 11 and the air 37 in contact with it to atmospheric pressure. In this state, by applying a voltage between the sample elution end 3 and the sample injection end 2 for a certain period of time, the sample can be electric field injected into the capillary 1 from the sample injection end 2.

<Improvement Example 3: Example 2>
11 to 13 show an example of the configuration of a capillary electrophoresis device (corresponding to device F) according to Improved Example 3 (Embodiment 2). Improved Example 3 (Embodiment 2) is configured by making modifications to Conventional Example 2 (FIGS. 4 to 6). Improved Example 3 has a different configuration from Improved Example 2 (FIGS. 8 to 10), but produces the same effects as Improved Example 2.

The capillary electrophoresis device according to the improved example 3 (embodiment 2) is equipped with an air-type pressure mechanism on the cathode side (sample injection end side) and a plunger-type pressure mechanism on the anode side (sample elution end side). Figure 11 shows the state in which atmospheric pressure electrophoresis is being performed in the capillary electrophoresis device according to the improved example 3 (embodiment 2). The configuration of the cathode side is the same as that of the conventional example 2. However, the cathode pressure valve 28 is closed and the cathode release valve 29 is opened to release the inside of the cathode buffer solution tank 8 to atmospheric pressure. On the anode side, first, the integrated sample elution end 3 and anode 5 are separated, and the anode 5 is changed from a pipe shape to a cylindrical shape. The sample elution end 3 is connected to an acrylic T-block 15 filled with a polymer solution 18 using a connector 14, and immersed in the polymer solution 18. An inverted T-shaped flow path is formed inside the T-block 15, and the flow path is filled with the polymer solution 18. A pressure-resistant syringe 16 and a polymer solution tube 19 are also connected to the T-block 15, and both are filled with the polymer solution 18 (they are connected using connectors, not shown). The material of the polymer solution tube 19 used in the present invention does not necessarily need to be flexible.

When applying high pressure to the internal polymer solution 18, a material with high pressure resistance must be used. Teflon (registered trademark) resin or PEEK can be used. Alternatively, as with the T-block 15, a flow path may be formed in a block of acrylic or the like. Each connection is sealed so that the contents do not leak out even if the pressure of the internal polymer solution increases. The anode buffer solution tank 9, in which the anode buffer solution 7 is stored, is fixed on the anode stage 13. The end of the polymer solution tube 19 opposite the T-block 15 is inserted into the anode buffer solution tank 9 and immersed in the anode buffer solution 7. A polymer solution valve 20 is installed at the end of the polymer solution tube 19, at the boundary between the polymer solution 18 and the anode buffer solution 7. The polymer solution valve 20 has a plunger part that moves up and down (Z-axis direction). When the plunger part moves up (+Z-axis direction), the polymer solution valve 20 is open, and when the plunger part moves down (-Z-axis direction), the polymer solution valve 20 is closed.

In FIG. 11, the polymer solution valve 20 is open. In FIG. 11, only the plunger part of the polymer solution valve 20 is drawn, but in reality, the polymer solution valve 20 includes a solenoid mechanism (not shown) for moving the plunger part up and down, and a connection mechanism (not shown) for interlocking the solenoid mechanism and the plunger part. The positive electrode 5 is inserted into the anode buffer solution tank 9 and immersed in the anode buffer solution 7. The positive electrode 5 and the negative electrode 4 are connected to a DC power source 21 by an electric wire 22. A voltage is applied between the positive electrode 5 and the negative electrode 4, that is, a voltage is applied between the sample elution end 3 and the sample injection end 2 of the capillary 1, and atmospheric pressure electrophoresis is performed. The vertical height (Z coordinate) of the interface 101 between the cathode buffer solution 6 and the air (i.e., the liquid level of the cathode buffer solution 6) and the interface 102 between the anode buffer solution 7 and the air (i.e., the liquid level of the anode buffer solution 7) are aligned. For example, it is advisable to set the difference in height between the two liquid levels to 1 mm or less. This will prevent the polymer solution 18 inside the capillary 1 from moving due to gravity during electrophoresis.

FIG. 12 shows the state in which the capillary 1 is filled with the polymer solution 18 at high pressure in the capillary electrophoresis device according to the improved example 3 (embodiment 2). This filling can also replace the polymer solution 18 already filled in the capillary 1 with the newly filled polymer solution 18. The polymer solution valve 20 is closed, and the plunger 17 of the pressure-resistant syringe 16 is mechanically pushed in by a motor to apply high pressure to the polymer solution 18 inside. The polymer solution 18 in the polymer solution tube 19, the polymer solution 18 in the T-block 15, and the polymer solution 18 in the pressure-resistant syringe 16 form a continuous liquid system, which fills an enclosed space and contains almost no air. The sample elution end 3 is immersed in this liquid system. In this state, the plunger 17, which is a solid, directly compresses the liquid polymer solution 18 at the interface 104 with the polymer solution 18, so that a high pressure of up to about 70 atmospheres can be obtained.

Even if a gas such as air is inserted between the plunger 17 and the polymer solution 18, if the volume of the gas is sufficiently small, the plunger 17 can indirectly pressurize the polymer solution 18, and a similar high pressure can be obtained. The high pressure is transmitted to the entire liquid system including the sample elution end 3 (in Figure 12, the liquid system in a high pressure state is shown with a checkered pattern). Since the sample injection end 2 is under atmospheric pressure, a high pressure difference is generated between the sample elution end 3 and the sample injection end 2, and the polymer solution 18 is filled at high pressure into the capillary 1 from the sample elution end 3 toward the sample injection end 2. The pressure applied to the polymer solution 18 can be controlled by controlling the force of the motor that pushes the plunger 17. In practice, a high pressure of 35 atmospheres is applied to the polymer solution 18 to fill the capillary 1 with the polymer solution 18 at high pressure. After filling the polymer solution, the mechanical pushing of the plunger 17 is stopped, the plunger 17 and the mechanical pushing mechanism are separated, and the pressure applied to the liquid system is reduced to almost zero. Open the polymer solution valve 20 to expose the sample elution end 3 and the above liquid system to atmospheric pressure.

Figure 13 shows the state in which a sample is pressure-injected into the capillary 1 in the capillary electrophoresis device of Improved Example 3 (Example 2). The XYZ-axis drive mechanism of the cathode stage 12 moves the sample injection end 2 into the sample solution tank 11, immersing it in the sample solution 10, while at the same time pressing the O-ring 33 against the fixed block 31 to compress it, sealing the sample solution tank 11. The pressure of the compressed air discharged from the compressed air source 25 is set to 1 atmosphere. When the cathode pressure valve 28 is opened and the cathode release valve 29 is closed, the pressure of the sample solution 10 inside the sample solution tank 11 and the air 37 in contact with it rises to 1 atmosphere. As a result, a low pressure difference of 1 atmosphere is created between the sample injection end 2 and the sample elution end 3, and the sample solution 10 is injected from the sample injection end 2 into the capillary 1. After a predetermined time has elapsed, the cathode pressure valve 28 is closed and the cathode release valve 29 is opened to release the pressure of the sample solution 10 and air 37 inside the sample solution tank 11 to atmospheric pressure.

After this, the XYZ-axis drive mechanism of the cathode stage 12 moves the sample injection end 2 into the cathode buffer solution tank 8, immersing it in the anode buffer solution 6 while simultaneously sealing the cathode buffer solution tank 8. However, by closing the cathode pressure valve 28 and opening the cathode release valve 29 to release the inside of the cathode buffer solution tank 8 to atmospheric pressure, the state of FIG. 11 is restored. In this state, a voltage is applied between the sample elution end 3 and the sample injection end 2 to perform atmospheric pressure electrophoresis.

Therefore, Improved Example 3 (Example 2) achieves the first objective of the present invention, which is to provide a capillary electrophoresis device that (1) "enables filling of a polymer solution in a practical time using a high-viscosity polymer solution for DNA sequencing and DNA fragment analysis" and (2) "enables pressure injection of a sample." However, Improved Example 3 (Example 2) does not achieve the second and third objectives of the present invention.

In improved example 3 (embodiment 2), not only pressure injection of the sample is possible, but also electric field injection. In FIG. 13, the cathode pressure valve 28 is closed and the cathode release valve 29 is opened to release the sample solution 10 inside the sample solution tank 11 and the air 37 in contact with it to atmospheric pressure. In this state, by applying a voltage between the sample elution end 3 and the sample injection end 2 for a certain period of time, the sample can be electric field injected into the capillary 1 from the sample injection end 2.

As described above, in the capillary electrophoresis device according to the improved example 3 (embodiment 2), the cathode side has the same configuration as the cathode side of the conventional example 2, while the anode side has the same configuration as the anode side of the conventional example 1. Such a combination has not existed in the past. One of the issues with the configuration of the capillary electrophoresis device according to the improved example 3 (embodiment 2) is whether the sample can be pressure-injected by the method shown in FIG. 13, as in the method shown in FIG. 6 of the conventional example 2. In FIG. 6, the sample elution end 3 is immersed in a sufficient amount of anode buffer solution 7, and air 32 under atmospheric pressure is present nearby. In contrast, in FIG. 13, the sample elution end 3 is immersed in a small amount of polymer solution 18, and air under atmospheric pressure is present at a distance via a long flow path 19. In other words, there is a fluid resistance between the sample elution end 3 and the air under atmospheric pressure, which may adversely affect sample pressure injection from the sample injection end 2, for example, making it impossible to perform sample pressure injection efficiently. Therefore, using the device according to Improved Example 3 (Example 2), we performed capillary electrophoresis analysis by electric field injection or pressure injection of the sample, and confirmed through experiments whether the expected results could be obtained.

Figure 29 shows four electropherograms obtained by fixing the field injection voltage at 1.8 kV and changing the field injection time to 15, 30, 45, and 60 seconds. The sample solution was a solution of GeneScan ^TM 500 ROX ^TM dye size standard (Thermo Fisher Scientific) diluted with Hi-Di ^TM Formamide (Thermo Fisher Scientific). Figure 30 shows a graph plotting the average peak area of the multiple peaks obtained in each electropherogram in Figure 29 against the field injection time. The field injection time and the peak area have a linear relationship passing through the origin, which confirms that the field injection is performed as expected. In contrast, Figure 31 shows four electropherograms obtained by fixing the pressure of the pressure injection at 0.5 atm and changing the pressure injection time to 15, 30, 45, and 60 seconds. The sample solution was the same as above. Figure 32 shows a graph in which the average peak area of multiple peaks obtained in each electropherogram in Figure 31 is plotted against the pressure injection time. However, the electropherogram corresponding to the plot for a pressure injection time of 90 seconds is not shown in Figure 31. Since the pressure injection time and the peak area have a linear relationship passing through the origin, it can be confirmed that the pressure injection was performed as expected.

From the above, it was found that according to Improved Example 3 (Example 2), electrophoretic analysis can be performed well without any problems whether electric field injection of the sample or pressure injection of the sample is used.

Figure 33 shows in detail an example of the steps in one capillary electrophoresis analysis using electric field injection. Figure 34 shows in detail an example of the steps in one capillary electrophoresis analysis using pressure injection. The leftmost column shows the step numbers, and the steps proceed in numerical order for each analysis. The next column shows the functions such as "polymer solution filling", "pre-electrophoresis", "electric field injection", "pressure injection", "main electrophoresis", etc., and the corresponding rows show the details of each step. "Reference" shows the reference state in the process. "Main electrophoresis" shows normal electrophoresis. The left half of the third column and after in the table shows the state or condition of "contact object", "closed/open", "pressure", and "electric potential" for the "sample elution end", and the right half shows the state or condition of "sample injection end". "Contact object" shows the liquid in which the capillary end is immersed in each step. "Closed/open" indicates whether the space containing the liquid system in which the capillary end is immersed is sealed or open to the atmosphere in each process. "Pressure" indicates the pressure applied to the capillary end in each process as either "-", "low pressure", or "high pressure". "-" means atmospheric pressure, i.e. 0 atmospheres, "low pressure" means pressure greater than 0 atmospheres and less than 7 atmospheres, and "high pressure" means pressure greater than 7 atmospheres. "Potential" indicates the potential applied to the capillary end in each process as either "-", "positive potential", or "negative potential". "-" at both ends of the capillary means that no voltage is applied between the two ends, and "positive potential" at the "sample injection end" and "negative potential" at the "sample elution end" means that voltage is applied between the two ends. "↓" indicates the same state or condition as the line above.

An example of the process of capillary electrophoresis analysis using electric field injection in Figure 33 will be explained in order. With some exceptions, it basically follows Figures 11 to 13. Since the sample elution end is connected to a T-block filled with a polymer solution, the contact object is always the polymer solution.
Step 1: The sample elution end is under atmospheric pressure, and the sample injection end is immersed in the cathode buffer solution under atmospheric pressure. This state is used as the reference.

Step 2: Move the cathode stage and insert the sample injection tip into the waste liquid tank and immerse it in the waste liquid (water) (the waste liquid tank that stores the waste liquid is not depicted on the cathode stage in Figure 12).

Step 3: Close the polymer solution valve to seal the space that contains the liquid system that the sample elution end comes into contact with (polymer solution tube, T-block, and polymer solution in the pressure-resistant syringe) from the atmosphere.

Step 4: The plunger (solid) of the pressure-resistant syringe is mechanically pushed in, applying high pressure to the liquid system and filling the capillary with the polymer solution from the sample elution end toward the sample injection end. The polymer solution overflowing from the sample injection end is discharged into the waste liquid. (In Figure 12, the polymer solution overflowing from the sample injection end is discharged into the cathode buffer solution. The discharged polymer solution contains components of the sample previously analyzed, which may be carried over to the next analysis. Therefore, it is preferable to discharge the polymer solution into the waste liquid rather than into the cathode buffer solution.)

Step 5: The mechanical pushing of the plunger is stopped, the plunger is separated from the mechanical pushing mechanism, the pressure applied to the liquid system is reduced to nearly zero, and the polymer solution filling is completed.

Step 6: The polymer solution valve is opened to open the space containing the liquid system that the sample elution end comes into contact with (the anode buffer solution, the polymer solution tube, the T-block, and the polymer solution in the pressure-resistant syringe) to the atmosphere.
Step 7: The cathode stage is moved to insert the sample injection end into the cathode buffer solution tank, immersed in the cathode buffer solution, and returned to the reference state.
Step 8: A voltage is applied between the sample elution end and the sample injection end to perform preliminary electrophoresis.
Step 9: Stop applying voltage, end pre-electrophoresis, and return to baseline.
Step 10: The cathode stage is moved so that the sample injection end is inserted into the sample solution tank and immersed in the sample solution.
Step 11: A voltage is applied between the sample elution end and the sample injection end to perform electric field injection of the sample.
Step 12: The voltage application is stopped, and the field injection is completed.
Step 13: The cathode stage is moved to insert the sample injection end into the cathode buffer solution tank, immersed in the cathode buffer solution, and returned to the reference state.
Step 14: A voltage is applied between the sample elution end and the sample injection end to perform the main electrophoresis.
Step 15: Stop the voltage application, end the electrophoresis, and return to the reference state. If multiple capillary electrophoresis analyses are to be performed, steps 1 to 15 may be repeated multiple times.

It is preferable to carry out the above steps 1 to 15 in the order listed. However, a unit of capillary electrophoresis analysis may be, for example, steps 7 to 15, followed by steps 1 to 7. Even in such a case, when carrying out multiple capillary electrophoresis analyses, the steps 1 to 15 are always carried out in order. Among steps 1 to 15, the features are step 4, filling with polymer solution, and step 11, electric field injection. That is, the present invention is characterized in that step A (step 4) is carried out in the order of step A, step B, in which the sample injection end is exposed to atmospheric pressure, the polymer solution of the separation medium is brought into contact with the sample elution end, a solid is pressed against the polymer solution, high pressure (above 7 atmospheres) is applied to the polymer solution, and part of the polymer solution is filled into the capillary, and step B (step 11) is carried out in the order of step A and step B, in which the sample injection end is brought into contact with the sample injection end, a voltage is applied between the sample elution end and the sample injection end, and part of the sample solution is injected into the capillary. Alternatively, a feature of the present invention is that the analysis includes a process in which steps A and B are performed in sequence.

An example of the steps of capillary electrophoresis analysis using pressure injection in Fig. 34 will be described in order. With some exceptions, the procedure basically follows Fig. 11 to Fig. 13. Steps 1 to 10 are the same as steps 1 to 10 in Fig. 33, so their description will be omitted.
Step 11: The space (the space inside the sample solution reservoir) that contains the liquid system (sample solution) that comes into contact with the sample injection end is sealed against the atmosphere.
Step 12: Compressed air is introduced into the space housing the liquid system that comes into contact with the sample injection end, and low pressure is applied to pressure-inject the sample.
Step 13: The pressure of the compressed air is set to atmospheric pressure, and the pressure of the space that contains the liquid system that comes into contact with the sample injection end is set to atmospheric pressure (step 13 can be omitted).
Step 14: The space containing the liquid system that comes into contact with the sample injection end is opened to the atmosphere.
Steps 15 to 17 are the same as steps 13 to 15 in FIG. 33, and therefore their explanation will be omitted.

When performing capillary electrophoresis analysis multiple times, steps 1 to 17 can be repeated multiple times. It is preferable to perform steps 1 to 17 in the order listed. However, a single unit of capillary electrophoresis analysis can also be, for example, performed steps 7 to 17, followed by steps 1 to 7. Even in such a case, when performing capillary electrophoresis analysis multiple times, a step of performing steps 1 to 17 will always be included.

The features of steps 1 to 17 are filling with the polymer solution in step 4 and pressure injection in step 12. That is, the present invention is characterized in that the following steps are carried out in the order of step A and step B: step A (step 4) is to fill the capillary with a part of the polymer solution by pressing a solid against the polymer solution and applying high pressure (a pressure of more than 7 atmospheres) to the polymer solution while the sample injection end is open to atmospheric pressure and the polymer solution of the separation medium is in contact with the sample elution end; and step B (step 12) is to apply low pressure (a pressure of 0 to 7 atmospheres) to the sample solution while the sample solution is in contact with the sample injection end by bringing compressed air into contact with the sample solution and injecting a part of the sample solution into the capillary. Alternatively, the present invention is characterized in that the analysis includes a step of carrying out steps A and B in order.

<Improvement Example 4 (Example 3)>
14 to 16 show the configuration of a capillary electrophoresis device according to Improved Example 4 (Example 3) and an analysis method using the same. Improved Example 4 (Example 3) is configured by adding modifications to Improved Example 2 (Example 1: Figures 8 to 10), and has a configuration that solves the problems of Improved Example 2 (Example 1).

The capillary electrophoresis device according to Improved Example 4 (Example 3) is equipped with an air pressure mechanism on the cathode side (sample injection end side), and a plunger pressure mechanism and an air pressure mechanism on the anode side (sample elution end side). Figure 14 shows the state in which double-end pressure electrophoresis is being performed in the capillary electrophoresis device according to Improved Example 4 (Example 3).

The configuration of the cathode side is the same as that of the improved example 2 (embodiment 1). However, the pressure of the compressed air discharged from the compressed air source 25 is set to 1 atmosphere, the cathode pressure valve 28 is opened, and the cathode release valve 29 is closed to pressurize the inside of the cathode buffer solution tank 8 to 1 atmosphere. As for the configuration of the anode side, the configuration of the polymer solution tank 32 to which the pressure-resistant syringe 16 containing the polymer solution 18 is connected is the same as that of the improved example 2 (embodiment 1). The configurations of the anode buffer solution tank 9 containing the anode buffer solution 7 and the fixing block 31 are the same as those of the conventional example 2 (Figures 4 to 6). The anode buffer solution tank 9 containing the anode buffer solution 7 and the polymer solution tank 32 to which the pressure-resistant syringe 16 containing the polymer solution 18 is connected are fixed on the anode stage 13, and the anode stage 13 is further connected to an XYZ axis drive mechanism (not shown). An O-ring 33 is installed at the upper end of each of the anode buffer solution tank 9 and the polymer solution tank 32. The anode stage 13 is moved by the XYZ-axis drive mechanism, and the sample elution end 3 is inserted into the anode buffer solution tank 9 together with the anode 5, and immersed in the anode buffer solution 7. At the same time, the anode stage 13 is moved in the +Z-axis direction, and the O-ring 33 is pressed against the flat surface of the fixed block 31, compressing it, and sealing the anode buffer solution tank 9. The anode pressure valve 26 is opened, and the anode release valve 27 is closed, so that the inside of the anode buffer solution tank 9 is pressurized to 1 atmosphere. In this state, a voltage is applied between the sample elution end 3 and the sample injection end 2, and double-ended pressurized electrophoresis is performed. This increases the pressure of the polymer solution 18 inside the capillary 1 to 1 atmosphere, suppressing the generation of bubbles inside the capillary 1 during electrophoresis, and high separation performance can be stably obtained.

In addition, the vertical height (Z coordinate) of the interface 101 between the cathode buffer solution 6 and the air (i.e., the liquid level of the cathode buffer solution 6) and the interface 102 between the anode buffer solution 7 and the air (i.e., the liquid level of the anode buffer solution 7) are aligned. For example, it is advisable to set the difference in height between the two liquid levels to 1 mm or less. This makes it possible to prevent the polymer solution 18 inside the capillary 1 from moving due to gravity during electrophoresis.

In this specification, the process of opening the anode pressure valve 26 to introduce compressed air to the sample elution end 3 side and the process of opening the cathode pressure valve 28 to introduce compressed air to the sample injection end 2 side are described separately, but it is preferable to carry out these processes simultaneously. In addition, the process of opening the anode release valve 27 to release the sample elution end 3 side to atmospheric pressure and the process of opening the cathode release valve 29 to release the sample injection end 2 side to atmospheric pressure are described separately, but it is preferable to carry out these processes simultaneously. If the timing of these processes is not synchronized, a time period will occur during which a pressure difference occurs between the sample elution end 3 and the sample injection end 2, during which the polymer solution 18 in the capillary 1 will move. To easily align the timing, it is effective to change the anode pressure valve 26 and the cathode release valve 29 to one valve, and to change the anode release valve 27 and the cathode release valve 29 to one valve. For the same reason, it is also preferable to align the speed of pressure increase and pressure decrease on the sample elution end 3 side and the sample injection end 2 side. To achieve this, it is advisable to make the air flow resistance equal on both sides. Alternatively, it is also effective to slowly increase and decrease the pressure of the compressed air discharged from the compressed air source 25, for example, at a speed of 0.5 atmospheres/second or less, or 0.1 atmospheres/second or less.

Figure 15 shows the state in which the capillary 1 is filled with the polymer solution 18 at high pressure in the capillary electrophoresis device according to Improved Example 4 (Example 3). The sample elution end 3 is inserted into the polymer solution tank 32 by moving the anode stage 13 using the XYZ axis drive mechanism, and is immersed in the polymer solution 18. At the same time, the O-ring 33 is pressed against the fixed block 31 and compressed, sealing the polymer solution tank 32. At this time, the structure of the O-ring 33 and the fixed block 31 is determined so that the polymer solution tank 32 and the air tube 30 are not connected, that is, so that the polymer solution 18 in the polymer solution tank 32 does not come into contact with the air in the air tube 30. The polymer solution tank 32 and the pressure-resistant syringe 16 are integrated and have a single internal space, which is filled with the polymer solution 18 and contains almost no air. While the cathode buffer solution 6 and air 34 inside the cathode buffer solution tank 8 are released to atmospheric pressure, the plunger 17 of the pressure-resistant syringe 16 is mechanically pushed in, increasing the pressure of the polymer solution inside the polymer solution tank 32 and the syringe 16 to 35 atmospheres, and the polymer solution 18 is filled into the capillary 1 at high pressure. After filling with the polymer solution, the mechanical pushing of the plunger 17 is stopped, the plunger 17 and the mechanical pushing mechanism are separated, and the pressure applied to the liquid system is reduced to almost zero.

FIG. 16 shows the state in which a sample is pressure-injected into the capillary 1 in the capillary electrophoresis apparatus according to the improved example 4 (embodiment 3). The anode stage 13 is moved by the XYZ-axis drive mechanism to insert the sample elution end 3 into the anode buffer solution tank 9 and immerse it in the anode buffer solution 7, while simultaneously sealing the anode buffer solution tank 9. The anode pressure valve 26 is closed and the anode release valve 27 is opened, and the pressure inside the anode buffer solution tank 9 is set to atmospheric pressure. Meanwhile, the cathode stage 12 is moved by the XYZ-axis drive mechanism to insert the sample injection end 2 into the sample solution tank 11 and immerse it in the sample solution 10, while simultaneously sealing the sample solution tank 11. The pressure of the compressed air discharged from the compressed air source 25 is set to 1 atmosphere. When the cathode pressure valve 28 is opened and the cathode release valve 29 is closed, the pressure of the sample solution 10 inside the sample solution tank 11 and the air 37 in contact with it rises to 1 atmosphere. As a result, a low pressure difference of 1 atmosphere is created between the sample injection end 2 and the sample elution end 3, and the sample solution 10 is injected into the capillary 1 from the sample injection end 2. After a predetermined time has passed, the cathode pressure valve 28 is closed and the cathode release valve 29 is opened to release the pressure of the sample solution 10 and air 37 inside the cathode sample solution tank 11 to atmospheric pressure.

After this, the XYZ-axis drive mechanism of the cathode stage 12 moves the sample injection end 2 into the cathode buffer solution tank 8, immersing it in the anode buffer solution 6 while sealing the cathode buffer solution tank 8. In addition, the anode pressure valve 26 is opened, the anode release valve 27 is closed, the cathode pressure valve 28 is opened, and the cathode release valve 29 is closed to pressurize the inside of the anode buffer solution tank 9 and the cathode buffer solution tank 8 to 1 atmosphere, returning to the state shown in FIG. 14. In this state, a voltage is applied between the sample elution end 3 and the sample injection end 2 to perform double-end pressure electrophoresis. Of course, atmospheric pressure electrophoresis can also be performed by closing the anode pressure valve 26, opening the anode release valve 27, closing the cathode pressure valve 28, and opening the cathode release valve 29 to release the inside of the anode buffer solution tank 9 and the cathode buffer solution tank 8 to atmospheric pressure, and applying a voltage between the sample elution end 3 and the sample injection end 2.

As described above, according to the fourth improvement example (embodiment 3), the first objective is to provide a capillary electrophoresis device that (1) "enables filling with a polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis" and (2) "enables pressure injection of a sample", the second objective is to provide a capillary electrophoresis device that (1) "enables filling with a polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis" and (3) "enables both ends to be pressurized during electrophoresis", and the third objective is to provide a capillary electrophoresis device that (1) "enables filling with a polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis", (2) "enables pressure injection of a sample", and (3) "enables both ends to be pressurized during electrophoresis". All of these objectives can be achieved.

Improvement Example 4 (Example 3) allows not only pressure injection of the sample but also electric field injection. In FIG. 16, the cathode pressure valve 28 is closed and the cathode release valve 29 is opened to release the sample solution 10 inside the sample solution tank 11 and the air 37 in contact with it to atmospheric pressure. In this state, by applying a voltage between the sample elution end 3 and the sample injection end 2 for a certain period of time, the sample can be electric field injected into the capillary 1 from the sample injection end 2.

<Improvement Example 5: Example 4>
17 to 19 show an example of the configuration of a capillary electrophoresis apparatus (corresponding to apparatus H) according to Improved Example 5 (Example 4) and an analysis method using the same. Improved Example 5 (Example 4) is configured by modifying Improved Example 3 (Example 2: Figs. 11 to 13) and has a configuration that solves the problems of Improved Example 3 (Example 2). An air-type pressurizing mechanism is provided on the cathode side (sample injection end side), and a plunger-type pressurizing mechanism and an air-type pressurizing mechanism are provided on the anode side (sample elution end side). Fig. 17 shows a state in which both-end pressurized electrophoresis is performed in Improved Example 5 (Example 4). The configuration on the cathode side is the same as that of Improved Example 3 (Example 2). However, the pressure of the compressed air discharged from the compressed air source 25 is set to 1 atmosphere, the cathode pressurizing valve 28 is opened, and the cathode release valve 29 is closed, so that the inside of the cathode buffer solution tank 8 is pressurized to 1 atmosphere.

The configuration of the anode side is the same as that of Improved Example 3 (Example 2), except for the sealed chamber and the mechanism for introducing compressed air into the sealed chamber, which will be described later. To provide an air-operated pressurizing mechanism on the anode side, a mechanism for sealing the anode buffer solution tank 9 and introducing compressed air into it is necessary. However, the polymer solution valve 20 of Improved Example 3 (Example 2) has a solenoid mechanism for moving the plunger part up and down, and a connection mechanism for linking the solenoid mechanism with the plunger part. In other words, the polymer solution valve 20 protrudes significantly from the anode buffer solution tank 9 and has a movable part. For this reason, it is difficult to seal the anode buffer solution tank 9 using the O-ring 33 and the fixed block 31, as in the conventional device 2 (Figures 4 to 6). Therefore, in Improved Example 5 (Example 4), the anode buffer solution tank 9 and the polymer solution valve 20 are entirely housed inside the sealed chamber 38. Furthermore, the sealed chamber 38 is connected to the compressed air source 25 or the atmosphere via the air tube 30 using the anode pressure valve 26 and the anode release valve 27.

As shown in FIG. 17, when the anode pressure valve 26 is opened and the anode release valve 27 is closed, compressed air flows from the compressed air source 25 into the sealed chamber 38 via the air tube 30, and the pressure of the air 35 inside the sealed chamber 38 and the anode buffer solution tank 9 rises to the pressure of the compressed air discharged from the compressed air source 25, i.e., 1 atmosphere. At the same time, the pressure of the anode buffer solution 7 contained in the anode buffer solution tank 9 is similarly pressurized to 1 atmosphere. As a result, both ends of the cathode and anode sides are pressurized. In this state, a voltage is applied between the sample elution end 3 and the sample injection end 2 to perform double-pressurized electrophoresis. This increases the pressure of the polymer solution 18 inside the capillary 1 to 1 atmosphere, suppressing the generation of bubbles inside the capillary 1 during electrophoresis, and high separation performance can be stably obtained. In addition, the vertical height (Z coordinate) of the interface 101 between the cathode buffer solution 6 and the air (i.e., the liquid level of the cathode buffer solution 6) and the interface 102 between the anode buffer solution 7 and the air (i.e., the liquid level of the anode buffer solution 7) are aligned. For example, it is advisable to set the difference in height between the two liquid levels to 1 mm or less. This makes it possible to prevent the polymer solution 18 inside the capillary 1 from moving due to gravity during electrophoresis.

Figure 18 shows the state in which the capillary 1 is filled with the polymer solution 18 at high pressure in the capillary electrophoresis device according to Improved Example 5 (Example 4). The anode pressure valve 26 is closed and the anode release valve 27 is opened to release the air 35 inside the sealed room 38 and the anode buffer solution tank 9 to atmospheric pressure. The cathode pressure valve 28 is closed and the cathode release valve 29 is opened to release the air 34 inside the cathode buffer solution tank 8 to atmospheric pressure. In this state, the polymer solution valve 20 is closed and the plunger 17 of the pressure-resistant syringe 16 is mechanically pushed in using a motor, applying a high pressure of 35 atmospheres to the polymer solution 18 inside where the sample elution end 2 is immersed.

Because the sample injection end 2 is under atmospheric pressure, a high pressure difference of 35 atmospheres is created between the sample elution end 3 and the sample injection end 2, and the polymer solution 18 is filled into the capillary 1 from the sample elution end 3 toward the sample injection end 2 at a high pressure of 35 atmospheres. After filling with the polymer solution, the mechanical pushing of the plunger 17 is stopped, the plunger 17 and the mechanical pushing mechanism are separated, and the pressure applied to the polymer solution 18 is reduced to almost zero. The polymer solution valve 20 is opened, and the polymer solution 18 and the sample elution end 3 are released to atmospheric pressure.

Figure 19 shows the state in which a sample is pressure-injected into the capillary 1 in the capillary electrophoresis device of Improvement Example 5 (Example 4). The XYZ-axis drive mechanism of the cathode stage 12 moves the sample injection end 2 into the sample solution tank 11, immersing it in the sample solution 10, while at the same time pressing the O-ring 33 against the fixed block 31 to compress it, sealing the sample solution tank 11. The pressure of the compressed air discharged from the compressed air source 25 is set to 1 atmosphere. When the cathode pressure valve 28 is opened and the cathode release valve 29 is closed, the pressure of the sample solution 10 inside the sample solution tank 11 and the air 37 in contact with it rises to 1 atmosphere. As a result, a low pressure difference of 1 atmosphere is created between the sample injection end 2 and the sample elution end 3, and the sample solution 10 is injected from the sample injection end 2 into the capillary 1. After a predetermined time has elapsed, the cathode pressure valve 28 is closed and the cathode release valve 29 is opened to release the pressure of the sample solution 10 and air 37 inside the sample solution tank 11 to atmospheric pressure.

After this, the XYZ-axis drive mechanism of the cathode stage 12 is used to move the sample injection end 2 into the cathode buffer solution tank 8 and immerse it in the anode buffer solution 6, while simultaneously sealing the cathode buffer solution tank 8. The pressure of the compressed air discharged from the compressed air source 25 is set to 1 atmosphere. Next, the anode pressure valve 26 is opened, the anode release valve 27 is closed, the cathode pressure valve 28 is opened, and the cathode release valve 29 is closed, pressurizing the sealed room 38, the inside of the anode buffer solution tank 9, and the inside of the cathode buffer solution tank 8 to 1 atmosphere, returning to the state shown in Figure 17. In this state, a voltage is applied between the sample elution end 3 and the sample injection end 2, and double-ended pressure electrophoresis is performed. Of course, atmospheric pressure electrophoresis can also be performed by closing the anode pressure valve 26, opening the anode release valve 27, closing the cathode pressure valve 28, and opening the cathode release valve 29 to expose the inside of the anode buffer solution tank 9 and the cathode buffer solution tank 8 to atmospheric pressure, and applying a voltage between the sample elution end 3 and the sample injection end 2.

As described above, according to the fifth improved example (fourth embodiment), the first object of the present invention is to provide a capillary electrophoresis device that (1) "enables filling with a polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis" and (2) "enables pressure injection of a sample", the second object is to provide a capillary electrophoresis device that (1) "enables filling with a polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis" and (3) "enables both ends to be pressurized during electrophoresis", and the third object is to provide a capillary electrophoresis device that (1) "enables filling with a polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis", (2) "enables pressure injection of a sample", and (3) "enables both ends to be pressurized during electrophoresis". All of these objects can be achieved.

Improvement Example 5 (Example 4) allows not only pressure injection of the sample but also electric field injection. In FIG. 19, the cathode pressure valve 28 is closed and the cathode release valve 29 is opened to release the sample solution 10 inside the sample solution tank 11 and the air 37 in contact with it to atmospheric pressure. In this state, by applying a voltage between the sample elution end 3 and the sample injection end 2 for a certain period of time, the sample can be electric field injected into the capillary 1 from the sample injection end 2.

<Improvement Example 6: Example 5>
Figure 20 shows an example of the configuration of a capillary electrophoresis device according to Improvement Example 6 (Example 5). Improvement Example 6 (Example 5) is configured by making modifications to Improvement Example 3 (Example 2: Figures 11 to 13), and is configured to solve the problems of Improvement Example 3 (Example 2). The capillary electrophoresis device according to Improvement Example 6 (Example 5) is equipped with an air-type pressure mechanism on the cathode side (sample injection end side), and is equipped with a plunger-type pressure mechanism and an air-type pressure mechanism on the anode side (sample elution end side).

　Figure 20 shows the state in which double-end pressurized electrophoresis is being performed in a capillary electrophoresis apparatus according to Improved Example 6 (Example 5). The configuration on the cathode side is the same as that of Improved Example 3. However, the pressure of the compressed air discharged from the compressed air source 25 is set to 1 atmosphere, the cathode pressurization valve 28 is opened, and the cathode release valve 29 is closed, so that the inside of the cathode buffer solution tank 8 is pressurized to 1 atmosphere.

In Improved Example 6 (Example 5), in order to provide an air-operated pressurizing mechanism on the anode side, the polymer solution valve 20 in Improved Example 3 (Example 2) is removed, and the anode buffer solution tank 9 is sealed using an O-ring 33 and a fixed block 31 as in Conventional Example 2 (Figures 4 to 6). Instead of the polymer solution valve 20 located at the boundary between the anode buffer solution 7 in the anode buffer solution tank 9 and the polymer solution 18 in the polymer solution tube 19, a compressed air side polymer solution valve 39 is installed on the polymer solution tube 19 between the anode buffer solution tank 9 and the T-block 15. The feature of this configuration is that the compressed air side polymer solution valve 39 is located between the sample elution end 3 and the interface 102 between the anode buffer solution 7 and the air (i.e., the liquid surface of the anode buffer solution 7).

In Figure 20, the polymer solution valve 39 on the compressed air side is open. Also, the anode buffer solution tank 9 is connected to the compressed air source 25 or the atmosphere using the anode pressure valve 26 and the anode release valve 27 via the air tube 30. However, the structure of the sealed anode buffer tank 9 differs from that of the capillary electrophoresis device of Conventional Example 2 in the following respects. In Conventional Example 2, the sample elution end 3 of the capillary 1 and the anode 5 are inserted into the sealed anode buffer tank 9 and immersed in the cathode buffer solution 7.

In contrast, in the capillary electrophoresis device according to the improved example 6 (Example 5), the sample elution end 3 of the capillary 1 is not inserted into the sealed anode buffer tank 9, but instead, the anode 5 and the polymer solution tube 19 are inserted into the sealed anode buffer tank 9 and immersed in the cathode buffer solution 7. As shown in FIG. 20, when the anode pressure valve 26 is opened and the anode release valve 27 is closed, compressed air flows from the compressed air source 25 into the anode buffer solution tank 9 via the air tube 30, and the pressure inside the anode buffer solution tank 9 rises to the pressure of the compressed air discharged from the compressed air source 25, that is, 1 atmosphere. As a result, both ends of the cathode side and the anode side are pressurized. In this state, a voltage is applied between the sample elution end 3 and the sample injection end 2 to perform both-end pressurized electrophoresis. As a result, the pressure of the polymer solution 18 inside the capillary 1 is raised to 1 atmosphere, the generation of bubbles inside the capillary 1 during electrophoresis is suppressed, and high separation performance can be stably obtained. In addition, the vertical height (Z coordinate) of the interface 101 between the cathode buffer solution 6 and the air (i.e., the liquid level of the cathode buffer solution 6) and the interface 102 between the anode buffer solution 7 and the air (i.e., the liquid level of the anode buffer solution 7) are aligned. This makes it possible to prevent the polymer solution 18 inside the capillary 1 from moving due to gravity during electrophoresis.

Improvement Example 6 (Example 5) can achieve the first to third objects of the present invention, but it has the following problem. When a pressure of 1 atmosphere is applied to the inside of the anode buffer solution tank 9 in the state shown in FIG. 20, the polymer solution 18 filled inside the polymer solution tube 19, the T-block 15, and the pressure-resistant syringe 16 is also pressurized to 1 atmosphere. At this time, the plunger 17 of the pressure-resistant syringe 16 is subjected to a force in a direction in which it is pulled out of the pressure-resistant syringe 16, and there is a possibility that the plunger 17 moves in a direction in which it is pulled out of the pressure-resistant syringe 16. When the plunger 17 actually moves, the pressure of the polymer solution 18 filled inside the polymer solution tube 19, the T-block 15, and the pressure-resistant syringe 16 changes, so the balance of the pressure at both ends of the capillary 1 is lost, the polymer solution 18 inside the capillary 1 moves, and the separation ability of the electrophoresis is reduced. Therefore, the above problem will be solved and the first to third objects of the present invention will be achieved.

<Improvement Example 7: Example 6>
21 shows an example of the configuration of a capillary electrophoresis device according to Improved Example 7 (Embodiment 6) (when performing double-end pressurized electrophoresis). Improved Example 7 (Embodiment 6) is configured by adding modifications to Improved Example 6 (Embodiment 5: FIG. 20) and has a configuration that solves the problems of Improved Example 6 (Embodiment 5).

Improvement Example 7 (Example 6) differs from Improvement Example 6 (Example 5) in that a plunger stopper 41 capable of fixing the plunger 17 of the pressure-resistant syringe 16 is added. An X-axis drive mechanism (not shown) including a stepping motor can be used as one of the means for mechanically pushing in the plunger 17 of the pressure-resistant syringe 16. To keep the polymer solution 18 inside the pressure-resistant syringe 16 at a desired pressure, the drive force in the -X-axis direction of the drive part of the X-axis drive mechanism can be set to a corresponding constant value (specifically, the relationship is made by "pressure" = "drive force" ÷ "internal cross-sectional area of the pressure-resistant syringe"). To do this, the drive force can be measured using a load sensor or the like, and the operation of the drive part can be controlled so that the drive force is a constant value. Alternatively, the current value driving the stepping motor can be controlled so that the stepping motor steps out when the drive force exceeds a certain value.

The most basic configuration of the plunger stopper 41 is in a state where the drive unit of the X-axis drive mechanism and the plunger 17 are connected. The plunger stopper 41 is operated by continuing to excite the stepping motor with the drive unit stationary. If the stepping motor is continued to be excited with the drive unit stationary, a static torque acts on the stepping motor, making it possible to stop the movement of the plunger 17. However, if the torque applied to the stepping motor by the force acting on the drive unit exceeds the maximum static torque, the drive unit will operate.

However, connecting the drive unit of the X-axis drive mechanism to the plunger 17 causes the following problem. In Fig. 21, the compressed air discharged from the compressed air source 25 is set to 1 atmosphere, the anode pressure valve 26 is opened, the anode release valve 27 is closed, the cathode pressure valve 28 is opened, and the cathode release valve 29 is closed to set the inside of the anode buffer solution tank 9 and the cathode buffer solution tank 8 to a low pressure of 1 atmosphere. From this state, the anode pressure valve 26 is closed, the anode release valve 27 is opened, the cathode pressure valve 28 is closed, and the cathode release valve 29 is opened to release the inside of the anode buffer solution tank 9 and the cathode buffer solution tank 8 to atmospheric pressure. Furthermore, the compressed air side polymer solution valve 39 is closed, and the section of the polymer solution tube 19 between the compressed air side polymer solution valve 39 and the T-block 15, the T-block 15, and the polymer solution 18 inside the pressure-resistant syringe 16 are connected into a single liquid system. By driving the X-axis drive mechanism, the plunger 17 of the pressure-resistant syringe 16 is mechanically pushed in, increasing the pressure of the liquid system to a high pressure of 35 atmospheres, and the polymer solution 18 can be filled into the capillary 1. Here, even if the drive of the X-axis drive mechanism is stopped and the plunger 17 is stopped to stop the polymer solution filling, the high pressure state of the liquid system is maintained (more precisely, the pressure slowly decreases as the polymer solution is filled), and the polymer solution filling continues. In this state, if the compressed air side polymer solution valve 39 is opened, the liquid system is released to atmospheric pressure and the polymer solution filling is stopped.

However, as soon as the compressed air side polymer solution valve 39 is opened, a large amount of the polymer solution 18 inside the polymer solution tube 19 is rapidly discharged into the cathode buffer solution 7, causing a sudden drop in pressure in the liquid system. These phenomena are undesirable because they lead to waste of the polymer solution 18, the generation of bubbles, and the like. In order to avoid these phenomena, it is necessary to move the drive unit of the X-axis drive mechanism in the +X-axis direction and move the plunger 17 in the direction of pulling it out of the pressure-resistant syringe 16 to reduce the pressure in the liquid system. However, it is unclear how far the drive unit needs to be moved in the +X-axis direction to make the pressure in the liquid system become atmospheric pressure, and it is difficult to predict this. Furthermore, if the movement distance is even slightly greater than the optimal value, the pressure in the liquid system becomes negative.

To solve the above problem, the drive unit of the X-axis drive mechanism and the plunger 17 can be uncoupled, and when the drive unit moves in the -X-axis direction, it comes into contact with the plunger 17, pushing the plunger 17 into the pressure-resistant syringe 16, while when the drive unit moves in the +X-axis direction, it separates from the plunger 17, avoiding the plunger 17 moving in the direction of pulling it out of the pressure-resistant syringe 16. By shifting the drive unit from a state in which it moves in the -X-axis direction to a state in which it moves in the +X-axis direction, it is possible to switch from a filled state with the polymer solution 18 to a non-filled state. Even if the drive unit separates from the plunger 17, some residual pressure may remain in the liquid system, but if the residual pressure is at that level, the effect of releasing it to atmospheric pressure by opening the compressed air side polymer solution valve 39 is small.

However, if the drive unit of the X-axis drive mechanism and the plunger 17 are not connected, the static torque of the stepping motor does not function as the plunger stopper 41. In this case, as shown in Figure 21, a device other than the X-axis drive mechanism functions as the plunger stopper 41 that directly suppresses the movement of the plunger 17.

As described above, Improved Example 7 (Example 6) can achieve the first to third objects of the present invention while solving the problems of Improved Example 6 (Example 5). Improved Example 7 (Example 6) allows not only pressure injection of the sample but also electric field injection.

<Improvement Example 8: Example 7>
22 to 24 show the configuration of a capillary electrophoresis device (corresponding to device I) according to Improved Example 8 (Example 7). Improved Example 8 (Example 7) is configured by modifying Improved Example 6 (Example 5: FIG. 20) and is configured to solve the problems of Improved Example 6 (Example 5). Improved Example 8 (Example 7) is different from Improved Example 6 (Example 5) in that the T-block 15 and the pressure-resistant syringe 16 are connected by a plunger-side polymer solution tube 43, and a plunger-side polymer solution valve 40 is added on the plunger-side polymer solution tube 43 between the T-block 15 and the pressure-resistant syringe 16. The configuration of Improved Example 8 (Example 7) is characterized in that the plunger-side polymer solution valve 40 is disposed between the sample elution end 3 and the interface 104 between the plunger 17 in the pressure-resistant syringe 16 and the polymer solution. The capillary electrophoresis apparatus according to Improvement Example 8 (Example 7) is provided with an air pressure mechanism on the cathode side (sample injection end side) and a plunger type pressure mechanism and an air pressure mechanism on the anode side (sample elution end side).

　Figure 22 shows the state in which double-end pressurized electrophoresis is being performed in the capillary electrophoresis device of Improved Example 8 (Example 7). In the first stage of Figure 22, the anode pressurization valve 26 is closed, the anode release valve 27 is opened, the compressed air side polymer solution valve 39 is opened, and the plunger side polymer solution valve 40 is opened to release the inside of the anode buffer tank 9, polymer solution tube 19, T-block 15, plunger side polymer solution tube 43, and pressure-resistant syringe 16 to atmospheric pressure. In this state, the plunger side polymer solution valve 40 is closed.

Next, the pressure of the compressed air discharged from the compressed air source 25 is set to 1 atmosphere, the anode pressure valve 26 is opened, and the anode release valve 27 is closed to pressurize the inside of the anode buffer tank 9, the polymer solution tube 19, and the T-block 15 to 1 atmosphere.

Meanwhile, the sample injection end 2 is inserted into the cathode buffer solution tank 8 and the cathode buffer solution tank 8 is sealed while immersed in the cathode buffer solution 6. The cathode pressure valve 28 is opened and the cathode release valve 29 is closed to pressurize the inside of the cathode buffer solution tank 8 to 1 atmosphere. This results in the both-end pressurized state shown in Figure 22. In this state, a voltage is applied between the sample elution end 3 and the sample injection end 2 to perform both-end pressurized electrophoresis. This increases the pressure of the polymer solution 18 inside the capillary 1 to 1 atmosphere, suppresses the generation of bubbles inside the capillary 1 during electrophoresis, and enables stable high separation performance to be obtained. In addition, the vertical heights (Z coordinates) of the interface 101 between the cathode buffer solution 6 and the air (i.e., the liquid level of the cathode buffer solution 6) and the interface 102 between the anode buffer solution 7 and the air (i.e., the liquid level of the anode buffer solution 7) are aligned. This prevents the polymer solution 18 inside the capillary 1 from moving due to gravity during electrophoresis.

Meanwhile, the polymer solution 18 in the T-block 15 in which the sample elution end 3 is immersed and the polymer solution 18 in the pressure-resistant syringe 16 are separated by the plunger-side polymer solution valve 40, so the pressure of one does not affect the other. In other words, the 1 atmosphere pressure of the polymer solution 18 in the T-block 15 does not move the plunger 17 of the pressure-resistant syringe 16. Conversely, the movement of the plunger 17 does not change the pressure of the polymer solution 18 in the T-block 15.

FIG. 23 shows the state in which the capillary 1 is filled with the polymer solution 18 at high pressure in the capillary electrophoresis device according to the eighth improved example (embodiment seven). The anode pressure valve 26 is closed and the anode release valve 27 is opened to open the inside of the anode buffer solution tank 9 to atmospheric pressure. The cathode pressure valve 28 is closed and the cathode release valve 29 is opened to open the inside of the cathode buffer solution tank 8 to atmospheric pressure. In this state, the compressed air side polymer solution valve 39 is closed and the plunger side polymer solution valve 40 is opened, and the plunger 17 of the pressure-resistant syringe 16 is mechanically pushed in using a motor to apply a high pressure of 35 atmospheres to the polymer solution 18 in which the sample elution end 2 is immersed. Since the sample injection end 2 is under atmospheric pressure, a high pressure difference of 35 atmospheres is generated between the sample elution end 3 and the sample injection end 2, and the polymer solution 18 is filled in the capillary 1 from the sample elution end 3 toward the sample injection end 2 at a high pressure of 35 atmospheres. After filling the polymer solution, the mechanical pushing of the plunger 17 is stopped, the plunger 17 and the mechanical pushing mechanism are separated, and the pressure applied to the polymer solution 18 is reduced to almost zero. The compressed air side polymer solution valve 39 is opened, and the sample elution end 3 is opened to atmospheric pressure.

FIG. 24 shows the state in which a sample is pressure-injected into the capillary 1 in the capillary electrophoresis device according to the eighth improved example (seventh embodiment). The XYZ-axis drive mechanism of the cathode stage 12 moves the sample injection end 2 into the sample solution tank 11, immersing it in the sample solution 10 and sealing the sample solution tank 11 at the same time. When the cathode pressure valve 28 is opened and the cathode release valve 29 is closed, the pressure of the sample solution 10 in the sample solution tank 11 and the air 37 in contact with it rises to 1 atmosphere. As a result, a low pressure difference of 1 atmosphere is created between the sample injection end 2 and the sample elution end 3, and the sample solution 10 is injected from the sample injection end 2 into the capillary 1. After a predetermined time has passed, the cathode pressure valve 28 is closed and the cathode release valve 29 is opened to release the pressure of the sample solution 10 and the air 37 in the sample solution tank 11 to atmospheric pressure.

After this, the XYZ-axis drive mechanism of the cathode stage 12 moves the sample injection tip 2 into the cathode buffer solution tank 8, immersing it in the anode buffer solution 6 while simultaneously sealing the cathode buffer solution tank 8.

Then, the plunger side polymer solution valve 40 is closed. After that, the anode pressure valve 26 is opened and the anode release valve 27 is closed to pressurize the inside of the anode buffer tank 9, polymer solution tube 19, and T-block 15 to 1 atmosphere. Meanwhile, the cathode pressure valve 28 is opened and the cathode release valve 29 is closed to pressurize the inside of the cathode buffer solution tank 8 to 1 atmosphere, returning to the state shown in Figure 22. In this state, a voltage is applied between the sample elution end 3 and the sample injection end 2 to perform double-ended pressure electrophoresis.

As described above, according to the improved example 8 (embodiment 7), the first object of the present invention is to provide a capillary electrophoresis device that (1) "enables filling of the polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis" and (2) "enables pressure injection of the sample", the second object is to provide a capillary electrophoresis device that (1) "enables filling of the polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis" and (3) "enables both ends to be pressurized during electrophoresis", and the third object is to provide a capillary electrophoresis device that (1) "enables filling of the polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis", (2) "enables pressure injection of the sample", and (3) "enables both ends to be pressurized during electrophoresis". All of these objects can be achieved. Of course, it is also possible to electric-field-inject a sample using the capillary electrophoresis device according to the improved example 8 (embodiment 7).

Figure 35 shows in detail an example of the steps of a single capillary electrophoresis analysis using pressure injection and double-ended pressure electrophoresis. The notation method is the same as in Figures 33 and 34. The steps are explained in order, following Figures 22 to 24 with some exceptions. Steps 1 to 15 are the same as steps 1 to 15 in Figure 34, so their explanation is omitted.

Step 16: The space (the space inside the cathode buffer solution tank) that contains the liquid system (cathode buffer solution) that the sample injection end comes into contact with is sealed from the atmosphere. At the same time, the space (the anode buffer solution tank, the polymer solution tube, the T-block, and the space inside the pressure-resistant syringe) that contains the liquid system (anode buffer solution, polymer solution tube, T-block, and polymer solution in the pressure-resistant syringe) that the sample elution end comes into contact with is sealed from the atmosphere.

Step 17: The same compressed air is introduced into the space housing the liquid system with which the sample injection end comes into contact and the space housing the liquid system with which the sample elution end comes into contact, a low pressure is applied, and both ends are pressurized.
Step 18: A voltage is applied between the sample elution end and the sample injection end to perform double-ended pressure electrophoresis.
Step 19: Stop applying voltage and return to applying pressure only to both ends.

Step 20: The pressure of the compressed air is set to atmospheric pressure, and the pressure in the space housing the liquid system with which the sample injection end comes into contact, and the pressure in the space housing the liquid system with which the sample elution end comes into contact, are set to atmospheric pressure (step 20 can be omitted).

Step 21: The space that contains the liquid system that the sample injection end comes into contact with is opened to the atmosphere. At the same time, the space that contains the liquid system that the sample elution end comes into contact with is opened to the atmosphere. This returns the system to the baseline.

When performing capillary electrophoresis analysis multiple times, steps 1 to 21 can be repeated multiple times. It is preferable to perform steps 1 to 21 in the order listed. However, a single unit of capillary electrophoresis analysis may be, for example, performed steps 7 to 21, followed by steps 1 to 7. Even in such a case, when performing capillary electrophoresis analysis multiple times, a step of performing steps 1 to 21 will always be included.

The key features of steps 1 to 21 are polymer solution filling in step 4, pressure injection in step 12, and double-end pressurized electrophoresis (main electrophoresis) in step 16. That is, the present invention is characterized in that the following steps are carried out in the order of steps A, B, and C: step A (step 4) of pressing a solid against the polymer solution while the sample injection end is open to atmospheric pressure and, with the polymer solution of the separation medium in contact with the sample elution end, applying high pressure (a pressure of more than 7 atmospheres) to the polymer solution and filling the capillary with a part of the polymer solution; step B (step 12) of bringing the sample solution into contact with the sample injection end and bringing compressed air into contact with it to apply low pressure (a pressure of 0 to 7 atmospheres) to the sample solution and injecting a part of the sample solution into the capillary; and step C3 (step 16) of bringing the cathode buffer solution into contact with the sample injection end and bringing compressed air into contact with the cathode buffer solution to apply low pressure (a pressure of 0 to 7 atmospheres) to the liquid system containing the polymer solution with which the sample elution end is in contact, with the polymer solution in contact with the sample elution end, bringing compressed air into contact with the liquid system to apply low pressure (a pressure of 0 to 7 atmospheres) to the liquid system, and applying a voltage between the sample elution end and the sample injection end. Alternatively, a feature of the present invention is that the analysis includes a process in which steps A and B are performed in sequence.

　By capillary electrophoresis, the components of the sample eluted from the sample elution end move slowly towards the positive electrode 5. Such components may be reinjected into the capillary when the polymer solution is filled for the subsequent analysis, resulting in carryover. The following configuration is effective in reducing such a possibility. When the location where the flow path from the anode buffer solution tank 9 to the sample elution end 3 and the flow path from the pressure-resistant syringe 16 to the sample elution end 3 intersect is called the intersection, it is desirable to make the distance between the sample elution end 3 and the intersection as small as possible. In addition, it is preferable to make the distance between the sample elution end 3 and the intersection 10 mm or less, more preferably 1 mm or less, and most preferably 0 mm.

<Improvement Example 9: Example 8>
25 to 27 show an example of the configuration of a capillary electrophoresis device according to Improved Example 9 (Embodiment 8). Improved Example 9 (Embodiment 8) is configured by adding modifications to Improved Example 8 (Embodiment 7: Figs. 22 to 24), and achieves the object of the present invention more effectively than Improved Example 8 (Embodiment 7). Improved Example 9 (Embodiment 8) differs from Improved Example 8 (Embodiment 7) in that the compressed air side polymer solution valve 39 and the plunger side polymer solution valve 40 in Improved Example 8 (Embodiment 7) are replaced with a rotary valve 42. This modification makes it possible to combine two valves into one valve, and facilitates automation of the device. The capillary electrophoresis device according to Improved Example 9 (Embodiment 8) is provided with an air pressure mechanism on the cathode side (sample injection end side), and a plunger pressure mechanism and an air pressure mechanism on the anode side (sample elution end side).

　Figure 25 shows the state in which double-ended pressurized electrophoresis is being performed in a capillary electrophoresis device according to Improved Example 9 (Example 8). The rotary valve 42 used in the capillary electrophoresis device according to Improved Example 9 (Example 8) has six ports. In the rotary valve 42 in Figure 25, the port at the 1 o'clock position is port 1, the port at the 3 o'clock position is port 2, the port at the 5 o'clock position is port 3, the port at the 7 o'clock position is port 4, the port at the 9 o'clock position is port 5, and the port at the 11 o'clock position is port 6.

The rotary valve 42 has an automatic rotation mechanism, and is rotated to take either position A or position B. In position A, port 2 and port 3, port 4 and port 5, and port 6 and port 1 are connected, respectively. In position B, port 1 and port 2, port 3 and port 4, and port 5 and port 6 are connected, respectively. As shown in Figure 25, the polymer solution tube 19 is connected to port 4 and port 5, the polymer solution tube 19 connected to port 4 is connected to the anode buffer solution tank 9, and the polymer solution tube 19 connected to port 5 is connected to the T-block 15. In addition, the plunger side polymer solution tube 43 is connected to port 1 and port 2, the plunger side polymer solution tube 43 connected to port 2 is connected to the pressure-resistant syringe 16, and the plunger side polymer solution tube 43 connected to port 1 is connected to the T-block 15. In addition, ports 3 and 6 are blocked. In Figure 25, the rotary valve 42 is in position A. This connects the two polymer solution tubes 19 and connects the anode buffer solution tank 9 and the T-block 15 with the polymer solution tube 19.

The two plunger side polymer solution tubes 43 are also disconnected, and the pressure-resistant syringe 16 and the T-block 15 are not connected. That is, the anode buffer solution 7 in the anode buffer solution tank 9, the polymer solution 18 in the polymer solution tube 19, the polymer solution 18 in the T-block 15, and the polymer solution 18 in the plunger side polymer solution tube 43 between the T-block 15 and the rotary valve 42 form a continuous liquid system A, and the sample elution end 3 is immersed in the liquid system A. The pressure of the compressed air discharged from the compressed air source 25 is set to 1 atmosphere. The sample injection end 2 is inserted into the cathode buffer solution tank 8, and the cathode buffer solution tank 8 is sealed while immersed in the cathode buffer solution 6. The cathode pressure valve 28 is opened, and the cathode release valve 29 is closed, and a pressure of 1 atmosphere is applied to the air 34 and the cathode buffer solution 6 inside the cathode buffer solution tank 8.

Meanwhile, the anode pressure valve 26 is opened and the anode release valve 27 is closed to apply a pressure of 1 atmosphere to the air 35 and the anode buffer solution 7 inside the cathode buffer solution tank 9. This also applies a pressure of 1 atmosphere to the liquid system A.

As a result of the above, both ends of the cathode and anode sides in Figure 25 are pressurized. In this state, a voltage is applied between the sample elution end 3 and the sample injection end 2 to perform both-end pressurized electrophoresis. This increases the pressure of the polymer solution 18 inside the capillary 1 to 1 atmosphere, suppressing the generation of bubbles inside the capillary 1 during electrophoresis and enabling stable high separation performance to be obtained.

In addition, the vertical height (Z coordinate) of the interface 101 between the cathode buffer solution 6 and the air (i.e., the liquid level of the cathode buffer solution 6) and the interface 102 between the anode buffer solution 7 and the air (i.e., the liquid level of the anode buffer solution 7) are aligned. This makes it possible to prevent the polymer solution 18 inside the capillary 1 from moving due to gravity during electrophoresis. Furthermore, since the polymer solution 18 in the T-block 15 in which the sample elution end 3 is immersed and the polymer solution 18 in the pressure-resistant syringe 16 are not connected, the pressure of one does not affect the other. In other words, the pressure of 1 atmosphere of the polymer solution 18 in the T-block 15 does not move the plunger 17 of the pressure-resistant syringe 16. Conversely, the movement of the plunger 17 does not change the pressure of the polymer solution 18 in the T-block 15.

Figure 26 shows the state in which the capillary 1 is filled with the polymer solution 18 at high pressure in a capillary electrophoresis apparatus according to improved example 9 (embodiment 8). The anode pressure valve 26 is closed and the anode release valve 27 is opened to atmospheric pressure inside the anode buffer solution tank 9. The cathode pressure valve 28 is closed and the cathode release valve 29 is opened to atmospheric pressure inside the cathode buffer solution tank 8. In this state, the rotary valve 42 is set to position B. This separates the two polymer solution tubes 19, and the anode buffer solution tank 9 and the T-block 15 are not connected. The two plunger side polymer solution tubes 43 are also linked, and the pressure-resistant syringe 16 and the T-block 15 are connected by the plunger side polymer solution tube 43. That is, the polymer solution 18 in the pressure-resistant syringe 16, the polymer solution 18 in the plunger side polymer solution tube 43, the polymer solution 18 in the T-block 15, and the polymer solution 18 in the polymer solution tube 19 between the T-block 15 and the rotary valve 42 form a continuous liquid system B, and the sample elution end 3 is immersed in the liquid system B.

In this state, the plunger 17 of the pressure-resistant syringe 16 is mechanically pushed in by the motor, applying a high pressure of 35 atmospheres to the liquid system B. Since the sample injection end 2 is under atmospheric pressure, a high pressure difference of 35 atmospheres is created between the sample elution end 3 and the sample injection end 2, and the polymer solution 18 is filled into the capillary 1 from the sample elution end 3 toward the sample injection end 2 at a high pressure of 35 atmospheres. After the polymer solution is filled, the mechanical pushing of the plunger 17 is stopped, the plunger 17 and the mechanical pushing mechanism are separated, and the pressure applied to the polymer solution 18 is reduced to almost zero. In this state, the rotary valve 42 is set to position A, and the liquid system A and the sample elution end 3 are released to atmospheric pressure. At this time, even if some pressure remains in the polymer solution 18 in the pressure-resistant syringe 16, this does not affect the sample elution end 3, so there is no problem.

FIG. 27 shows the state in which a sample is pressure-injected into the capillary 1 in the capillary electrophoresis device according to the ninth improvement (embodiment 8). The XYZ-axis drive mechanism of the cathode stage 12 moves the sample injection end 2 into the sample solution tank 11, immersing it in the sample solution 10 and simultaneously sealing the sample solution tank 11. When the cathode pressure valve 28 is opened and the cathode release valve 29 is closed, the pressure of the sample solution 10 in the sample solution tank 11 and the air 37 in contact with it rises to 1 atmosphere. As a result, a low pressure difference of 1 atmosphere is created between the sample injection end 2 and the sample elution end 3, and the sample solution 10 is injected from the sample injection end 2 into the capillary 1. After a predetermined time has passed, the cathode pressure valve 28 is closed and the cathode release valve 29 is opened to release the pressure of the sample solution 10 and the air 37 in the sample solution tank 11 to atmospheric pressure.

After this, the XYZ-axis drive mechanism of the cathode stage 12 is used to move the sample injection end 2 into the cathode buffer solution tank 8, immersing it in the anode buffer solution 6 while simultaneously sealing the cathode buffer solution tank 8. Next, the anode pressure valve 26 is opened and the anode release valve 27 is closed, pressurizing the air 35 in the anode buffer solution tank 9 and the liquid system A to 1 atmosphere. Meanwhile, the cathode pressure valve 28 is opened and the cathode release valve 29 is closed, pressurizing the air 34 in the cathode buffer solution tank 8 and the cathode buffer solution 6 to 1 atmosphere, returning to the state shown in FIG. 25. In this state, a voltage is applied between the sample elution end 3 and the sample injection end 2, and double-ended pressurized electrophoresis is performed.

As described above, according to the 9th improvement (Example 8), the first object of the present invention is to provide a capillary electrophoresis device that (1) "enables filling with a polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis" and (2) "enables pressure injection of a sample", the second object is to provide a capillary electrophoresis device that (1) "enables filling with a polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis" and (3) "enables both ends to be pressurized during electrophoresis", and the third object is to provide a capillary electrophoresis device that (1) "enables filling with a polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis", (2) "enables pressure injection of a sample", and (3) "enables both ends to be pressurized during electrophoresis". All of these objects can be achieved.

In the 9th improvement (Example 8), not only pressure injection of the sample is possible, but also electric field injection. In FIG. 27, the cathode pressure valve 28 is closed and the cathode release valve 29 is opened to release the sample solution 10 inside the sample solution tank 11 and the air 37 in contact with it to atmospheric pressure. In this state, by applying a voltage between the sample elution end 3 and the sample injection end 2 for a certain period of time, the sample can be electric field injected into the capillary 1 from the sample injection end 2.

<Improvement Example 10: Example 9>
In the above-mentioned improved examples, for the sake of simplicity, each device has one capillary, and each device performs electrophoretic analysis using one capillary. However, the present invention is also applicable to the case where each device has multiple capillaries and performs electrophoretic analysis in parallel using the multiple capillaries. As an example, FIG. 28 shows a configuration example of a capillary electrophoresis device according to improved example 10 (embodiment 9) that handles three capillaries in improved example 8 (embodiment 7) (when performing electrophoresis with both ends pressurized). Of course, the number of capillaries is only a specific example, and the number of capillaries can be any number such as 4, 8, 12, ...

The sample injection ends 2 of the three capillaries 1 are inserted into the three pipe-shaped cathode electrodes 4, respectively, and the two are integrated. The three sample injection ends 2 are oriented in the -Z-axis direction, aligned in the Y-axis and Z-axis directions, and arranged at equal intervals in the X-axis direction. The interval in the X-axis direction is, for example, 9 mm, which is the same as the interval between wells in a microtiter plate. The cathode buffer solution tanks 8, which contain the three cathode buffer solutions 6, are aligned in the Y-axis and Z-axis directions, and fixed onto the cathode stage 12 at intervals in the X-axis direction that are the same as the intervals between the three sample injection ends 2 in the X-axis direction, and the cathode stage 12 is further connected to an XYZ-axis drive mechanism (not shown).

A sample solution tank 11, which contains at least three sample solutions 10, is also fixed on the cathode stage 12, but is omitted in Fig. 28. An O-ring 33 is placed on the edge of the upper end of each of the three cathode buffer solution tanks 8. By moving the cathode stage 12 by the XYZ axis drive mechanism, the three sample injection ends 2 are inserted into each of the three cathode buffer solution tanks 8 and immersed in the three cathode buffer solutions 6. At the same time, by moving the cathode stage 12 in the +Z direction, the three O-rings 33 are pressed against the flat surface of the fixed block 31 and compressed, sealing the three cathode buffer solution tanks 8. The gaps between the three capillaries 1 and the three cathodes 4 and the fixed block 31 are sealed. The three cathode buffer solution tanks 8 contain the three cathode buffer solutions 6 and a common air 34 that contacts the three cathode buffer solutions 6. The air 34 is integrated inside the fixed block 31 and connected to the compressed air source 25 or the atmosphere via the air tube 30 through the cathode pressure valve 28 and the cathode release valve 29. The pressure of the compressed air discharged from the compressed air source 25 is set to 1 atmosphere.

As shown in Figure 28, when the cathode pressure valve 28 is opened and the cathode release valve 29 is closed, compressed air flows into the three cathode buffer solution tanks 8 via the air tube 30, and the pressure of the air 34 rises to 1 atmosphere. At the same time, the pressure of the three cathode buffer solutions 6 contained in the three cathode buffer solution tanks 8 also rises to 1 atmosphere.

The three detection positions on the three capillaries 1 are arranged in a straight line, and the laser beam 24 emitted from the laser light source 23 is irradiated to the three detection positions simultaneously, and the fluorescence emitted from the three detection positions is detected simultaneously by a fluorescence detection device (not shown).

The sample elution ends 3 of the three capillaries 1 are bundled together and connected to a T-block 15 using a connector 14, and immersed in the polymer solution 18 in the T-block 15. The other components on the anode side (polymer solution tube 19 containing polymer solution 18, compressed air side polymer solution valve 39, anode buffer solution tank 9 containing anode buffer solution 7 and air 35, fixed block 31, plunger side polymer solution tube 43, plunger side polymer solution valve 40, pressure-resistant syringe 16 containing polymer solution 18, etc.) are the same as those in Improved Example 8 (Example 7).

As shown in Figure 28, when the anode pressure valve 26 is opened and the anode release valve 27 is closed, compressed air flows into the anode buffer solution tank 9 via the air tube 30, and the pressure of the air 35 rises to 1 atmosphere. At the same time, the pressures of the anode buffer solution 7 contained in the anode buffer solution tank 9, the polymer solution 18 contained in the polymer solution tube 19, and the polymer solution 18 contained in the T-block 15 also rise to 1 atmosphere. In this state, a voltage is applied between the sample elution end 3 and the sample injection end 2 to perform double-ended pressurized electrophoresis. This increases the pressure of the polymer solution 18 inside the capillary 1 to 1 atmosphere, suppressing the generation of bubbles inside the capillary 1 during electrophoresis, and achieving stable high separation performance.

In addition, the vertical heights (Z coordinates) of the interface 101 between the three cathode buffer solutions 6 and the air (i.e., the liquid levels of the three cathode buffer solutions 6) and the interface 102 between the anode buffer solution 7 and the air (i.e., the liquid level of the anode buffer solution 7) are aligned. This makes it possible to prevent the polymer solution 18 inside the capillary 1 from moving due to gravity during electrophoresis. The methods of polymer solution filling, sample pressure injection, etc. are the same as those in Improved Example 8 (Example 7).

As described above, according to the improvement example 10 (Example 9), the first object of the present invention is to provide a capillary electrophoresis device that (1) "enables filling of the polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis" and (2) "enables pressure injection of the sample" for each of the three capillaries, the second object of the present invention is to provide a capillary electrophoresis device that (1) "enables filling of the polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis" and (3) "enables both ends to be pressurized during electrophoresis", and the third object is to provide a capillary electrophoresis device that (1) "enables filling of the polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis", (2) "enables pressure injection of the sample", and (3) "enables both ends to be pressurized during electrophoresis". All of these objects can be achieved. In the improvement example 10 (Example 9), not only pressure injection of the sample but also electric field injection is possible.

Summary of the embodiment
First, in device B (conventional example 2), the air-type pressure mechanism provided at the sample elution end is replaced with a plunger-type pressure mechanism (improved example 1: device D). The configuration on the sample injection end side is not changed. The sample elution end is connected to a container filled with a polymer solution, and a syringe filled with the polymer solution is further connected to the container. The internal spaces of the container and the syringe form a single sealed internal space filled with the polymer solution. The sample elution end and the positive electrode integrated with the sample elution end are immersed in the polymer solution in the above internal space. With the sample injection end open to atmospheric pressure, the pressure of the internal polymer solution can be increased to 35 atmospheres by mechanically pushing in the plunger of the syringe, and the high-viscosity polymer solution can be filled into the capillary. By immersing the sample injection end in the sample solution and applying a voltage to both ends of the capillary, the sample can be injected into the capillary by electric field. However, even if the sample injection end is immersed in the sample solution and a low pressure of 1 atmosphere is applied by the air pressure mechanism, the sample cannot be pressure-injected into the capillary. This is because in Device D, the sample elution end cannot be released to atmospheric pressure, so no pressure difference occurs between both ends of the capillary. This is the same problem as Device C. However, electrophoresis can be performed if the sample injection end of the capillary is immersed in the cathode buffer solution and a voltage is applied to both ends of the capillary. Therefore, the configuration of Device D cannot achieve any of the objectives of the present invention.

Next, in device B, the air-type pressure mechanism provided at the sample elution end is replaced with the plunger-type pressure mechanism of device A (Reference Example 1: Device E). The configuration on the sample injection end side is not changed. The sample elution end integrated with the anode is connected to a flow path containing the polymer solution, and a syringe containing the polymer solution is further connected to the flow path. In addition, an anode buffer tank containing an anode buffer solution is also connected to the flow path, and a valve is installed at the boundary between the anode buffer solution and the polymer solution. With the sample injection end open to atmospheric pressure and the valve closed, the pressure of the internal polymer solution is increased to 35 atmospheres by mechanically pushing the syringe plunger, thereby filling the capillary with a high viscosity polymer solution. When the valve is opened, the sample elution end is opened to atmospheric pressure, so that the sample injection end is immersed in the sample solution and a low pressure of 1 atmosphere is applied by the air-type pressure mechanism, and the sample can be pressure-injected into the capillary. Alternatively, the sample can be injected into the capillary by immersing the sample injection end in the sample solution and applying a voltage to both ends of the capillary. Electrophoresis can be performed by immersing the sample injection end of the capillary in the cathode buffer solution and applying a voltage to both ends of the capillary. However, immersing the anode integrated with the sample elution end in the polymer solution contained in the flow path has the following adverse effects on electrophoretic analysis. Unlike the anode buffer tank, the flow path has a small inner diameter and a small internal volume per unit length in the electrophoretic direction. As a result, the ion composition of the polymer solution near the sample elution end changes with electrophoresis, eventually resulting in ion depletion. In addition, gas generated on the surface of the anode during electrophoresis remains in the flow path as bubbles, resulting in large electrical resistance. Furthermore, sample components eluted from the sample elution end during electrophoresis remain in the flow path, and such components flow back into the capillary when the polymer solution is filled in the subsequent analysis, causing a carryover problem.

In the device E, the integrated positive electrode and sample elution end are separated, and the sample elution end is connected to a flow path containing a polymer solution, while the positive electrode is immersed in an anode buffer solution (Improvement Example 3 (Example 2): Device F). With the configuration of Device F, the position of the positive electrode does not adversely affect the electrophoretic analysis. Therefore, it is possible to fill the polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis, and it is also possible to pressure-inject the sample. Therefore, Device F can achieve the first object of the present invention, which is to provide a capillary electrophoresis device that (1) "enables filling the polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis" and (2) "enables pressure-injection of the sample." However, since the sample elution end does not have an air-type pressure mechanism, it is not possible to perform pressure application at both ends during electrophoresis.

In order to realize double-ended pressurized electrophoresis, in Device A (Conventional Example 1), a plunger-type pressurizing mechanism is installed at the sample injection end, as well as at the sample elution end (Reference Example 2: Device G). The configuration of the sample elution end side is not changed. The sample injection end integrated with the cathode is connected to a flow path containing a polymer solution, and a syringe containing the polymer solution is connected to the flow path. In addition, a cathode buffer tank containing a cathode buffer solution is also connected to the flow path, and a valve is installed at the boundary between the cathode buffer solution and the polymer solution. The plunger-type pressurizing mechanism increases the pressure of the polymer solution inside by mechanically pushing the plunger of the syringe with the valve closed. In addition, when the valve is opened, the sample injection end is released to atmospheric pressure. As a result, since independent plunger-type pressurizing mechanisms are installed at both ends of the capillary, it is expected that double-ended pressurization during electrophoresis will be possible. However, compared to the air-type pressurizing mechanism, the plunger-type pressurizing mechanism is suitable for applying high pressure, but is not suitable for applying low pressure with high precision and stability. For example, the sliding resistance when pushing the plunger into the syringe is not constant, so even if the pushing force is constant, the internal pressure fluctuates. In addition, when an air-type pressurizing mechanism is used at both ends of the capillary, as in device B, it is possible to equalize the pressure at both ends by branching the same compressed air and using it for each. In contrast, when a plunger-type pressurizing mechanism is used at both ends of the capillary, as in device G, it is difficult to equalize the pressure at both ends because each is an independent mechanism. If a difference in pressure occurs at both ends, the polymer solution in the capillary moves from the side with higher pressure to the side with lower pressure, which has a negative effect on the separation ability. In addition, because the sample injection end is connected to the flow path containing the polymer solution, it is difficult to separate the sample injection end from the flow path and immerse it in the sample solution. Therefore, it is difficult to perform both pressure injection and electric field injection of the sample.

In consideration of the above, in the device F, a sealed room containing the anode buffer tank, the valve, and a set of mechanisms for driving the valve is installed, and an air-operated pressurizing mechanism is installed by introducing compressed air into the sealed room (Improvement Example 5 (Example 4): Device H). The air in the sealed room can also be released to atmospheric pressure. The valve and the mechanism for driving the valve protrude far from the anode buffer tank and move spatially, so it is difficult to seal only the anode buffer tank as in device B. For this reason, a relatively large sealed room as described above is installed. With the sample injection end open to atmospheric pressure, the sealed room open to atmospheric pressure, and the valve closed, the syringe plunger is mechanically pushed in, increasing the pressure of the polymer solution inside to 35 atmospheres, and the high-viscosity polymer solution can be filled into the capillary. When the valve is opened, the sample elution end is released to atmospheric pressure, so that the sample injection end is immersed in the sample solution and a low pressure of 1 atmosphere is applied by the air-operated pressurizing mechanism, and the sample can be pressure-injected into the capillary. Alternatively, the sample can be injected into the capillary by immersing the sample injection end in the sample solution and applying a voltage to both ends of the capillary. Electrophoresis can be performed by immersing the sample injection end of the capillary in the cathode buffer solution and applying a voltage to both ends of the capillary. During electrophoresis, a low pressure of 1 atmosphere is applied to the sample elution end by an air pressure mechanism that introduces compressed air into a sealed chamber with the valve open, and at the same time, a low pressure of 1 atmosphere is applied to the sample injection end by an air pressure mechanism that seals the cathode buffer tank and introduces compressed air, thereby achieving pressurization at both ends.

As described above, device H can achieve the third objective of the present invention, which is to provide a capillary electrophoresis device that (1) "enables filling of a polymer solution in a practical time using a high-viscosity polymer solution for DNA sequencing and DNA fragment analysis," (2) "enables pressure injection of a sample," and (3) "enables pressure application to both ends during electrophoresis." However, there is an issue that the structure of the sealed chamber is large and complex. Another issue is that because the internal volume of the sealed chamber is large, it takes a long time for the internal pressure to reach a specified pressure when compressed air is introduced.

Furthermore, based on the above, in device F, the valve at the boundary between the anode buffer solution and the polymer solution is removed, valve 1 is installed on the flow path between the sample elution end and the anode buffer tank, and valve 2 is also installed on the flow path between the sample elution end and the syringe. At the same time, as in device B, the anode buffer tank is made to have a sealed structure, and an air-type pressurizing mechanism is installed by introducing compressed air into the anode buffer tank (Improvement example 8 (Example 7): device I). However, unlike device B, the anode buffer tank does not have the sample elution end of the capillary inserted, and instead a flow path containing the polymer solution that connects to the sample elution end is inserted. The anode buffer tank can also be opened to atmospheric pressure. The configuration on the sample injection end side is not changed. With the sample injection end opened to atmospheric pressure, valve 1 is closed, and valve 2 is open, the pressure of the internal polymer solution can be increased to 35 atmospheres, and the high-viscosity polymer solution can be filled into the capillary by mechanically pushing in the plunger of the syringe. When the anode buffer tank is opened to atmospheric pressure and valve 1 is opened, the sample elution end is opened to atmospheric pressure. In this state, the sample injection end is immersed in the sample solution and a low pressure of 1 atmosphere is applied by the air pressure mechanism to pressure-inject the sample into the capillary. Alternatively, the sample injection end is immersed in the sample solution and a voltage is applied to both ends of the capillary to electric field-inject the sample into the capillary. Electrophoresis can be performed by immersing the sample injection end of the capillary in the cathode buffer solution and applying a voltage to both ends of the capillary. Furthermore, during electrophoresis, both-side pressure electrophoresis can be performed by opening valve 1 and closing valve 2, applying a low pressure of 1 atmosphere to the anode buffer tank and sample elution end by the air pressure mechanism, and simultaneously applying a low pressure of 1 atmosphere to the sample injection end by the air pressure mechanism.

As a result, Device I can provide a capillary electrophoresis device that (1) "enables filling of a polymer solution in a practical time using a high-viscosity polymer solution for DNA sequencing and DNA fragment analysis," (2) "enables pressure injection of a sample," and (3) "enables pressure application to both ends during electrophoresis," which is the third objective of the present invention.

1 Capillary
2 Sample injection port
3. Sample elution end
4 negative electrode
5 positive electrode
6. Cathode Buffer Solution
7. Anode Buffer Solution
8 Cathode buffer solution tank
9 Anode buffer solution tank
10 Sample solution
11 Sample solution tank
12 Cathode Stage
13 Anode Stage
14 Connectors
15 T-Block
16 Pressure-resistant syringe
17 Plunger
18 Polymer Solution
19 Polymer solution tube
20 Polymer Solution Valve
21 DC power supply
22 Electric wire
23 Laser or lamp light source
24 Laser beam or lamp light
25 Compressed Air Source
26 Anode pressure valve
27 Anode Release Valve
28 Cathode pressure valve
29 Cathode Release Valve
30 Air tube
31 Fixed Block
32 Polymer solution tank
33 O-ring
34 Air in contact with the cathode buffer solution
35 Air in contact with the anode buffer solution
36 Air in contact with polymer solutions
37 Air in contact with sample solution
38 Sealed Room
39 Compressed air side polymer solution valve
40 Plunger side polymer solution valve
41 Plunger stopper
42 Rotary Valve
43 Plunger side polymer solution tube
101 Cathode buffer solution/air interface
102 Anode buffer solution/air interface
103 Interface between anode buffer solution and polymer solution
104 Interface between polymer solution and plunger
105 Sample solution/air interface
106 Polymer solution/air interface

Claims (20)

a first liquid system in contact with a first capillary end of the capillary;
a second liquid system in contact with a second capillary end of the capillary;
a power source that applies a voltage between the first liquid system and the second liquid system;
a first pressurizing mechanism that increases a first pressure in the first liquid system by compressing a first gas that is in direct or indirect contact with a first interface of the first liquid system;
a first pressure reducing mechanism for reducing the first pressure in the first liquid system;
a second pressurizing mechanism that increases a second pressure in the second liquid system by moving a solid that is in direct or indirect contact with a second interface of the second liquid system;
a second pressure reducing mechanism for reducing the second pressure in the second liquid system;
A capillary electrophoresis device, wherein the second liquid system has a second gas in direct or indirect contact with a third interface of the second liquid system. In claim 1,
the first pressurizing mechanism is a pressurizing mechanism that seals a first space in which the first liquid system is contained from the atmosphere and supplies compressed air to the first space;
a second pressurizing mechanism that seals a second space in which the second liquid system is contained from the atmosphere and reduces a volume of the second space; In claim 2,
the first pressure reduction mechanism is a first atmospheric pressure release mechanism that opens the first space to the atmosphere and reduces the first pressure to atmospheric pressure,
a second pressure reducing mechanism that opens the second space to the atmosphere and reduces the second pressure to atmospheric pressure; In claim 3,
a portion of the second liquid system that contacts the second capillary end is a polymer solution of a separation medium;
a capillary electrophoresis device, wherein, while the first pressure is set to atmospheric pressure by the first atmospheric pressure release mechanism, the second pressurizing mechanism increases the second pressure to fill a portion of the polymer solution into the capillary. In claim 3,
a portion of the first liquid system that contacts the end of the first capillary is a sample solution;
a first pressure mechanism for increasing the first pressure and injecting a portion of the sample solution into the capillary while the second pressure is set to atmospheric pressure by the second atmospheric pressure release mechanism; In claim 3,
the first pressure is made atmospheric pressure by the first atmospheric pressure release mechanism,
In a state where the second pressure is set to atmospheric pressure by the second atmospheric pressure release mechanism,
a capillary electrophoresis device, wherein when a voltage is applied to the first capillary end and the second capillary end to perform electrophoresis, the first interface and the third interface are aligned in vertical height; A method for capillary electrophoresis performed by applying a voltage between a first capillary end and a second capillary end of a capillary, comprising:
a step 1 of pressing a solid against the polymer solution to pressurize the polymer solution while the end of the first capillary is open to atmospheric pressure and the end of the second capillary is in contact with the polymer solution of a separation medium, thereby filling the capillary with a part of the polymer solution;
and step 2 of pressurizing the sample solution by bringing compressed air into contact with the sample solution while the sample solution is in contact with the end of the first capillary, thereby injecting a portion of the sample solution into the capillary,
The method is a capillary electrophoresis analysis method, in which step 1 and step 2 are carried out in this order. A method for capillary electrophoresis performed by applying a voltage between a first capillary end and a second capillary end of a capillary, comprising:
A step 1 of applying a pressure of 0 atmospheres to the end of the first capillary and a pressure of more than 7 atmospheres to the end of the second capillary while the end of the second capillary is in contact with a polymer solution of a separation medium, thereby filling the capillary with a part of the polymer solution;
and step 2 of applying a pressure of 7 atmospheres or less to the end of the first capillary while the end of the first capillary is in contact with a sample solution, applying a pressure of 0 atmospheres to the end of the second capillary, and injecting a portion of the sample solution into the capillary,
The method is a capillary electrophoresis analysis method, wherein the step 1 and the step 2 are carried out in this order. a first liquid system in contact with a first capillary end of the capillary;
a second liquid system in contact with a second capillary end of the capillary;
a power source that applies a voltage between the first liquid system and the second liquid system;
a first pressurizing mechanism that increases a first pressure in the first liquid system by compressing a first gas that is in direct or indirect contact with a first interface of the first liquid system;
a second pressurizing mechanism that increases a second pressure in the second liquid system by moving a solid that is in direct or indirect contact with a second interface of the second liquid system;
a third pressurizing mechanism that increases a second pressure in the second liquid system by compressing a second gas that is in direct or indirect contact with a third interface of the second liquid system;
A capillary electrophoresis apparatus comprising: In claim 9, further comprising:
a first pressure reducing mechanism for reducing the first pressure in the first liquid system;
a second pressure reducing mechanism for reducing the second pressure in the second liquid system;
A capillary electrophoresis apparatus comprising: In claim 10,
the first pressurizing mechanism is a pressurizing mechanism that seals a first space in which the first liquid system is contained from the atmosphere and supplies compressed air to the first space;
A capillary electrophoresis apparatus, wherein the second pressurizing mechanism is a pressurizing mechanism that seals a second space in which the second liquid system is contained from the atmosphere and reduces a volume of the second space, and the third pressurizing mechanism is a pressurizing mechanism that seals the second space from the atmosphere and supplies compressed air to the second space. In claim 11,
the first pressure reduction mechanism is a first atmospheric pressure release mechanism that opens the first space to the atmosphere and reduces the first pressure to atmospheric pressure,
a second pressure reducing mechanism that opens the second space to the atmosphere and reduces the second pressure to atmospheric pressure; In claim 12,
a portion of the second liquid system that contacts the second capillary end is a polymer solution of a separation medium;
a capillary electrophoresis device, wherein, while the first pressure is set to atmospheric pressure by the first atmospheric pressure release mechanism, the second pressurizing mechanism increases the second pressure to fill a portion of the polymer solution into the capillary. In claim 12,
a portion of the first liquid system that contacts the end of the first capillary is a sample solution;
a first pressure mechanism for increasing the first pressure and injecting a portion of the sample solution into the capillary while the second pressure is set to atmospheric pressure by the second atmospheric pressure release mechanism; In claim 12,
The first pressure mechanism and the third pressure mechanism use compressed air of equal pressure,
The first pressure mechanism increases the first pressure, and the third pressure mechanism increases the second pressure, thereby making the first pressure equal to the second pressure;
a capillary electrophoresis device that performs capillary electrophoresis by applying a voltage between the first capillary end and the second capillary end; In claim 12, further comprising:
a capillary electrophoresis apparatus comprising a first valve between the third interface and the second capillary end, the first valve dividing or connecting the second liquid system; In claim 12, further comprising:
a capillary electrophoresis apparatus comprising a second valve between the second interface and the second capillary end, the second valve dividing or joining the second liquid system; In claim 12,
A capillary electrophoresis device, wherein when a voltage is applied to the first capillary end and the second capillary end to perform electrophoresis, the height of the first interface and the vertical height of the third interface are aligned. A method for capillary electrophoresis performed by applying a voltage between a first capillary end and a second capillary end of a capillary, comprising:
a step 1 of pressing a solid against the polymer solution to pressurize the polymer solution while the end of the first capillary is open to atmospheric pressure and the end of the second capillary is in contact with the polymer solution of a separation medium, thereby filling the capillary with a part of the polymer solution;
a step 2 of pressurizing the sample solution by bringing a first compressed air into contact with the sample solution while the sample solution is in contact with the end of the first capillary, and injecting a portion of the sample solution into the capillary;
a step 3 of bringing a second compressed air into contact with the buffer solution while the first capillary end is in contact with the buffer solution to pressurize the buffer solution, bringing the second compressed air into contact with a liquid system containing the polymer solution while the second capillary end is in contact with the polymer solution to pressurize the liquid system, and applying a voltage between the first capillary end and the second capillary end;
The method is a capillary electrophoresis analysis method, wherein the step 1, the step 2, and the step 3 are carried out in this order. A method for capillary electrophoresis performed by applying a voltage between a first capillary end and a second capillary end of a capillary, comprising:
A step 1 of applying a first pressure of 0 atmospheres to the end of the first capillary and applying a second pressure of more than 7 atmospheres to the end of the second capillary while the end of the second capillary is in contact with a polymer solution of a separation medium, thereby filling the capillary with a part of the polymer solution;
a step 2 of applying a third pressure of 7 atmospheres or less to the end of the first capillary while the end of the first capillary is in contact with a sample solution, applying the first pressure to the end of the second capillary, and injecting a portion of the sample solution into the capillary;
and step 3 of applying a fourth pressure of 7 atmospheres or less to the first capillary end while the first capillary end is in contact with a buffer solution, applying the fourth pressure to the second capillary end while the second capillary end is in contact with a polymer solution, and applying a voltage between the first capillary end and the second capillary end to perform double-end pressure electrophoresis,
The method is a capillary electrophoresis analysis method, wherein the step 1, the step 2, and the step 3 are carried out in this order.

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