Capillary electrophoresis device and capillary electrophoresis analysis method

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

[0001] The present invention relates to an apparatus and method for analyzing components contained in a sample solution by capillary electrophoresis.

[0002] 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 pressures, and pressures above 7 atmospheres are called high pressures. 1 atmosphere is approximately 0.1 MPa.

[0003] A capillary electrophoresis system is an automated device that separates various components contained in a sample solution by electrophoresis based on charge and size, and then analyzes them. For example, the Applied Biosystems capillary electrophoresis system sold by Thermo Fisher Scientific is available. TM SeqStudio TMAs described in Non-Patent Document 1, the Flex Series Genetic Analyzers (hereinafter referred to as "System A") can perform DNA sequencing and DNA fragment analysis. System A can perform parallel analysis using eight or 24 capillaries, but this specification mainly describes one capillary (similar explanations apply to other capillaries). Because DNA fragments are negatively charged, the sample injection end of the capillary is the cathode, and the sample elution end is the anode, and electrophoresis occurs from the cathode to the anode. System A can apply high pressure to the sample elution end of the capillary using a plunger-type pressure mechanism. The sample elution end is connected to a flow channel containing a polymer solution, and a syringe containing the polymer solution is also connected to the flow channel. An anode buffer solution tank containing an anode buffer solution is also connected to the flow channel, and the anode buffer solution in the anode buffer solution tank is connected to the polymer solution in the flow channel, with a valve installed at the boundary between them. The plunger-type pressure mechanism mechanically pushes in the syringe plunger while the valve is closed, increasing the pressure of the internal polymer solution to a maximum of approximately 70 atmospheres, practically a high pressure of 35 atmospheres. At this time, the sample injection end is open to atmospheric pressure, allowing the high-viscosity polymer solution to be filled into the capillary.

[0004] On the other hand, when the valve is opened, the inside of the anode buffer solution reservoir and the sample elution end are exposed to atmospheric pressure. The cathode is pipe-shaped, and the sample injection end of the capillary is inserted through the cathode, integrating the two. The anode is not integrated with the sample elution end of the capillary; it is inserted into the anode buffer solution reservoir and immersed in the anode buffer solution. With both ends of the capillary 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. Furthermore, with both ends of the capillary open to atmospheric pressure, electrophoresis (called atmospheric pressure electrophoresis) can be performed by immersing the sample injection end in the cathode buffer solution and applying a voltage to both ends of the capillary.

[0005] The PA 800 Plus Pharmaceutical Analysis System (hereinafter referred to as "Apparatus B") sold by Sciex is a different type of capillary electrophoresis apparatus from Apparatus A, and as described in Non-Patent Document 2, it is capable of analyzing proteins such as antibodies. Apparatus B can analyze both negatively and positively charged components. When analyzing negatively charged components, the sample injection end of the capillary is the cathode, and the sample elution end is the anode, and electrophoresis is performed from the cathode to the anode.

[0006] On the other hand, when analyzing positively charged components, the sample injection end of the capillary is the anode and the sample elution end is the cathode, and electrophoresis proceeds from the anode to the cathode. For simplicity's sake, the following describes the analysis of samples containing negatively charged components. In System B, low pressure can be applied independently to both the sample injection end and the sample elution end of the capillary using an air pressure mechanism. Each end can also be independently opened to atmospheric pressure. The negative electrode is pipe-shaped and is integrated with the sample injection end of the capillary, penetrating it. The positive electrode is also pipe-shaped and is integrated with the sample elution end of the capillary, penetrating it. The air pressure mechanism inserts one of the capillary ends into a container containing the solution, immersing it in the solution. Compressed air is then introduced into the sealed container, increasing the pressure inside the container to a maximum of approximately 7 atmospheres. The polymer solution can be filled into the capillary 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 using the air pressure mechanism, and releasing the sample injection end to atmospheric pressure. (Alternatively, the polymer solution can be filled into the capillary 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 using the air pressure mechanism, and releasing the sample elution end to atmospheric pressure.) The sample can also 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 using 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 and 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), the generation of bubbles in the polymer solution inside the capillary can be suppressed, stabilizing the electrophoretic analysis.

[0007] The device shown in Figure 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 oriented, 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. The cathode buffer tank has a sealed structure to prevent the polymer solution in the capillary from falling due to gravity. Similar to Device A, a high pressure of 35 atmospheres can be applied to the sample elution end of the capillary using a plunger-type pressure mechanism, allowing the capillary to be filled with a highly viscous polymer solution. A low pressure of 1 atmosphere can also be applied to the sample injection end using an air-type pressure mechanism. Furthermore, the sealed cathode buffer tank effectively prevents the polymer solution from falling due to gravity. In this state, the sample injection end can be immersed in the sample solution and a voltage applied to both ends of the capillary to allow the sample to be injected into the capillary by electric field.

[0008] A plunger-type pressurizing mechanism compresses a liquid stored in a sealed container by moving a solid that is in direct contact with the liquid, thereby increasing its pressure. Compared to gases, the volumetric change in solids and liquids relative to pressure is small, so high pressure can be achieved with even small movements of solids. In other words, a plunger-type pressurizing mechanism is a suitable method for applying high pressure to a liquid 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 subsequent increase in pressure due to the movement of solids will be significant. Furthermore, 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, high pressure can be similarly achieved with a plunger-type pressurizing mechanism.

[0009] In contrast, air-operated pressurization mechanisms compress the liquid by compressing the gas that comes into direct contact with the liquid (increasing the gas density). Because gas volume changes significantly with pressure, a large device is required to generate compressed air. Therefore, the airtight container and the large device must be located far apart, and they must be connected using air tubes, connectors, valves, etc. As a result, the volume and surface area of ​​the compressed air handled are large, so even a small pressure generates a large force, making it more likely to leak or burst. For these reasons, it is generally difficult to produce high-pressure compressed air, and in practice, low-pressure compressed air of 5 atmospheres or less is used. In other words, air-operated pressurization mechanisms are suitable for applying low pressure to liquids stored in airtight containers. Furthermore, air-operated pressurization mechanisms can also be used to obtain low pressures when compressing gas that is in indirect contact with the liquid stored in an airtight container because a small amount of solid is sandwiched between the liquid and the gas.

[0010] Japanese Patent Application Laid-Open No. 2001-124736

[0011] SeqStudioTM Flex Series Genetic Analyzer with Instrument Software v1.1, USER GUIDEhttps: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / 100104689_SeqStudioFlex_v1_RUO_UG.pdfPA 800 Plus Pharmaceutical Analysis System, Mehods Development Guidehttps: / / sciex.com / content / dam / SCIEX / pdf / customer-docs / user-guide / pa-800-plus-methods-development-guide.pdf

[0012] In capillary electrophoresis, the sample injection volume using field injection is proportional to the sample concentration when the sample concentration is low, but saturates at high concentrations. On the other hand, the sample injection volume using pressure injection is proportional to the sample concentration regardless of the sample concentration. Therefore, pressure injection may be superior to field injection for preparing high-concentration samples and obtaining larger injection volumes. Furthermore, compared to field injection, pressure injection provides a linear relationship between sample concentration and peak intensity over a wide range of sample concentrations. Based on these findings, pressure injection is expected to be superior to field injection in terms of improving sensitivity and quantitative accuracy. Furthermore, the double-end pressurization function during electrophoresis suppresses bubble generation by increasing the pressure of the polymer solution inside the capillary, contributing to the stable achievement of high-resolution separation performance. Improving resolution due to bubble generation is particularly important when injecting large amounts of highly concentrated samples.

[0013] The pressure injection of the sample and the pressure application at both ends during electrophoresis are expected to have similar effects when performing DNA sequencing and DNA fragment analysis. TM Polymer, for 3500 / SeqStudio TM DNA sequencing and DNA fragment analysis were performed on System A using the Flex (Thermo Fisher Scientific, hereafter referred to as POP-7). The polymer solution was filled by applying a high pressure of 35 atmospheres using a plunger-type pressure mechanism, which allowed the polymer solution to be filled within a practical time of approximately 5 minutes, despite the high viscosity of the polymer solution. However, because System A cannot use an air-type pressure mechanism, it was not possible to pressure-inject the sample or to apply pressure to both ends during electrophoresis (double-end pressure electrophoresis).

[0014] Therefore, we attempted DNA sequencing and DNA fragment analysis using Device B, which is equipped with air pressure mechanisms on both ends of the capillary. The sample elution end of the capillary was immersed in a highly viscous polymer solution, POP-7, and a low pressure of 5 atmospheres was applied using the air pressure mechanism. The sample elution end was then released to atmospheric pressure. However, the viscosity of POP-7 was higher than that of the polymer solution typically used in Device B for protein analysis (e.g., Sciex's SDS-MW Gel Buffer), significantly reducing the polymer solution filling speed. 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 Device B.

[0015] Next, DNA sequencing and DNA fragment analysis were performed using Device C. As with Device A, a high pressure of 35 atmospheres was applied using the plunger-type pressure mechanism attached to the sample elution end, allowing the highly viscous polymer solution POP-7 to be filled within a practical time frame. On the other hand, although a low pressure of 1 atmosphere could be applied to the sample injection end using the air-type pressure mechanism attached to the sample injection end, it was found that pressure injection of the sample was not possible. This was because the anode buffer tank connected to the sample elution end has a sealed structure that cannot be released to atmospheric pressure, so the pressure applied to the sample injection end was transmitted to the entire polymer solution inside the capillary, preventing a pressure difference from occurring at both ends of the capillary.

[0016] Based on the above, the present invention proposes a capillary electrophoresis technology that achieves either of the following: (1) to provide a capillary electrophoresis device that "enables filling of 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" (first objective); (1) to provide a capillary electrophoresis device that "enables filling of a polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis" and (3) "enables pressure injection of both ends during electrophoresis" (second objective); or (1) to provide a capillary electrophoresis device that "enables filling of 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" and (3) "enables pressure injection of both ends during electrophoresis" (third objective).

[0017] 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 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 decompression mechanism that reduces the first pressure 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; and a second decompression mechanism that reduces 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 a liquid when the liquid is filled into the capillaries.

[0018] Further features related to the present disclosure will become apparent from the description of this specification and the accompanying drawings. Also, aspects of the present disclosure are achieved and realized by the elements and combinations of various elements and the aspects of the following detailed description and the appended claims. The description of this specification is merely exemplary and does not limit the scope or application of the claims of the present disclosure in any way.

[0019] According to one embodiment of the present invention (the configuration of Apparatus F; the details of Apparatus F are described below), it is possible to fill the polymer solution within a practical time frame using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis, and it is also possible to pressure-inject the sample. As a result, in DNA sequence and DNA fragment analysis, it is possible to improve sensitivity by injecting a larger amount of sample than before, while also enabling highly quantitative analysis. Furthermore, according to another embodiment of the present invention (the configuration of Apparatus I; the details of Apparatus I are described below), in addition to the effects of Apparatus F described above, it is possible to perform double-ended pressure electrophoresis. This makes it possible to suppress the generation of bubbles and consistently achieve high separation performance.

[0020] FIG. 1 is a diagram showing an example of the configuration of a conventional apparatus 1 performing atmospheric pressure electrophoresis. FIG. 2 is a diagram showing an example of the configuration of a conventional apparatus 1 performing high-pressure polymer solution filling. FIG. 3 is a diagram showing an example of the configuration of a conventional apparatus 1 performing sample field injection. FIG. 4 is a diagram showing an example of the configuration of a conventional apparatus 2 performing double-end pressure electrophoresis. FIG. 5 is a diagram showing an example of the configuration of a conventional apparatus 2 performing low-pressure polymer solution filling. FIG. 6 is a diagram showing an example of the configuration of a conventional apparatus 2 performing sample field injection. FIG. 7 is a diagram showing an example of the configuration of a capillary electrophoresis apparatus according to improved example 1 performing atmospheric pressure electrophoresis. FIG. 8 is a diagram showing an example of the configuration of a capillary electrophoresis apparatus (embodiment 1) according to improved example 2 performing atmospheric pressure electrophoresis. FIG. 9 is a diagram showing an example of the configuration of a capillary electrophoresis apparatus (embodiment 1) according to improved example 2 performing high-pressure polymer solution filling. FIG. 10 is a diagram showing an example of the configuration of a capillary electrophoresis apparatus (embodiment 1) according to improved example 2 performing sample pressure injection. FIG. 11 is a diagram showing an example of the configuration of a capillary electrophoresis apparatus (embodiment 2) according to improved example 3 performing atmospheric pressure electrophoresis. FIG. 12 is a diagram showing an example of the configuration of a capillary electrophoresis apparatus (embodiment 2) according to improved example 3 performing high-pressure polymer solution filling. FIG. 1 is a diagram showing an example of the configuration of a capillary electrophoresis apparatus (Example 2) according to Improvement Example 3 in which sample pressure injection is performed. FIG. 2 is a diagram showing an example of the configuration of a capillary electrophoresis apparatus (Example 2) according to Improvement Example 3 in which double-ended pressure electrophoresis is performed. FIG. 3 is a diagram showing an example of the configuration of a capillary electrophoresis apparatus (Example 3) according to Improvement Example 4 in which high-pressure polymer solution filling is performed. FIG. 4 is a diagram showing an example of the configuration of a capillary electrophoresis apparatus (Example 3) according to Improvement Example 4 in which sample pressure injection is performed. FIG. 5 is a diagram showing an example of the configuration of a capillary electrophoresis apparatus (Example 4) according to Improvement Example 5 in which double-ended pressure electrophoresis is performed. FIG. 6 is a diagram showing an example of the configuration of a capillary electrophoresis apparatus (Example 4) according to Improvement Example 5 in which high-pressure polymer solution filling is performed. FIG. 7 is a diagram showing an example of the configuration of a capillary electrophoresis apparatus (Example 5) according to Improvement Example 6 in which double-ended pressure electrophoresis is performed. FIG. 8 is a diagram showing an example of the configuration of a capillary electrophoresis apparatus (Example 6) according to Improvement Example 7 in which double-ended pressure electrophoresis is performed.29 is a diagram showing an example of the configuration of a capillary electrophoresis apparatus (Example 7) according to Improvement Example 8 in which double-ended pressure electrophoresis is performed. FIG. 30 is a diagram showing an example of the configuration of a capillary electrophoresis apparatus (Example 7) according to Improvement Example 8 in which high-pressure polymer solution filling is performed. FIG. 31 is a diagram showing an example of the configuration of a capillary electrophoresis apparatus (Example 7) according to Improvement Example 8 in which sample pressure injection is performed. FIG. 32 is a diagram showing an example of the configuration of a capillary electrophoresis apparatus (Example 8) according to Improvement Example 9 in which double-ended pressure electrophoresis is performed. FIG. 33 is a diagram showing an example of the configuration of a capillary electrophoresis apparatus (Example 8) according to Improvement Example 9 in which high-pressure polymer solution filling is performed. FIG. 34 is a diagram showing an example of the configuration of a capillary electrophoresis apparatus (Example 8) according to Improvement Example 9 in which sample pressure injection is performed. FIG. 35 is a diagram showing an example of the configuration of a capillary electrophoresis apparatus (Example 9) according to Improvement Example 10 in which double-ended pressure electrophoresis is performed. FIG. 36 is an electropherogram obtained by field injection using a capillary electrophoresis apparatus (Example 2) according to Improvement Example 3. FIG. 37 is a diagram showing the relationship between field injection time and peak area in FIG. 38. FIG. 39 is an electropherogram obtained by pressure injection using Improvement Example 3 (Example 2). Fig. 32 is a diagram showing the relationship between pressure injection time and peak area in Fig. 31. Fig. 33 is a diagram showing the process of capillary electrophoresis analysis using electric field injection. Fig. 34 is a diagram showing the process of capillary electrophoresis analysis using pressure injection. Fig. 35 is a diagram showing the process of capillary electrophoresis analysis using pressure injection and double-ended pressure electrophoresis.

[0021] The configuration of the capillary electrophoresis apparatus and the characteristics of the analytical method of the present invention (improved example) will be explained in comparison with the conventional method. For simplicity, the following description will mainly focus on a capillary electrophoresis apparatus using a single capillary, but this method may also be applied to a multi-capillary electrophoresis apparatus using multiple capillaries. In the following embodiments, multiple apparatuses with different configurations will be referred to as Apparatus A, Apparatus B, etc., and each will be explained (however, Apparatus A, B, and C are also described in the background art above). Furthermore, since the following description deals with multiple different apparatuses, the multiple apparatuses with different configurations will be referred to as Conventional Example 1, Conventional Example 2, etc. for the conventional method (conventional example), and as Improved Example 1, Improved Example 2, etc. for the new method (improved example), and will be explained in detail using figures. However, the correspondence between them will be noted in each case.

[0022] 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 capillary temperature to 60°C.

[0023] As shown in the upper left corner of each figure in this specification, a right-handed XYZ Cartesian coordinate system is defined in each figure. That is, in each figure, the X axis is taken horizontally, with the right 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 assigned the same reference numerals.

[0024] In this specification, the liquid system is defined as the entire set of liquids (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 series of liquids (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).

[0025] <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 the analytical method using it. A single capillary 1 is positioned with its sample injection end 2 on the right and its sample elution end 3 on the left, 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 electrophoresis (atmospheric pressure electrophoresis) being 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 polymer solution 18 (POP-7). The sample then electrophoreses through the capillary toward the sample elution end 3, electrophoretically separating the multiple different components contained in the sample based on their charge and size. The sample injection end 2 is inserted into the pipe-shaped negative electrode 4, integrating the two. A cathode buffer solution reservoir 8 containing a cathode buffer solution 6 and a sample solution reservoir 11 containing a sample solution 10 are fixed on a cathode stage 12, which is further connected to an XYZ-axis drive mechanism (not shown). Of course, other containers, such as multiple sample solution reservoirs containing different samples, are also fixed on the cathode stage 12, but for simplicity, these are not shown. The sample injection end 2 equipped with a cathode 4 is inserted into the cathode buffer solution reservoir 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 channel is formed inside the T-block 15, and the channel 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 (each is connected using a connector, not shown). Each connection is sealed to prevent leakage of the contents even if the pressure of the polymer solution inside increases (it has pressure-resistant properties). The anode buffer solution tank 9, which stores the anode buffer solution 7, 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 opens, and when the plunger part moves down (-Z-axis direction), the polymer solution valve 20 closes.

[0026] Although Figure 1 shows only the plunger portion of the polymer solution valve 20, the polymer solution valve 20 actually includes a solenoid mechanism (not shown) for moving the plunger portion up and down and a connection mechanism (not shown) for interlocking the solenoid mechanism with the plunger portion. 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 supply 21 via an electric wire 22. A voltage is applied between the anode 5 and cathode 4, i.e., 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 air (i.e., the liquid level of the cathode buffer solution 6) and the interface 102 between the anode buffer solution 7 and air (i.e., the liquid level of the anode buffer solution 7) are aligned. For example, the difference in height between the two liquid levels should be 1 mm or less. This prevents the polymer solution 18 inside the capillary 1 from moving due to gravity during electrophoresis. A position on the capillary 1 that is a certain distance from the sample injection end 2 after electrophoresis 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 fluorescence emitted from the detection position is detected by a fluorescence detection device (not shown).

[0027] FIG. 2 shows the state in which the capillary 1 is being filled with a polymer solution 18 at high pressure in a capillary electrophoresis apparatus according to Conventional Example 1. This filling also allows the polymer solution 18 already filled in the capillary 1 to be replaced 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 using a motor, applying 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 a sealed space, and this sealed space contains almost no air. The sample elution end 3 is immersed in this liquid system. In this state, the solid plunger 17 directly pressurizes the liquid polymer solution 18 at the interface 104 with the polymer solution 18, thereby achieving a high pressure of approximately 70 atmospheres. 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, thereby obtaining a similar high pressure. The high pressure is transmitted to the entire liquid system including the sample elution end 3.

[0028] In each figure in this specification, polymer solutions under atmospheric pressure are indicated by vertical dotted lines, and polymer solutions under high pressure are indicated by checkered patterns. Therefore, in Figure 1, the liquid system under atmospheric pressure is indicated by vertical dotted lines, and in Figure 2, the liquid system under high pressure is indicated by checkered patterns. Because the sample injection end 2 is under atmospheric pressure, a high pressure difference is created between the sample elution end 3 and the sample injection end 2, and the polymer solution 18 fills 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, filling the capillary 1 with the polymer solution 18 at high pressure. After filling the capillary 1 with the polymer solution, the mechanical pushing of the plunger 17 is stopped, and the plunger 17 and the mechanical pushing mechanism are separated, reducing the pressure applied to the liquid system to almost zero. However, in reality, some pressure may remain. Then, the polymer solution valve 20 is opened to release the sample elution end 3 and the above liquid system to atmospheric pressure.

[0029] Figure 3 shows the state in which a sample is being electrochemically injected into a capillary 1 in Conventional Example 1. The cathode stage 12 is moved by an XYZ-axis drive mechanism, and the sample injection tip 2 is inserted into a sample solution reservoir 11 and immersed in a sample solution 10. In this state, a voltage is applied between the sample elution tip 3 and the sample injection tip 2 of the capillary 1 for a certain period of time, thereby electrochemically injecting the sample into the capillary 1 through the sample injection tip 2. In this case, 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) do not necessarily need to be aligned. If these heights are misaligned, the polymer solution 18 inside the capillary 1 may move due to gravity, but this effect is small if the electrochemical injection time is short.

[0030] After this, the cathode stage 12 is moved by its XYZ-axis drive mechanism to insert the sample injection tip 2 into the cathode buffer solution tank 8 and immerse it in the cathode buffer solution 6. Returning to the state shown in Figure 1, a voltage is applied between the sample elution tip 3 and the sample injection tip 2, and electrophoresis (atmospheric pressure electrophoresis) is performed with both ends of the capillary 1 under atmospheric pressure. The vertical heights (Z coordinates) of the interface 101 between the cathode buffer solution 6 and air (i.e., the liquid level of the cathode buffer solution 6) and the interface 102 between the anode buffer solution 7 and 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.

[0031] As described above, the capillary electrophoresis apparatus according to Conventional Example 1 does not have an air pressure device, and therefore cannot perform pressure injection of a sample, and cannot perform pressure application on both ends during electrophoresis.

[0032] 4 to 6 show the configuration of a capillary electrophoresis apparatus (corresponding to apparatus B) according to conventional example 2 and an analytical method using the same. Hereinafter, we will omit some of the explanations that overlap with conventional example 1, and focus on the differences from conventional example 1. An air-operated pressurizing mechanism is provided on the cathode side (sample injection end), and another air-operated pressurizing mechanism is provided on the anode side (sample elution end).

[0033] Figure 4 shows the state in which electrophoresis (double-ended pressure electrophoresis) is being performed in the capillary electrophoresis apparatus 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 reservoir 8 containing a cathode buffer solution 6 and a sample solution reservoir 11 containing a sample solution 10 are fixed on the cathode stage 12, which is further connected to an XYZ-axis drive mechanism (not shown). Of course, other containers may also be fixed on the cathode stage 12, such as multiple sample solution reservoirs containing different samples, a polymer solution reservoir containing a polymer solution, or a waste liquid reservoir containing waste liquid, but these are not shown for simplicity.

[0034] O-rings 33 are placed on the edges of the upper ends of the cathode buffer solution reservoir 8 and the sample solution reservoir 11. The fixing block 31 is parallel to the XY plane and has a flat surface facing the -Z direction. By moving the cathode stage 12 using the XYZ-axis drive mechanism, the sample injection tip 2 together with the cathode 4 is inserted into the cathode buffer solution reservoir 8 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 fixing block 31 and compressed, sealing the cathode buffer solution reservoir 8.

[0035] 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.

[0036] As shown in Figure 4, when the cathode pressure valve 28 is opened and the cathode release valve 29 is closed, air 34 is connected to the compressed air source 25, and compressed air flows into the cathode buffer solution tank 8 via the air tube 30. 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 indicated by a blank space, and air under low pressure is indicated by a dotted space. Furthermore, buffer solution under atmospheric pressure is indicated by a dotted horizontal line, and buffer solution under low pressure is indicated by a solid horizontal line. Therefore, in Figure 4, air 34 under low pressure is indicated by a dotted space, and in Figure 5, air 34 under atmospheric pressure is indicated by a blank space. Furthermore, cathode buffer solution 6 under low pressure is indicated by a solid horizontal line in Figure 4, and cathode buffer solution 6 under atmospheric pressure is indicated by a dotted horizontal line in Figure 5. These patterns are also used in other drawings.

[0037] The anode side has a similar device configuration to the cathode side. The sample elution tip 3 is inserted into the pipe-shaped anode 5, and the two are integrated. An anode buffer solution tank 9 containing an anode buffer solution 7 and a polymer solution tank 32 containing a polymer solution 18 are fixed on 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 tank containing waste liquid, can also be fixed on the anode stage 13, but for simplicity, these are not shown in the figure.

[0038] O-rings 33 are placed on the edges of the upper ends of the anode buffer solution reservoir 9 and the polymer solution reservoir 32. The fixed block 31 is parallel to the XY plane and has a flat surface facing the -Z direction. By moving the cathode stage 13 using the XYZ-axis drive mechanism, the sample elution tip 3 together with the anode 5 is inserted into the anode buffer solution reservoir 9 and immersed in the anode buffer solution 7. At the same time, by moving the anode stage 13 in the +Z direction, the O-ring 33 is pressed against the flat surface of the fixed block 31 and compressed, sealing the anode buffer solution reservoir 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 reservoir 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 anode pressure valve 26 and the anode release valve 27 via the air tube 30.

[0039] As shown in Figure 4, when anode pressure valve 26 is opened and anode release valve 27 is closed, air 35 is connected to compressed air source 25, and compressed air flows into anode buffer solution tank 9 via air tube 30. The pressure of air 35 rises until it becomes equal to the pressure of the compressed air discharged from compressed air source 25. At the same time, the pressure of anode buffer solution 7 contained in anode buffer solution tank 9 similarly rises until it becomes equal to the pressure of the compressed air discharged from compressed air source 25. As with the cathode side, air 35 under low pressure is shown with a dotted pattern in Figure 4, and air 35 under atmospheric pressure is shown with a blank pattern in Figure 5. Furthermore, anode buffer solution 7 under low pressure is shown with a horizontal solid line in Figure 4, and anode buffer solution 7 under atmospheric pressure is shown with a horizontal dotted line in Figure 5.

[0040] 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 the sample injection end 2 and the sample elution end 3 of the capillary 1. In this state, a voltage is applied between the anode electrode 5 and the cathode electrode 4, and a voltage is applied to both the sample injection end 2 and the sample elution end 3 of the capillary 1, performing double-ended pressure 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. Furthermore, 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. For example, it is recommended that the difference in height between the two liquid levels be 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.

[0041] 5 shows the state in which the capillary 1 is being filled with the polymer solution 18 at low pressure in the capillary electrophoresis apparatus of Conventional Example 2. The sample elution tip 3 is inserted into the polymer solution tank 32 together with the anode 5 by moving the anode stage 13 using the XYZ-axis drive mechanism. The sample elution tip 3 is immersed in the polymer solution 18, and 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. This increases the pressure of the polymer solution 18 in the polymer solution tank 32 and the air 36 in contact with it to 5 atmospheres.

[0042] In Figure 5, air 36 under a low pressure of 5 atmospheres is shown with a dotted pattern. In addition, in each figure in this specification, a polymer solution under atmospheric pressure is shown with a vertical dotted line pattern, and a polymer solution under low pressure is shown with a vertical solid line pattern. Therefore, in Figure 5, a polymer solution 18 under low pressure is shown with a vertical solid line pattern. These patterns are also used in other figures.

[0043] 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 atmospheres). The air 34 is shown with a blank pattern, and the cathode buffer solution 6 is shown with a horizontal dotted line pattern, indicating that it is at atmospheric pressure. As a result, a low pressure difference of 5 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 at low pressure 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 release part of the air 36 inside the anode buffer solution tank 9 into the atmosphere via the air tube 30, and the pressure of the polymer solution 18 and the air 36 is set to atmospheric pressure.

[0044] Figure 6 shows the state in which a sample is pressure-injected into a capillary 1 in a capillary electrophoresis apparatus according to Conventional Example 2. The anode stage 13 is moved by the XYZ-axis drive mechanism to insert the sample elution tip 3 into the anode buffer solution reservoir 9 and immerse it in the anode buffer solution 7. The pressure of the anode buffer solution 7 and air 32 inside the anode buffer solution reservoir 9 is atmospheric pressure. Meanwhile, the cathode stage 12 is moved by the XYZ-axis drive mechanism to insert the sample injection tip 2 into the sample solution reservoir 11 and immerse it 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 reservoir 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 reservoir 11 and the air 37 in contact with it rises to 1 atmosphere.

[0045] In each figure in this specification, sample solutions under atmospheric pressure are indicated by horizontal dotted lines, and sample solutions under low pressure are indicated by horizontal solid lines. Therefore, in Figure 6, the sample solution 10 under low pressure is indicated by a horizontal solid line. These patterns are also used in other figures. Air 37, which is also under low pressure, is indicated by a dotted pattern. As a result, a low pressure difference of 1 atmosphere is created between the sample injection port 2 and the sample elution port 3, and the sample solution 10 is injected into the capillary 1 from the sample injection port 2. After a predetermined time has passed, the cathode pressure valve 28 is closed and the cathode release valve 29 is opened, releasing the pressure of the sample solution 10 and air 37 inside the cathode sample solution reservoir 11 to atmospheric pressure. In this state, a voltage can be applied between the sample elution port 3 and the sample injection port 2 to perform electric field injection of the sample.

[0046] After this, the XYZ-axis drive mechanism of the cathode stage 12 moves the sample injection tip 2 into the cathode buffer solution reservoir 8, immersing it in the anode buffer solution 6 while simultaneously sealing the cathode buffer solution reservoir 8. Opening the anode pressure valve 26, closing the anode release valve 27, opening the cathode pressure valve 28, and closing the cathode release valve 29 pressurizes the cathode buffer solution 7 and air 35 in the anode buffer solution reservoir 9, and the anode buffer solution 6 and air 34 in the cathode buffer solution reservoir 8, to 1 atmosphere, returning to the state shown in Figure 4. Because the compressed air introduced into the anode buffer solution reservoir 9 and the cathode buffer solution reservoir 8 comes from the same compressed air source 25, the pressures inside the anode buffer solution reservoir 9 and the cathode buffer solution reservoir 8 are strictly equal. In this state, a voltage is applied between the sample elution tip 3 and the sample injection tip 2, and double-ended pressure 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. Furthermore, 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.

[0047] 7 shows an example of the configuration of a capillary electrophoresis apparatus (corresponding to apparatus D) according to improved example 1. Improved example 1 is configured by adding modifications to conventional example 2. However, as a result of investigation, it has been found that improved example 1, like apparatus C of Patent Document 1, is unable to achieve any of the objects of the present invention.

[0048] Improved Example 1 is equipped with an air-operated 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 a capillary electrophoresis device based on Improved Example 1. The cathode side configuration 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 open, releasing the inside of the cathode buffer solution tank 8 to atmospheric pressure. As with Conventional Example 2, the sample elution end 3 is inserted into the pipe-shaped anode 5, and the two are integrated. A pressure-resistant syringe 16 containing the polymer solution 18 is connected to a polymer solution tank 32 that also contains the polymer solution 18 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. At the same time, 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 removed.

[0049] The polymer solution reservoir 32 and the pressure-resistant syringe 16 form a single internal space, which is filled with the polymer solution 18 and forms 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. Furthermore, by releasing the cathode buffer solution 6 and air 34 inside the cathode buffer solution reservoir 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 reservoir 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).

[0050] However, it was found that even if the sample injection end 2 is inserted into the sample solution reservoir 11, the sample solution reservoir 11 is sealed, and the internal pressure is increased to 1 atmosphere after the mechanical pushing of the plunger 17 is stopped, as in Conventional Example 2, the sample is not pressure-injected. This is because the sealed structure of the polymer solution reservoir 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 between the two 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.

[0051] 8 to 10 show an example of the configuration of a capillary electrophoresis device according to Improved Example 2 (Embodiment 1) and an analytical method using the same. Improved Example 2 (Embodiment 1) is configured by adding modifications to Conventional Example 2, and is configured to solve the problems of Improved Example 1.

[0052] The capillary electrophoresis apparatus according to Improved Example 2 (Example 1) is equipped with an air-operated pressure mechanism on the cathode side (sample injection end) and a plunger-operated pressure mechanism on the anode side (sample elution end). Figure 8 shows the state in which atmospheric pressure electrophoresis is being performed in Improved Example 2 (Example 1). The cathode side configuration is the same as that of Conventional Example 2. However, unlike Figure 4, the cathode pressure valve 28 is closed and the cathode release valve 29 is open, releasing 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 anode buffer solution tank 9 containing the anode buffer solution 7 and the polymer solution tank 32, which is connected to a 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 an XYZ-axis drive mechanism (not shown).

[0053] Although no O-ring 33 is installed on the upper edge of the anode buffer solution reservoir 9, an O-ring 33 is installed on the upper end of the polymer solution reservoir 32. The cathode stage 13 is moved by the XYZ-axis drive mechanism to insert the sample elution end 3 into the anode buffer solution reservoir 9 and immerse it in the anode buffer solution 7. Unlike Conventional Example 2 and Improved Example 1, the anode buffer solution reservoir 9 is not sealed, and the anode buffer solution 7 inside is exposed 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 coordinates) of the interface 101 between the cathode buffer solution 6 and air (i.e., the liquid level of the cathode buffer solution 6) and the interface 102 between the anode buffer solution 7 and air (i.e., the liquid level of the anode buffer solution 7) are aligned. For example, the difference in height between the two liquid levels should be 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.

[0054] 9 shows the state in which the capillary 1 is being filled with the polymer solution 18 at high pressure in a capillary electrophoresis apparatus according to Improved Example 2 (Example 1). The anode stage 13 is moved by the XYZ-axis drive mechanism to insert the sample elution end 3 into the polymer solution reservoir 32 and immerse it 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 reservoir 32. The polymer solution reservoir 32 and the pressure-resistant syringe 16 are integrated and form a single internal space. This internal space is filled with the polymer solution 18 and contains almost no air. While the cathode buffer solution 6 and air 34 in the cathode buffer solution reservoir 8 are released to atmospheric pressure, the plunger 17 of the pressure-resistant syringe 16 is mechanically pressed in, thereby increasing the pressure of the polymer solution 18 in the polymer solution reservoir 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, and the plunger 17 and the mechanical pushing mechanism are separated, reducing the pressure applied to the polymer solution 18 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, thereby releasing the compression of the O-ring and releasing the polymer solution 18 to atmospheric pressure.

[0055] 10 shows the state in which a sample is being pressure-injected into the capillary 1 in the capillary electrophoresis apparatus according to Improved Example 2 (Example 1). By moving the anode stage 13 using the XYZ-axis drive mechanism, the sample elution end 3 is inserted into the anode buffer solution tank 9 and immersed in the anode buffer solution 7. The pressure of the anode buffer solution 7 inside the anode buffer solution tank 9 is atmospheric pressure.

[0056] Meanwhile, the XYZ-axis drive mechanism of the cathode stage 12 moves the sample injection tip 2 into the sample solution reservoir 11, immersing it 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 reservoir 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 reservoir 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 tip 2 and the sample elution tip 3, and the sample solution 10 is injected from the sample injection tip 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, releasing the pressure of the sample solution 10 and air 37 inside the cathode sample solution reservoir 11 to atmospheric pressure.

[0057] After this, the cathode stage 12 is moved by the XYZ-axis drive mechanism to insert the sample injection end 2 into the cathode buffer solution reservoir 8 and immerse it in the anode buffer solution 6, while simultaneously sealing the cathode buffer solution reservoir 8. However, by closing the cathode pressure valve 28 and opening the cathode release valve 29, the inside of the cathode buffer solution reservoir 8 is released to atmospheric pressure, returning to the state shown in Figure 8. In this state, a voltage is applied between the sample elution end 3 and the sample injection end 2, and atmospheric pressure electrophoresis is performed.

[0058] Therefore, Improved Example 2 (Embodiment 1) achieves the first object of the present invention, which is to provide a capillary electrophoresis device that (1) "enables filling of a polymer solution using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis within a practical time" and (2) "enables pressure injection of a sample." However, Improved Example 2 (Embodiment 1) does not achieve the second and third objects of the present invention.

[0059] In the second improved example (first embodiment), not only pressure injection of the sample but also electric field injection is possible. In Fig. 10, the cathode pressure valve 28 is closed and the cathode release valve 29 is opened, releasing the sample solution 10 in the sample solution reservoir 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.

[0060] 11 to 13 show an example of the configuration of a capillary electrophoresis apparatus (corresponding to apparatus F) according to Improved Example 3 (Example 2). Improved Example 3 (Example 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.

[0061] The capillary electrophoresis device according to Improved Example 3 (Example 2) 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 11 shows the state in which atmospheric pressure electrophoresis is being performed in the capillary electrophoresis device according to Improved Example 3 (Example 2). The cathode side configuration is the same as that of Conventional Example 2. However, the cathode pressure valve 28 is closed and the cathode release valve 29 is opened to release atmospheric pressure inside the cathode buffer solution tank 8. 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 polymer solution 18 using a connector 14 and immersed in the polymer solution 18. An inverted T-shaped channel is formed inside the T-block 15, and the channel is filled with 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 have to be flexible.

[0062] To apply high pressure to the polymer solution 18 inside, a high-pressure-resistant material must be used. Materials such as Teflon (registered trademark) resin and PEEK can be used. Alternatively, a flow path formed in an acrylic block, similar to the T-block 15, can be used. Each connection is sealed to prevent leakage of the contents even if the pressure of the internal polymer solution increases. The anode buffer solution tank 9, which contains the anode buffer solution 7, 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 that moves up and down (in the Z-axis direction). When the plunger moves up (in the +Z-axis direction), the polymer solution valve 20 opens, and when the plunger moves down (in the -Z-axis direction), the polymer solution valve 20 closes.

[0063] In Figure 11, the polymer solution valve 20 is open. While Figure 11 shows only the plunger portion of the polymer solution valve 20, the polymer solution valve 20 actually includes a solenoid mechanism (not shown) for moving the plunger portion up and down and a connection mechanism (not shown) for interlocking the solenoid mechanism with the plunger portion. The 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 supply 21 via an electric wire 22. A voltage is applied between the anode 5 and cathode 4, i.e., 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 coordinates) of the interface 101 between the cathode buffer solution 6 and air (i.e., the liquid level of the cathode buffer solution 6) and the interface 102 between the anode buffer solution 7 and air (i.e., the liquid level of the anode buffer solution 7) are aligned. For example, the difference in height between the two liquid levels should be 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.

[0064] FIG. 12 shows a state in which the capillary 1 is being filled with a polymer solution 18 under high pressure in a capillary electrophoresis apparatus according to Improved Example 3 (Embodiment 2). This filling also allows the polymer solution 18 already filled in the capillary 1 to be replaced 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 using a motor, applying 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, filling a sealed space with almost no air. The sample elution end 3 is immersed in this liquid system. In this state, the solid plunger 17 directly compresses the liquid polymer solution 18 at the interface 104 with the polymer solution 18, thereby achieving a high pressure of approximately 70 atmospheres.

[0065] 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, thereby achieving a similar high pressure. This high pressure is transmitted to the entire liquid system, including the sample elution end 3 (the liquid system under high pressure is indicated by a checkered pattern in Figure 12). Because the sample injection end 2 is under atmospheric pressure, a high pressure difference is created 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, filling the capillary 1 with the polymer solution 18 at high pressure. After filling the capillary 1 with the polymer solution, the mechanical pushing of the plunger 17 is stopped, and the plunger 17 and the mechanical pushing mechanism are separated, reducing the pressure applied to the liquid system to almost zero. The polymer solution valve 20 is opened to expose the sample elution end 3 and the above liquid system to atmospheric pressure.

[0066] 13 shows a state in which a sample is pressure-injected into the capillary 1 in a capillary electrophoresis apparatus according to Improved Example 3 (Example 2). The XYZ-axis drive mechanism of the cathode stage 12 moves the sample injection tip 2 into the sample solution reservoir 11 and immerses it in the sample solution 10. At the same time, the O-ring 33 is pressed against the fixed block 31, compressing it and sealing the sample solution reservoir 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 reservoir 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 tip 2 and the sample elution tip 3, and the sample solution 10 is injected into the capillary 1 from the sample injection tip 2. 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.

[0067] After this, the cathode stage 12 is moved by the XYZ-axis drive mechanism to insert the sample injection end 2 into the cathode buffer solution reservoir 8 and immerse it in the anode buffer solution 6, while simultaneously sealing the cathode buffer solution reservoir 8. However, when the cathode pressure valve 28 is closed and the cathode release valve 29 is opened to release the inside of the cathode buffer solution reservoir 8 to atmospheric pressure, the state returns to that shown in Figure 11. In this state, a voltage is applied between the sample elution end 3 and the sample injection end 2, and atmospheric pressure electrophoresis is performed.

[0068] Therefore, Improved Example 3 (Embodiment 2) achieves the first object of the present invention, which is to provide a capillary electrophoresis device that (1) "enables filling of a polymer solution using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis within a practical time" and (2) "enables pressure injection of a sample." However, Improved Example 3 (Embodiment 2) does not achieve the second and third objects of the present invention.

[0069] In the improved example 3 (embodiment 2), not only pressure injection of the sample but also electric field injection is possible. In Fig. 13, the cathode pressure valve 28 is closed and the cathode release valve 29 is opened, and the sample solution 10 inside the sample solution reservoir 11 and the air 37 in contact with it are released 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.

[0070] As described above, in the capillary electrophoresis device according to Improved Example 3 (Example 2), the cathode side has the same configuration as the cathode side of Conventional Example 2, while the anode side has the same configuration as the anode side of Conventional Example 1. This combination has not existed in the past. One of the challenges in the configuration of the capillary electrophoresis device according to Improved Example 3 (Example 2) is whether or not the method shown in FIG. 13 allows pressure injection of a sample, similar to the method shown in FIG. 6 of 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, fluid resistance exists 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, preventing efficient sample pressure injection. Therefore, using the device according to the third improved example (embodiment 2), capillary electrophoresis analysis was carried out by electric field injection or pressure injection of the sample, and an experiment was carried out to confirm whether the expected results could be obtained.

[0071] Figure 29 shows four electropherograms obtained by changing the field injection time to 15, 30, 45, and 60 seconds while fixing the field injection voltage at 1.8 kV. The sample solution was analyzed by GeneScan TM 500 ROX TM Dye Size Standard (Thermo Fisher Scientific) was used. TMThe solution was diluted with 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 relationship between field injection time and peak area is linear, passing through the origin, confirming that field injection was performed as expected. Figure 31 shows four electropherograms obtained by fixing the pressure injection pressure at 0.5 atmospheres and varying the pressure injection time to 15, 30, 45, and 60 seconds. The sample solution was the same as above. Figure 32 shows a graph plotting the average peak area of ​​the multiple peaks obtained in each electropherogram in Figure 31 against the field injection time. However, the electropherogram corresponding to the plot for a 90-second field injection time is not shown in Figure 31. The relationship between field injection time and peak area is linear, passing through the origin, confirming that field injection was performed as expected.

[0072] From the above, it was found that according to the improved example 3 (embodiment 2), electrophoretic analysis can be performed satisfactorily without any problems whether electric field injection of the sample or pressure injection of the sample is used.

[0073] Figure 33 shows in detail an example of the steps in a single capillary electrophoresis analysis using field injection. Figure 34 shows in detail an example of the steps in a single capillary electrophoresis analysis using pressure injection. The leftmost column lists the step numbers, and each analysis proceeds in numerical order. The next column lists the functions, such as "polymer solution filling," "pre-electrophoresis," "field injection," "pressure injection," and "main electrophoresis," with the corresponding rows showing details of each step. "Reference" indicates the reference state for the step. "Main electrophoresis" indicates normal electrophoresis. From the third column onwards, the left half of the table shows the state or condition of "contact object," "sealed / open," "pressure," and "potential" for the "sample elution end," and the right half shows the state or condition of "contact object," "sealed / open," "pressure," and "potential" for the "sample injection end." "Contact object" indicates the liquid into which the capillary end is immersed in each step. "Sealed / open" indicates whether the space containing the liquid system into which the capillary end is immersed is sealed or open to the atmosphere in each step. "Pressure" indicates the pressure applied to the capillary end in each step as either "-", "low pressure", or "high pressure". "-" means atmospheric pressure, i.e., 0 atmospheres. "Low pressure" means pressure greater than 0 atmospheres but less than 7 atmospheres. "High pressure" means pressure greater than 7 atmospheres. "Potential" indicates the potential applied to the capillary end in each step as either "-", "positive potential", or "negative potential". "-" at both ends of the capillary means that no voltage is applied between the two ends, while "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 conditions as the line above.

[0074] An example of the steps in capillary electrophoresis analysis using field injection in Figure 33 will be explained in order. With some exceptions, the process basically follows Figures 11 to 13. The sample elution end is connected to a T-block filled with polymer solution, so the polymer solution is always the object of contact. 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.

[0075] Step 2: The cathode stage is moved, and the sample injection tip is inserted into the waste tank and immersed in the waste liquid (water) (the waste liquid tank for storing the waste liquid is not depicted on the cathode stage in Figure 12).

[0076] Step 3: The polymer solution valve is closed, and the space containing the liquid system (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.

[0077] 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 previously analyzed sample, 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.)

[0078] 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 almost zero, and the filling of the polymer solution is completed.

[0079] Step 6: Open the polymer solution valve, and open the space containing 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 to the atmosphere. Step 7: Move the cathode stage, insert the sample injection end into the cathode buffer solution reservoir, immerse it in the cathode buffer solution, and return to the reference state. Step 8: Apply voltage between the sample elution end and the sample injection end to perform a preliminary run. Step 9: Stop applying voltage, end the preliminary run, and return to the reference state. Step 10: Move the cathode stage, insert the sample injection end into the sample solution reservoir, and immerse it in the sample solution. Step 11: Apply voltage between the sample elution end and the sample injection end to perform field injection of the sample. Step 12: Stop applying voltage, end field injection. Step 13: Move the cathode stage, insert the sample injection end into the cathode buffer solution reservoir, immerse it in the cathode buffer solution, and return to the reference state. Step 14: Apply voltage between the sample elution end and the sample injection end to perform the main electrophoresis. Step 15: Stop applying voltage, end the main electrophoresis, and return to the reference. If multiple capillary electrophoresis analyses are to be performed, simply repeat steps 1 to 15 multiple times.

[0080] It is preferable to perform the above steps 1 to 15 in this order. However, a single unit of capillary electrophoresis analysis may be, for example, performed by performing steps 7 to 15, followed by steps 1 to 7. Even in such a case, performing multiple capillary electrophoresis analyses always includes performing steps 1 to 15 in order. Among steps 1 to 15, the key features are step 4 (filling with polymer solution) and step 11 (electric field injection). That is, the present invention is characterized by the following: step A (step 4) in which the sample injection end is exposed to atmospheric pressure, and while the polymer solution of the separation medium is in contact with the sample elution end, a solid is pressed against the polymer solution to apply high pressure (a pressure greater than 7 atmospheres) to the polymer solution, thereby filling the capillary with a portion of the polymer solution; and step B (step 11) in which, while the sample solution is in contact with the sample injection end, a voltage is applied between the sample elution end and the sample injection end, and a portion of the sample solution is injected into the capillary. Alternatively, the present invention is characterized in that the analysis includes a step of carrying out step A and step B in that order.

[0081] An example of the steps of capillary electrophoresis analysis using pressure injection in Figure 34 will be explained in order. With some exceptions, the process basically follows Figures 11 to 13. Steps 1 to 10 are the same as steps 1 to 10 in Figure 33, so their explanation will be omitted. Step 11: The space (the space inside the sample solution tank) containing the liquid system (sample solution) that comes into contact with the sample injection end is sealed from the atmosphere. Step 12: Compressed air is introduced into the space containing 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 containing 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 Figure 33, so their explanation will be omitted.

[0082] 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 this order. However, a single capillary electrophoresis analysis unit may be configured to perform, for example, steps 7 to 17 and then steps 1 to 7. Even in such a case, when performing capillary electrophoresis analysis multiple times, steps 1 to 17 will always be included.

[0083] Among steps 1 to 17, the key features are step 4, filling with the polymer solution, and step 12, pressure injection. That is, the present invention is characterized by the following steps: step A (step 4), in which the sample injection end is exposed to atmospheric pressure, and while the polymer solution of the separation medium is in contact with the sample elution end, a solid is pressed against the polymer solution to apply high pressure (a pressure of more than 7 atmospheres) to the polymer solution, thereby filling a portion of the polymer solution into the capillary; and step B (step 12), in which while the sample solution is in contact with the sample injection end, compressed air is brought into contact with the sample solution to apply low pressure (a pressure of 0 to 7 atmospheres) to the sample solution, thereby injecting a portion of the sample solution into the capillary. Alternatively, the present invention is characterized by the inclusion of a step in which step A and step B are carried out in this order during analysis.

[0084] 14 to 16 show the configuration of a capillary electrophoresis device according to Improved Example 4 (Embodiment 3) and an analytical method using the same. Improved Example 4 (Embodiment 3) is configured by modifying Improved Example 2 (Embodiment 1: Figures 8 to 10) and is configured to solve the problems of Improved Example 2 (Embodiment 1).

[0085] The capillary electrophoresis apparatus according to the fourth improved embodiment (third embodiment) is equipped with an air pressure mechanism on the cathode side (sample injection end) and a plunger pressure mechanism and an air pressure mechanism on the anode side (sample elution end). Figure 14 shows the state in which double-ended pressure electrophoresis is being performed in the capillary electrophoresis apparatus according to the fourth improved embodiment (third embodiment).

[0086] The cathode side configuration is the same as in Improved Example 2 (Example 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, so that the cathode buffer solution tank 8 is pressurized to 1 atmosphere. Regarding the anode side configuration, the polymer solution tank 32, to which the pressure-resistant syringe 16 containing the polymer solution 18 is connected, is the same as in Improved Example 2 (Example 1). The anode buffer solution tank 9, to which the anode buffer solution 7 is stored, and the fixing block 31 are configured the same as in Conventional Example 2 (Figures 4 to 6). The anode buffer solution tank 9, to which the anode buffer solution 7 is stored, 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). O-rings 33 are installed at the top ends of the anode buffer solution tank 9 and the polymer solution tank 32, respectively. The anode stage 13 is moved by the XYZ-axis drive mechanism, inserting the sample elution end 3 together with the anode 5 into the anode buffer solution tank 9 and immersing it in the anode buffer solution 7. At the same time, the anode stage 13 is moved in the +Z-axis direction, pressing the O-ring 33 against the flat surface of the fixed block 31 and compressing it, sealing the anode buffer solution tank 9. The anode pressure valve 26 is opened and the anode release valve 27 is closed, creating a pressurized state of 1 atmosphere inside the anode buffer solution tank 9. 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 enabling stable high separation performance.

[0087] 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 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.

[0088] Although the present specification separately describes the steps of opening the anode pressure valve 26 to introduce compressed air into the sample elution end 3 and the cathode pressure valve 28 to introduce compressed air into the sample injection end 2, it is preferable to perform these steps simultaneously. Furthermore, although the description separately describes the steps of opening the anode release valve 27 to release the sample elution end 3 to atmospheric pressure and the cathode release valve 29 to release the sample injection end 2 to atmospheric pressure, it is preferable to perform these steps simultaneously. If the timing of these steps is not synchronized, a period of time will occur during which a pressure difference will occur between the sample elution end 3 and the sample injection end 2, causing the polymer solution 18 in the capillary 1 to move during this period. To easily synchronize the timing, it is effective to combine the anode pressure valve 26 and the cathode release valve 29 into a single valve, and to combine the anode release valve 27 and the cathode release valve 29 into a single valve. For the same reason, it is also preferable to synchronize the rates of pressure increase and decrease between the sample elution end 3 and the sample injection end 2. For this purpose, it is preferable to make the air flow resistance equal on both sides. Alternatively, it is effective to slowly increase and decrease the pressure of the compressed air discharged from the compressed air source 25, for example, at a rate of 0.5 atmospheres / second or less, or 0.1 atmospheres / second or less.

[0089] 15 shows a state in which the capillary 1 is being filled with the polymer solution 18 at high pressure in a capillary electrophoresis apparatus according to Improved Example 4 (Example 3). By moving the anode stage 13 using the XYZ-axis drive mechanism, the sample elution end 3 is inserted into the polymer solution tank 32 and immersed in the polymer solution 18. At the same time, the O-ring 33 is pressed against the fixing block 31 and compressed, sealing the polymer solution tank 32. At this time, the structures of the O-ring 33 and the fixing block 31 are 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, thereby increasing the pressure of the polymer solution inside the polymer solution tank 32 and syringe 16 to 35 atmospheres, and polymer solution 18 is filled at high pressure into the capillary 1. After the polymer solution is filled, the mechanical pushing of the plunger 17 is stopped, and the plunger 17 and the mechanical pushing mechanism are separated, reducing the pressure applied to the liquid system to almost zero.

[0090] Figure 16 shows the state in which a sample is pressure-injected into the capillary 1 in a capillary electrophoresis apparatus according to Improved Example 4 (Example 3). The anode stage 13 is moved by the XYZ-axis drive mechanism to insert the sample elution tip 3 into the anode buffer solution reservoir 9 and immerse it in the anode buffer solution 7, while simultaneously sealing the anode buffer solution reservoir 9. The anode pressure valve 26 is closed and the anode release valve 27 is opened, and the pressure inside the anode buffer solution reservoir 9 is set to atmospheric pressure. Meanwhile, the cathode stage 12 is moved by the XYZ-axis drive mechanism to insert the sample injection tip 2 into the sample solution reservoir 11 and immerse it in the sample solution 10, while simultaneously sealing the sample solution reservoir 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 reservoir 11 and the air 37 in contact with it rises to 1 atmosphere. As a result of the above, a low pressure difference of 1 atmosphere is created between the sample injection port 2 and the sample elution port 3, and the sample solution 10 is injected into the capillary 1 from the sample injection port 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 reservoir 11 to atmospheric pressure.

[0091] 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 reservoir 8, immersing it in the anode buffer solution 6 while simultaneously sealing the cathode buffer solution reservoir 8. Furthermore, 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, and the inside of the anode buffer solution reservoir 9 and the cathode buffer solution reservoir 8 are pressurized to 1 atmosphere, returning to the state shown in Figure 14. In this state, a voltage is applied between the sample elution end 3 and the sample injection end 2, and double-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, thereby releasing the inside of the anode buffer solution reservoir 9 and the cathode buffer solution reservoir 8 to atmospheric pressure and applying a voltage between the sample elution end 3 and the sample injection end 2.

[0092] 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 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 a sample." The second objective 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 pressure injection of both ends during electrophoresis." The third objective 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 a sample," and (3) "enables pressure injection of both ends during electrophoresis." All of these objectives can be achieved.

[0093] According to the fourth improvement (third embodiment), not only pressure injection of the sample but also electric field injection is possible. In Fig. 16, the cathode pressure valve 28 is closed and the cathode release valve 29 is opened, releasing the sample solution 10 in the sample solution reservoir 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.

[0094] <Improvement Example 5: Example 4> Figures 17 to 19 show an example of the configuration of a capillary electrophoresis apparatus (corresponding to Apparatus H) according to Improvement Example 5 (Example 4) and an analytical method using the same. Improvement Example 5 (Example 4) is constructed by modifying Improvement Example 3 (Example 2: Figures 11 to 13) and is configured to solve the problems of Improvement Example 3 (Example 2). An air-operated pressurizing mechanism is provided on the cathode side (sample injection end), and a plunger-operated pressurizing mechanism and an air-operated pressurizing mechanism are provided on the anode side (sample elution end). Figure 17 shows the state in which double-ended pressurized electrophoresis is being performed in Improvement Example 5 (Example 4). The configuration on the cathode side is the same as in Improvement 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, creating a pressurized state of 1 atmosphere inside the cathode buffer solution tank 8.

[0095] The anode side configuration 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 below. To provide an air-operated pressurization 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 up and down and a connection mechanism for interlocking the solenoid mechanism with the plunger. In other words, the polymer solution valve 20 protrudes significantly from the anode buffer solution tank 9 and has a movable part. This makes it difficult to seal the anode buffer solution tank 9 using the O-ring 33 and 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 an air tube 30 using an anode pressure valve 26 and an anode release valve 27 .

[0096] As shown in Figure 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. This causes the pressure of the air 35 inside the sealed chamber 38 and the anode buffer solution tank 9 to rise 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 increased to 1 atmosphere. As a result, both 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 enabling stable high separation performance. 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 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.

[0097] 18 shows a state in which the capillary 1 is being filled with the polymer solution 18 at high pressure in a capillary electrophoresis apparatus according to Improved Example 5 (Example 4). The anode pressure valve 26 is closed and the anode release valve 27 is opened, releasing the air 35 inside the sealed chamber 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, releasing 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 pressed in using a motor, applying a high pressure of 35 atmospheres to the polymer solution 18 inside, in which the sample elution end 2 is immersed.

[0098] Because the sample injection port 2 is under atmospheric pressure, a high pressure difference of 35 atmospheres is created between the sample elution port 3 and the sample injection port 2, and the polymer solution 18 is filled into the capillary 1 at a high pressure of 35 atmospheres from the sample elution port 3 toward the sample injection port 2. After the polymer solution is filled, the mechanical pushing of the plunger 17 is stopped, and the plunger 17 and the mechanical pushing mechanism are separated, reducing the pressure applied to the polymer solution 18 to almost zero. The polymer solution valve 20 is opened, and the polymer solution 18 and the sample elution port 3 are released to atmospheric pressure.

[0099] 19 shows a state in which a sample is pressure-injected into the capillary 1 in a capillary electrophoresis apparatus according to Improved Example 5 (Example 4). The XYZ-axis drive mechanism of the cathode stage 12 moves the sample injection tip 2 into the sample solution reservoir 11 and immerses it in the sample solution 10. At the same time, the O-ring 33 is pressed against the fixed block 31, compressing it and sealing the sample solution reservoir 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 reservoir 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 tip 2 and the sample elution tip 3, and the sample solution 10 is injected into the capillary 1 from the sample injection tip 2. 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.

[0100] 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 reservoir 8, immersing it in the anode buffer solution 6 while simultaneously sealing the cathode buffer solution reservoir 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, thereby pressurizing the inside of the sealed chamber 38, the anode buffer solution reservoir 9, and the cathode buffer solution reservoir 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-end 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 insides of the anode buffer solution reservoir 9 and the cathode buffer solution reservoir 8 to atmospheric pressure, and applying a voltage between the sample elution end 3 and the sample injection end 2.

[0101] As described above, according to the fifth improved example (fourth embodiment), the first object of the present invention can be achieved: (1) to provide a capillary electrophoresis device that "enables filling of 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 of the present invention can be achieved: (1) to provide a capillary electrophoresis device that "enables filling of a polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis" and (3) "enables pressure injection of both ends during electrophoresis." The third object of the present invention can be achieved: (1) to provide a capillary electrophoresis device that "enables filling of 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 ...3) to provide a capillary electrophoresis device that "enables filling of a polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis" and (3) "enables pressure injection of a sample."

[0102] According to the fifth improvement (fourth embodiment), not only pressure injection of the sample but also electric field injection is possible. In Fig. 19, the cathode pressure valve 28 is closed and the cathode release valve 29 is opened, releasing the sample solution 10 in the sample solution reservoir 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.

[0103] <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 modifying 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 a plunger-type pressure mechanism and an air-type pressure mechanism on the anode side (sample elution end side).

[0104] 20 shows the state in which double-ended 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.

[0105] In Improved Example 6 (Example 5), an air-operated pressurizing mechanism is provided on the anode side, so in Improved Example 3 (Example 2), the polymer solution valve 20 is removed, and the anode buffer solution tank 9 is sealed using an O-ring 33 and a fixing 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 midway between the anode buffer solution tank 9 and the T-block 15. A 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).

[0106] 20, the compressed air side polymer solution valve 39 is open. Also, the anode buffer solution tank 9 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. However, the structure of the sealed anode buffer tank 9 differs from that of the capillary electrophoresis apparatus 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.

[0107] In contrast, in the capillary electrophoresis apparatus according to Improved Example 6 (Example 5), the sample elution end 3 of the capillary 1 is not inserted into the sealed anode buffer tank 9. Instead, the anode 5 and 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, i.e., 1 atmosphere. As a result, both 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 enabling stable high separation performance. In addition, the vertical height (Z coordinate) of the interface 101 between the cathode buffer solution 6 and air (i.e., the liquid level of the cathode buffer solution 6) and the interface 102 between the anode buffer solution 7 and air (i.e., the liquid level of the anode buffer solution 7) are aligned, thereby preventing the polymer solution 18 inside the capillary 1 from moving due to gravity during electrophoresis.

[0108] Although the first to third objects of the present invention can be achieved with the sixth improved example (fifth embodiment), the following problem has been discovered. In the state shown in FIG. 20 , when a pressure of 1 atmosphere is applied to the inside of the anode buffer solution tank 9, the polymer solution 18 filling 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 may be subjected to a force in a direction that causes it to be pulled out of the pressure-resistant syringe 16, potentially causing the plunger 17 to move in the direction that causes it to be pulled out of the pressure-resistant syringe 16. If the plunger 17 actually moves, the pressure of the polymer solution 18 filling the inside of the polymer solution tube 19, the T-block 15, and the pressure-resistant syringe 16 changes, causing the balance of the pressures at both ends of the capillary 1 to be disrupted. This causes the polymer solution 18 inside the capillary 1 to move, resulting in a decrease in the electrophoretic resolution. Therefore, the first to third objects of the present invention will be achieved by solving the above problem as described below.

[0109] <Improvement Example 7: Example 6> Figure 21 shows an example of the configuration of a capillary electrophoresis device according to Improvement Example 7 (Example 6) (when performing double-ended pressure electrophoresis). Improvement Example 7 (Example 6) is configured by adding modifications to Improvement Example 6 (Example 5: Figure 20), and is configured to solve the problems of Improvement Example 6 (Example 5).

[0110] Improved Example 7 (Example 6) differs from Improved 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 means for mechanically pushing the plunger 17 of the pressure-resistant syringe 16. To maintain the desired pressure of the polymer solution 18 inside the pressure-resistant syringe 16, the drive force in the -X-axis direction of the drive unit of the X-axis drive mechanism can be set to a corresponding constant value (specifically, this is determined by the equation "pressure" = "drive force" ÷ "internal cross-sectional area of ​​the pressure-resistant syringe"). To achieve this, the drive force can be measured using a load sensor or the like, and the operation of the drive unit can be controlled so that the drive force remains constant. Alternatively, the current value driving the stepping motor can be controlled so that the stepping motor loses synchronization when the drive force exceeds a certain value.

[0111] The most basic configuration of the plunger stopper 41 is a state in which 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 while the drive unit is stationary. If the stepping motor is continued to be excited while the drive unit is 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.

[0112] However, connecting the drive unit of the X-axis drive mechanism to the plunger 17 raises the following problem. In Figure 21, the compressed air discharged from the compressed air source 25 is set to 1 atmosphere, and 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, creating a low pressure of 1 atmosphere inside the anode buffer solution tank 9 and the cathode buffer solution tank 8. 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, releasing 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 in the liquid system to a high pressure of 35 atmospheres, thereby filling the capillary 1 with the polymer solution 18. 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.

[0113] However, upon opening the compressed air-side polymer solution valve 39, 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 can lead to waste of the polymer solution 18, the generation of bubbles, and other problems. To avoid these problems, it is necessary to move the drive unit of the X-axis drive mechanism in the +X-axis direction, moving the plunger 17 in a direction that pulls it out of the pressure-resistant syringe 16, thereby lowering the pressure in the liquid system. However, it is unknown how far the drive unit needs to be moved in the +X-axis direction to return the pressure in the liquid system to atmospheric pressure, and this is difficult to predict. Furthermore, even a slight movement distance greater than the optimal value can cause the pressure in the liquid system to become negative.

[0114] To solve the above problem, the drive unit of the X-axis drive mechanism and plunger 17 can be uncoupled so that when the drive unit moves in the −X-axis direction, the drive unit comes into contact with plunger 17 and can push plunger 17 into pressure-resistant syringe 16, while when the drive unit moves in the +X-axis direction, the drive unit separates from plunger 17, preventing the plunger 17 from moving in a direction to be pulled out of 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 state in which polymer solution 18 is filled to a state in which it is not filled. Even when the drive unit separates from plunger 17, some residual pressure may remain in the liquid system. However, if this level of residual pressure is small, the effect of releasing atmospheric pressure by opening compressed air-side polymer solution valve 39 is small.

[0115] 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 fulfill the function of the plunger stopper 41. In this case, as shown in FIG. 21 , a device separate from the X-axis drive mechanism functions as the plunger stopper 41 that directly stops the movement of the plunger 17.

[0116] 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) enables not only pressure injection of the sample but also electric field injection.

[0117] 22 to 24 show the configuration of a capillary electrophoresis apparatus (corresponding to apparatus 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) differs 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 to the plunger-side polymer solution tube 43 midway between the T-block 15 and the pressure-resistant syringe 16. 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 and the polymer solution in the pressure-resistant syringe 16. The capillary electrophoresis apparatus according to the eighth improved example (seventh embodiment) 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).

[0118] Figure 22 shows the state in which double-ended pressurized electrophoresis is being performed in a capillary electrophoresis apparatus according to 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, thereby releasing 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.

[0119] 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, and the inside of the anode buffer tank 9, the polymer solution tube 19, and the T-block 15 are pressurized to 1 atmosphere.

[0120] Meanwhile, the sample injection end 2 is inserted into the cathode buffer solution tank 8 and, while immersed in the cathode buffer solution 6, the cathode buffer solution tank 8 is sealed. The cathode pressure valve 28 is opened and the cathode release valve 29 is closed, pressurizing the inside of the cathode buffer solution tank 8 to 1 atmosphere. This results in the double-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 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 enabling stable high separation performance. In addition, the vertical heights (Z coordinates) of the interface 101 between the cathode buffer solution 6 and air (i.e., the liquid level of the cathode buffer solution 6) and the interface 102 between the anode buffer solution 7 and 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.

[0121] On the other hand, 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 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.

[0122] 23 shows a state in which the polymer solution 18 is being filled into the capillary 1 at high pressure in a capillary electrophoresis apparatus according to Improved Example 8 (Example 7). The anode pressure valve 26 is closed and the anode release valve 27 is opened to release 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 release 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 motor is used to mechanically push the plunger 17 of the pressure-resistant syringe 16, applying a high pressure of 35 atmospheres to the polymer solution 18 in which 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 the polymer solution is filled, the mechanical pushing of the plunger 17 is stopped, the plunger 17 is separated from the mechanical pushing mechanism, 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 released to atmospheric pressure.

[0123] 24 shows a state in which a sample is pressure-injected into the capillary 1 in a capillary electrophoresis apparatus according to Improved Example 8 (Example 7). The cathode stage 12 is moved by the XYZ-axis drive mechanism to insert the sample injection tip 2 into the sample solution reservoir 11 and immerse it in the sample solution 10, while simultaneously sealing the sample solution reservoir 11. 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 reservoir 11 and the air 37 in contact with it rises to 1 atmosphere. This creates a low pressure difference of 1 atmosphere between the sample injection tip 2 and the sample elution tip 3, and the sample solution 10 is injected into the capillary 1 from the sample injection tip 2. 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 reservoir 11 to atmospheric pressure.

[0124] Thereafter, the cathode stage 12 is moved by the XYZ axis drive mechanism to insert the sample injection tip 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.

[0125] Next, 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, pressurizing 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, pressurizing 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, and double-end pressure electrophoresis is performed.

[0126] As described above, according to the eighth improved example (seventh embodiment), the first object of the present invention is to provide a capillary electrophoresis device that (1) "enables polymer solution filling within a practical time using a high-viscosity polymer solution for DNA sequencing and DNA fragment analysis" and (2) "enables sample pressure injection." The second object is to provide a capillary electrophoresis device that (1) "enables polymer solution filling within a practical time using a high-viscosity polymer solution for DNA sequencing and DNA fragment analysis" and (3) "enables both-end pressure application during electrophoresis." The third object is to provide a capillary electrophoresis device that (1) "enables polymer solution filling within a practical time using a high-viscosity polymer solution for DNA sequencing and DNA fragment analysis," (2) "enables sample pressure injection," and (3) "enables both-end pressure application during electrophoresis." Of course, the capillary electrophoresis device according to the eighth improved example (seventh embodiment) can also be used for electric field injection of a sample.

[0127] 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 is the same as in Figures 33 and 34. The steps will be explained in order, basically 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 will be omitted.

[0128] Step 16: The space (the space inside the cathode buffer solution reservoir) containing the liquid system (cathode buffer solution) that the sample injection end contacts is sealed from the atmosphere. At the same time, the space (the space inside the anode buffer solution reservoir, the polymer solution tube, the T-block, and the pressure-resistant syringe) containing the liquid system (anode buffer solution, polymer solution tube, T-block, and pressure-resistant syringe) that the sample elution end contacts is sealed from the atmosphere.

[0129] Step 17: The same compressed air is introduced into the space containing the liquid system that the sample injection end contacts and the space containing the liquid system that the sample elution end contacts, applying low pressure and performing double-end pressurization. Step 18: A voltage is applied between the sample elution end and the sample injection end to perform double-end pressurization electrophoresis. Step 19: The voltage application is stopped and the system is returned to double-end pressurization only.

[0130] Step 20: The pressure of the compressed air is set to atmospheric pressure, and the pressure in the space containing the liquid system that comes into contact with the sample injection end and the space containing the liquid system that comes into contact with the sample elution end is set to atmospheric pressure (step 20 can be omitted).

[0131] Step 21: The space containing the liquid system that the sample injection end contacts is opened to the atmosphere. At the same time, the space containing the liquid system that the sample elution end contacts is opened to the atmosphere. This returns the sample to the baseline.

[0132] When performing capillary electrophoresis analysis multiple times, steps 1 to 21 can be repeated multiple times. Steps 1 to 21 are preferably performed in this order. However, a single capillary electrophoresis analysis unit may be configured to, for example, perform steps 7 to 21 and then perform steps 1 to 7. Even in such a case, multiple capillary electrophoresis analyses will always include steps 1 to 21.

[0133] Among steps 1 to 21, the key features are step 4, polymer solution filling, step 12, pressure injection, and step 16, double-end pressure electrophoresis (main electrophoresis). 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) in which the sample injection end is exposed to atmospheric pressure, and with the polymer solution of the separation medium in contact with the sample elution end, a solid is pressed against the polymer solution to apply high pressure (a pressure of more than 7 atmospheres) to the polymer solution, thereby filling a part of the polymer solution into the capillary; step B (step 12) in which, with the sample solution in contact with the sample injection end, compressed air is brought into contact with the sample solution to apply low pressure (a pressure of 0 to 7 atmospheres) to the sample solution, thereby injecting a part of the sample solution into the capillary; and step C3 (step 16) in which, with the cathode buffer solution in contact with the sample injection end, compressed air is brought into contact with the cathode buffer solution to apply low pressure (a pressure of 0 to 7 atmospheres), and with the polymer solution in contact with the sample elution end, compressed air is brought into contact with a liquid system containing the polymer solution with which the sample elution end is in contact, thereby applying low pressure (a pressure of 0 to 7 atmospheres) to the liquid system, thereby applying a voltage between the sample elution end and the sample injection end. Alternatively, the present invention is characterized in that the analysis includes a step of carrying out step A and step B in that order.

[0134] During capillary electrophoresis, sample components eluted from the sample elution end slowly move toward the anode electrode 5. These components may be reinjected into the capillary when the polymer solution for subsequent analysis is filled, resulting in carryover. The following configuration is effective in reducing this possibility. The intersection point is the point where the flow path from the anode buffer solution reservoir 9 to the sample elution end 3 intersects with the flow path from the pressure-resistant syringe 16 to the sample elution end 3. It is desirable to minimize the distance between the sample elution end 3 and the intersection point. Furthermore, the distance between the sample elution end 3 and the intersection point is preferably 10 mm or less, more preferably 1 mm or less, and most preferably 0 mm.

[0135] <Improvement Example 9: Example 8> Figures 25 to 27 show an example of the configuration of a capillary electrophoresis device according to Improvement Example 9 (Example 8). Improved Example 9 (Example 8) is configured by making modifications to Improvement Example 8 (Example 7: Figures 22 to 24), and achieves the object of the present invention more effectively than Improved Example 8 (Example 7). Improved Example 9 (Example 8) differs from Improved Example 8 (Example 7) in that the compressed air-side polymer solution valve 39 and the plunger-side polymer solution valve 40 in Improved Example 8 (Example 7) are replaced with a rotary valve 42. This modification not only reduces the two valves to one valve, but also facilitates automation of the device. The capillary electrophoresis device according to Improvement Example 9 (Example 8) is equipped with an air-operated pressure mechanism on the cathode side (sample injection end) and a plunger-operated pressure mechanism and an air-operated pressure mechanism on the anode side (sample elution end).

[0136] 25 shows a state in which double-ended pressure electrophoresis is being performed in a capillary electrophoresis apparatus according to Improved Example 9 (Embodiment 8). The rotary valve 42 used in the capillary electrophoresis apparatus according to Improved Example 9 (Embodiment 8) has six ports. In the rotary valve 42 in FIG. 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.

[0137] The rotary valve 42 has an automatic rotation mechanism and can be positioned either at Position A or Position B by rotation. In Position A, Port 2 is connected to Port 3, Port 4 to Port 5, and Port 6 to Port 1. In Position B, Port 1 is connected to Port 2, Port 3 to Port 4, and Port 5 to Port 6. 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. Furthermore, 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 the anode buffer solution tank 9 and the T-block 15 are connected by the polymer solution tube 19 .

[0138] The two plunger-side polymer solution tubes 43 are also disconnected, and the pressure-resistant syringe 16 is not connected to the T-block 15. That is, the anode buffer solution 7 in the anode buffer solution reservoir 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 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 reservoir 8, and the cathode buffer solution reservoir 8 is sealed with the sample injection end 2 immersed in the cathode buffer solution 6. The cathode pressure valve 28 is opened, and the cathode release valve 29 is closed, applying a pressure of 1 atmosphere to the air 34 and the cathode buffer solution 6 inside the cathode buffer solution reservoir 8.

[0139] 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 inside the cathode buffer solution tank 9 and the anode buffer solution 7. This also applies a pressure of 1 atmosphere to the liquid system A.

[0140] As a result of the above, both ends of the cathode and anode are pressurized as shown in Figure 25. 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 enabling stable high separation performance.

[0141] In addition, the vertical height (Z coordinate) of the interface 101 between the cathode buffer solution 6 and air (i.e., the liquid level of the cathode buffer solution 6) and the interface 102 between the anode buffer solution 7 and 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. Furthermore, 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, so 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.

[0142] 26 shows a state in which the capillary 1 is being filled with the polymer solution 18 at high pressure in a capillary electrophoresis apparatus according to Improved Example 9 (Example 8). The anode pressure valve 26 is closed and the anode release valve 27 is opened to release 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 release the inside of the cathode buffer solution tank 8 to atmospheric pressure. In this state, the rotary valve 42 is set to Position B. This separates the two polymer solution tubes 19, disconnecting the anode buffer solution tank 9 from the T-block 15. The two plunger-side polymer solution tubes 43 are connected, 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.

[0143] In this state, the plunger 17 of the pressure-resistant syringe 16 is mechanically pushed in using a motor, applying a high pressure of 35 atmospheres to liquid system B. Because the sample injection port 2 is under atmospheric pressure, a high pressure difference of 35 atmospheres is created between the sample elution port 3 and the sample injection port 2, and the polymer solution 18 is filled into the capillary 1 at a high pressure of 35 atmospheres from the sample elution port 3 toward the sample injection port 2. After the polymer solution is filled, the mechanical pushing of the plunger 17 is stopped, and the plunger 17 and the mechanical pushing mechanism are separated, reducing the pressure applied to the polymer solution 18 to almost zero. In this state, the rotary valve 42 is set to position A, and liquid system A and the sample elution port 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 port 3 and does not pose a problem.

[0144] 27 shows a state in which a sample is pressure-injected into the capillary 1 in a capillary electrophoresis apparatus according to Improved Example 9 (Example 8). The cathode stage 12 is moved by the XYZ-axis drive mechanism to insert the sample injection tip 2 into the sample solution reservoir 11 and immerse it in the sample solution 10, while simultaneously sealing the sample solution reservoir 11. 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 reservoir 11 and the air 37 in contact with it rises to 1 atmosphere. This creates a low pressure difference of 1 atmosphere between the sample injection tip 2 and the sample elution tip 3, and the sample solution 10 is injected into the capillary 1 from the sample injection tip 2. 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 reservoir 11 to atmospheric pressure.

[0145] After this, the cathode stage 12 is moved by the XYZ-axis drive mechanism to insert the sample injection tip 2 into the cathode buffer solution reservoir 8 and immerse it in the anode buffer solution 6, while simultaneously sealing the cathode buffer solution reservoir 8. Next, the anode pressure valve 26 is opened and the anode release valve 27 is closed, and the air 35 in the anode buffer solution reservoir 9 and the liquid system A are pressurized to 1 atmosphere. Meanwhile, the cathode pressure valve 28 is opened and the cathode release valve 29 is closed, and the air 34 in the cathode buffer solution reservoir 8 and the cathode buffer solution 6 are pressurized to 1 atmosphere, returning to the state shown in Figure 25. In this state, a voltage is applied between the sample elution tip 3 and the sample injection tip 2, and double-ended pressure electrophoresis is performed.

[0146] As described above, according to the ninth improvement example (embodiment 8), the first object of the present invention can be achieved: (1) to provide a capillary electrophoresis device that "enables filling of 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 of the present invention can be achieved: (1) to provide a capillary electrophoresis device that "enables filling of a polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis" and (3) "enables pressure injection of a sample at both ends during electrophoresis." The third object of the present invention can be achieved: (1) to provide a capillary electrophoresis device that "enables filling of 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 ...3) to provide a capillary electrophoresis device that "enables filling of a polymer solution within a practical time using a high-viscosity polymer solution for DNA sequence and DNA fragment analysis" and (3) "enables pressure injection of a sample at both ends during electrophoresis."

[0147] In the ninth improvement (embodiment eight), not only pressure injection of the sample but also electric field injection is possible. In Fig. 27, the cathode pressure valve 28 is closed and the cathode release valve 29 is opened, releasing the sample solution 10 in the sample solution reservoir 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.

[0148] <Improvement Example 10: Example 9> In the above improvements, for simplicity of explanation, each device is equipped with one capillary, and electrophoretic analysis is performed using one capillary in each device. However, the present invention also applies when each device is equipped with multiple capillaries and performs electrophoretic analysis in parallel using multiple capillaries. As an example, Figure 28 shows an example configuration of a capillary electrophoresis device according to Improvement Example 10 (Example 9) that uses three capillaries in Improvement Example 8 (Example 7) (when performing double-ended pressure electrophoresis). Of course, the number of capillaries is only a specific example, and any number, such as four, eight, twelve, etc., can be used.

[0149] The sample injection ends 2 of the three capillaries 1 are inserted into 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 spacing in the X-axis direction is, for example, 9 mm, which matches the spacing between wells in a microtiter plate. The cathode buffer solution reservoirs 8 containing three cathode buffer solutions 6 are aligned in the Y-axis and Z-axis directions and fixed on the cathode stage 12 at intervals in the X-axis direction that are the same as the spacing between the three sample injection ends 2 in the X-axis direction. The cathode stage 12 is further connected to an XYZ-axis drive mechanism (not shown).

[0150] Sample solution reservoirs 11, each containing at least three sample solutions 10, are also fixed on the cathode stage 12, but are omitted from Figure 28. O-rings 33 are attached to the upper edge of each of the three cathode buffer solution reservoirs 8. By moving the cathode stage 12 using the XYZ-axis drive mechanism, the three sample injection tips 2 are inserted into each of the three cathode buffer solution reservoirs 8 and immersed in each of 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 fixing block 31, compressing them and sealing the three cathode buffer solution reservoirs 8. This seals the gaps between the three capillaries 1 and the three cathodes 4 and the fixing block 31. The three cathode buffer solution reservoirs 8 contain three cathode buffer solutions 6 and a common air 34 in contact with 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.

[0151] 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.

[0152] The three detection positions on the three capillaries 1 are arranged in a straight line, a laser beam 24 emitted from a laser light source 23 is irradiated onto the three detection positions simultaneously, and the fluorescence emitted from the three detection positions is detected simultaneously by a fluorescence detection device (not shown).

[0153] 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 inside the T-block 15. The other components on the anode side (polymer solution tube 19 containing the polymer solution 18, compressed air side polymer solution valve 39, anode buffer solution tank 9 containing anode buffer solution 7 and air 35, fixing 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).

[0154] 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, raising the pressure of the air 35 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, and double-ended pressure electrophoresis is performed. This raises 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.

[0155] Furthermore, the vertical height (Z coordinate) 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 prevents the polymer solution 18 inside the capillary 1 from moving due to gravity during electrophoresis. The methods for filling the polymer solution, pressure-injecting the sample, etc. are the same as those in Improved Example 8 (Example 7).

[0156] As described above, according to Improved Example 10 (Example 9), the first object of the present invention 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" for each of the three capillaries. The second object 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 (3) "enables pressure injection of a sample at both ends during electrophoresis." The third object 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" and (3) "enables pressure injection of a sample at both ends during electrophoresis." Note that Improved Example 10 (Example 9) allows not only pressure injection of a sample but also electric field injection.

[0157] 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 of the sample injection end is unchanged. 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 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 internal space. With the sample injection end open to atmospheric pressure, mechanically pushing the syringe plunger increases the pressure of the internal polymer solution to 35 atmospheres, allowing the high-viscosity polymer solution to 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 using an 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 the two ends of the capillary. This is the same problem as Device C. However, electrophoresis can be performed by immersing the sample injection end of the capillary in a cathode buffer solution and applying a voltage to both ends of the capillary. Therefore, the configuration of Device D cannot achieve any of the objectives of the present invention.

[0158] Next, in Device B, the air-operated pressure mechanism at the sample elution end is replaced with the plunger-operated pressure mechanism of Device A (Reference Example 1: Device E). The configuration of the sample injection end is unchanged. The sample elution end, which is 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. An anode buffer tank containing the 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 syringe plunger is mechanically pushed in, increasing the pressure of the internal polymer solution to 35 atmospheres, thereby filling the capillary with a highly viscous polymer solution. When the valve is opened, the sample elution end is exposed to atmospheric pressure. Therefore, by immersing the sample injection end in the sample solution and applying a low pressure of 1 atmosphere using the air-operated pressure mechanism, 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 a 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 an anode buffer tank, the flow path has a small inner diameter and a small internal volume per unit length in the electrophoresis direction. As a result, the ionic composition of the polymer solution near the sample elution end changes during electrophoresis, ultimately resulting in ion depletion. Furthermore, gas generated on the surface of the anode during electrophoresis remains in the flow path as bubbles, resulting in high electrical resistance. Furthermore, sample components eluted from the sample elution end during electrophoresis remain in the flow path. These components then flow back into the capillary when the polymer solution is filled in the subsequent analysis, resulting in carryover problems.

[0159] Therefore, in Device E, the integrated anode and sample elution end are separated, and the sample elution end is connected to a channel containing a polymer solution, while the anode is immersed in an anode buffer solution (Improvement 3 (Example 2): Device F). With the configuration of Device F, the position of the anode does not adversely affect electrophoresis analysis. Therefore, it is possible to fill the polymer solution with a high-viscosity polymer solution for DNA sequence and DNA fragment analysis within a practical time, and it is also possible to pressure-inject the sample. Therefore, Device F achieves the first object of the present invention: (1) to provide a capillary electrophoresis device that "enables filling the polymer solution with a high-viscosity polymer solution for DNA sequence and DNA fragment analysis within a practical time" and (2) "enables pressure-injection of the sample." However, because the sample elution end does not have an air-operated pressure mechanism, it is not possible to apply pressure to both ends during electrophoresis.

[0160] To achieve double-pressurized electrophoresis, a plunger-type pressure mechanism is installed at the sample injection end of Device A (Conventional Example 1), similar to the sample elution end (Reference Example 2: Device G). The configuration of the sample elution end is unchanged. The sample injection end, integrated with the cathode, is connected to a flow path containing the polymer solution, and a syringe containing the polymer solution is further connected to the flow path. A cathode buffer tank containing the 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 pressure mechanism mechanically pushes in the syringe plunger with the valve closed, increasing the pressure of the internal polymer solution. Furthermore, opening the valve releases the sample injection end to atmospheric pressure. As a result, independent plunger-type pressure mechanisms are installed at both ends of the capillary, which is expected to enable double-pressurization during electrophoresis. However, compared to air-type pressure mechanisms, plunger-type pressure mechanisms are suitable for applying high pressure, but are not suitable for applying low pressure with high precision and stability. For example, because the sliding resistance when pushing the plunger into the syringe is not constant, the internal pressure fluctuates even when the pushing force is constant. Furthermore, when using an air-type pressure mechanism 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 to each end. In contrast, when using a plunger-type pressure mechanism at both ends of the capillary, as in Device G, it is difficult to equalize the pressure at both ends because each mechanism is independent. If a difference in pressure occurs at both ends, the polymer solution in the capillary will move from the side with higher pressure to the side with lower pressure, adversely affecting separation ability. Furthermore, 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. This makes both pressure injection and electric field injection of samples difficult.

[0161] Based on the above, in Apparatus F, a sealed chamber containing the anode buffer tank, valve, and valve drive mechanism is installed, and an air-operated pressurization mechanism is installed that introduces compressed air into the sealed chamber (Improvement Example 5 (Example 4): Apparatus H). The air inside the sealed chamber can also be released to atmospheric pressure. Because the valve and valve drive mechanism protrude significantly from the anode buffer tank and move spatially, it is difficult to seal only the anode buffer tank, as in Apparatus B. For this reason, a relatively large sealed chamber as described above is installed. With the sample injection end open to atmospheric pressure, the sealed chamber is opened to atmospheric pressure, and the valve is closed, the syringe plunger is mechanically pushed in. This increases the pressure of the polymer solution inside to 35 atmospheres, allowing the high-viscosity polymer solution to be filled into the capillary. When the valve is opened, the sample elution end is exposed to atmospheric pressure. Therefore, by immersing the sample injection end in the sample solution and applying a low pressure of 1 atmosphere using the air-operated pressurization mechanism, 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 in the cathode buffer solution and applying a voltage to both ends of the capillary. Furthermore, during electrophoresis, a low pressure of 1 atmosphere can be applied to the sample elution end using an air-operated pressurization mechanism that introduces compressed air into a sealed chamber with the valve open. At the same time, a low pressure of 1 atmosphere can be applied to the sample injection end using an air-operated pressurization mechanism that seals the cathode buffer tank and introduces compressed air. This achieves double-sided pressurization.

[0162] As a result, Device H achieves the third objective of the present invention: (1) "enabling the filling of a high-viscosity polymer solution for DNA sequencing and DNA fragment analysis within a practical time frame using the polymer solution," (2) "enabling the pressure injection of a sample," and (3) "enabling the application of pressure to both ends during electrophoresis." However, the large and complex structure of the sealed chamber is a problem. Another problem is that the internal volume of the sealed chamber is large, so it takes a long time for the internal pressure to reach the specified pressure after introducing compressed air.

[0163] Furthermore, based on the above, in Apparatus F, the valve at the boundary between the anode buffer solution and the polymer solution was removed, and Valve 1 was installed in the flow path intermediate between the sample elution end and the anode buffer tank. Valve 2 was also installed in the flow path intermediate between the sample elution end and the syringe. At the same time, as in Apparatus B, the anode buffer tank was made to have a sealed structure, and an air-operated pressurization mechanism was installed by introducing compressed air into the anode buffer tank (Improvement Example 8 (Example 7): Apparatus I). However, unlike Apparatus B, the anode buffer tank does not have the sample elution end of the capillary inserted into it. Instead, a flow path containing the polymer solution connected 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 remains unchanged. 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, allowing the high-viscosity polymer solution to be filled into the capillary. When the anode buffer tank is opened to atmospheric pressure and valve 1 is opened, the sample elution end is exposed to atmospheric pressure. In this state, the sample injection end can be immersed in the sample solution and a low pressure of 1 atmosphere applied using the air pressure mechanism to inject the sample into the capillary. Alternatively, the sample injection end can be immersed in the sample solution and a voltage applied to both ends of the capillary to inject the sample into the capillary using an electric field. 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, double-ended pressure electrophoresis can be performed by opening valve 1 and closing valve 2, and simultaneously applying a low pressure of 1 atmosphere to the anode buffer tank and sample elution end using the air pressure mechanism, while simultaneously applying a low pressure of 1 atmosphere to the sample injection end using the air pressure mechanism.

[0164] As a result, Device I can provide a capillary electrophoresis device that achieves the third object of the present invention: (1) "enabling filling of a polymer solution within a practical time using a high-viscosity polymer solution for DNA sequencing and DNA fragment analysis," (2) "enabling pressure injection of a sample," and (3) "enabling pressure application to both ends during electrophoresis."

[0165] 1 Capillary 2 Sample injection port 3 Sample elution port 4 Cathode 5 Anode 6 Cathode buffer solution 7 Anode buffer solution 8 Cathode buffer solution reservoir 9 Anode buffer solution reservoir 10 Sample solution 11 Sample solution reservoir 12 Cathode stage 13 Anode stage 14 Connector 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 Electrical 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 Fixing block 32 Polymer solution reservoir 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 the polymer solution 37 Air in contact with the sample solution 38 Sealed chamber 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 Interface between the cathode buffer solution and air 102 Interface between the anode buffer solution and air 103 Interface between the anode buffer solution and polymer solution 104 Interface between the polymer solution and plunger 105 Interface between the sample solution and air 106 Interface between the polymer solution and air

Claims

1. A capillary electrophoresis device comprising: a first liquid system with which a first capillary end of a capillary contacts; a second liquid system with which a second capillary end of the capillary contacts; 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 of the first liquid system by compressing a first gas that directly or indirectly contacts a first interface of the first liquid system; a first decompression mechanism that reduces the first pressure of the first liquid system; a second pressurizing mechanism that increases a second pressure of the second liquid system by moving a solid that directly or indirectly contacts a second interface of the second liquid system; and a second decompression mechanism that reduces the second pressure of the second liquid system, wherein the second liquid system has a second gas that directly or indirectly contacts a third interface of the second liquid system.

2. A capillary electrophoresis device according to claim 1, wherein 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, and 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 the volume of the second space.

3. A capillary electrophoresis device according to claim 2, wherein 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, and the second pressure reduction mechanism is a second atmospheric pressure release mechanism that opens the second space to the atmosphere and reduces the second pressure to atmospheric pressure.

4. A capillary electrophoresis device according to claim 3, wherein the portion of the second liquid system that comes into contact with the end of the second capillary is a polymer solution of a separation medium, and while the first pressure is set to atmospheric pressure by the first atmospheric pressure release mechanism, the second pressure application mechanism increases the second pressure to fill a portion of the polymer solution into the capillary.

5. A capillary electrophoresis device according to claim 3, wherein the portion of the first liquid system that comes into contact with the end of the first capillary is a sample solution, and while the second pressure is set to atmospheric pressure by the second atmospheric pressure release mechanism, the first pressure mechanism increases the first pressure to inject a portion of the sample solution into the capillary.

6. A capillary electrophoresis device according to claim 3, wherein when the first pressure is made atmospheric pressure by the first atmospheric pressure release mechanism and the second pressure is made atmospheric pressure by the second atmospheric pressure release mechanism, and when a voltage is applied to the first capillary end and the second capillary end to perform electrophoresis, the vertical heights of the first interface and the third interface are aligned.

7. A capillary electrophoresis analysis method performed by applying a voltage between a first capillary end and a second capillary end of a capillary, comprising: step 1 of opening the first capillary end to atmospheric pressure, pressing a solid against the polymer solution while bringing a polymer solution of a separation medium into contact with the second capillary end, to pressurize the polymer solution, and filling the capillary with a portion of the polymer solution; and step 2 of bringing a sample solution into contact with the first capillary end and bringing compressed air into contact with the sample solution to pressurize the sample solution, and injecting a portion of the sample solution into the capillary, wherein the method is performed in the order of step 1 and step 2.

8. A capillary electrophoresis analysis method performed by applying a voltage between a first capillary end and a second capillary end of a capillary, comprising: step 1 of applying a pressure of 0 atmospheres to the first capillary end, and applying a pressure of more than 7 atmospheres to the second capillary end while it is in contact with a polymer solution of a separation medium, thereby filling the capillary with a portion of the polymer solution; and step 2 of applying a pressure of 7 atmospheres or less to the first capillary end while it is in contact with a sample solution, and applying a pressure of 0 atmospheres to the second capillary end, thereby injecting a portion of the sample solution into the capillary, wherein the method is performed in the order of step 1 and step 2.

9. 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 the capillary is in contact; a power source that applies a voltage between the first liquid system and the second liquid system; a first pressure mechanism that increases 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 second pressure mechanism that increases 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 third pressure mechanism that increases the second pressure of 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.

10. A capillary electrophoresis apparatus according to claim 9, further comprising: a first pressure reduction mechanism for reducing the first pressure in the first liquid system; and a second pressure reduction mechanism for reducing the second pressure in the second liquid system.

11. A capillary electrophoresis device according to claim 10, wherein 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, 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 the 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.

12. A capillary electrophoresis device according to claim 11, wherein 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, and the second pressure reduction mechanism is a second atmospheric pressure release mechanism that opens the second space to the atmosphere and reduces the second pressure to atmospheric pressure.

13. A capillary electrophoresis device as described in claim 12, wherein the portion of the second liquid system that comes into contact with the end of the second capillary is a polymer solution of a separation medium, and while the first pressure is set to atmospheric pressure by the first atmospheric pressure release mechanism, the second pressure mechanism increases the second pressure to fill a portion of the polymer solution into the capillary.

14. A capillary electrophoresis device as described in claim 12, wherein the portion of the first liquid system that comes into contact with the end of the first capillary is a sample solution, and when the second pressure is set to atmospheric pressure by the second atmospheric pressure release mechanism, the first pressure mechanism increases the first pressure to inject a portion of the sample solution into the capillary.

15. A capillary electrophoresis device according to claim 12, wherein 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 and the second pressure equal, and capillary electrophoresis is performed by applying a voltage between the end of the first capillary and the end of the second capillary.

16. A capillary electrophoresis device according to claim 12, further comprising a first valve between the third interface and the second capillary end for dividing or connecting the second liquid system.

17. A capillary electrophoresis device according to claim 12, further comprising a second valve between the second interface and the second capillary end for dividing or connecting the second liquid system.

18. A capillary electrophoresis device according to claim 12, 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.

19. A capillary electrophoresis analysis method performed by applying a voltage between a first capillary end and a second capillary end of a capillary, comprising: step 1 of opening the first capillary end to atmospheric pressure, pressing a solid against the polymer solution while the second capillary end is in contact with a polymer solution of a separation medium, to pressurize the polymer solution, and filling the capillary with a portion of the polymer solution; and step 2 of bringing a sample solution into contact with the first capillary end, bringing a first compressed air into contact with the sample solution, to pressurize the sample solution, and injecting a portion of the sample solution into the capillary, and step 3 of bringing a buffer solution into contact with the first capillary end and a second compressed air into contact with the buffer solution to pressurize the buffer solution, bringing the polymer solution into contact with the second capillary end and a liquid system containing the polymer solution into contact with the second compressed air to pressurize the liquid system, and applying a voltage between the first capillary end and the second capillary end, wherein the method is performed in the order of steps 1, 2, and 3.

20. A method of capillary electrophoresis analysis performed by applying a voltage between a first capillary end and a second capillary end of a capillary, comprising: step 1 of applying a first pressure of 0 atmospheres to the first capillary end, applying a second pressure of more than 7 atmospheres to the second capillary end while the second capillary end is in contact with a polymer solution of a separation medium, and filling the capillary with a portion of the polymer solution; and step 2 of applying a third pressure of 7 atmospheres or less to the first capillary end while the second capillary end is in contact with a sample solution, applying the first pressure to the second capillary end, 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 it is in contact with a buffer solution, applying the fourth pressure to the second capillary end while it 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, wherein the method is performed in the order of steps 1, 2, and 3.

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