Simple liquid chromatograph

Abstract

To provide a simple liquid chromatograph capable of further simplifying a low pressure gradient mechanism and also simplifying a sample injection mechanism.SOLUTION: A pump of a simple liquid chromatograph includes: a pump chamber 20 having a plurality of through-holes; and a liquid feeding part composed of a plunger 25 performing reciprocating operation and rotating operation in the pump chamber. A polygon formed by a connecting line of the through-hole is a regular polygon inscribed in a cross section of the pump chamber, and the plunger has grooves matched with the through-holes in a horizontal direction.SELECTED DRAWING: Figure 13

Description

[Technical field]

[0001] Liquid chromatography (HPLC) is a widely used method for separating and quantifying samples consisting of multiple components using columns filled with various gels. The principles of separation in HPLC include separation based on differences in hydrophobicity, separation based on differences in ionic strength, separation based on specific interactions, and separation based on differences in molecular size, and are used according to the purpose. The purpose of separation may be to simply perform qualitative / quantitative analysis, or to separate the separated components. The latter is called preparative chromatography, and the main purpose is often to purify the sample. Targets include a variety of substances such as food, proteins, medicines, viruses, and nucleic acids. When purifying these substances using preparative chromatography, the general method is to first conduct condition studies on a small scale, gradually scale up, and finally scale up to a commercial scale.

[0002] In HPLC for analytical purposes, separation ability is often important, and columns packed with high-performance gel are required. Since gels with particle sizes of a few μm to 10 μm are mainly used, the pressure during measurement is high, at 10 to 100 MPa. In addition, to ensure separation, a "gradient elution method" that gradually changes the buffer composition is often used, which makes the device configuration more complex.

[0003] On the other hand, for HPLC with the purpose of separation, in consideration of the final commercialization, it is desirable to simplify the equipment as much as possible, and high-pressure columns are not suitable. In addition, it is necessary to suppress the purification cost, so in most cases, gels with relatively large particle size and low operating pressure are used. When examining conditions on a small scale in preparative chromatography with commercialization in mind, a column tube with an inner diameter of a few centimeters is filled with relatively large gels with particle sizes of tens to hundreds of microns.

[0004] In general analytical HPLC, separation is often performed using a "high-pressure gradient elution method" in which multiple liquid delivery mechanisms are set up in parallel as shown in Figure 1, and the composition of the buffer delivered to the analytical column is gradually changed by changing the liquid delivery flow rate of each mechanism. While this method has the advantage of allowing the buffer composition to be freely changed, it also increases the cost of the equipment and makes its control more complex. In addition, the use of multiple liquid delivery mechanisms tends to increase the frequency of trouble.

[0005] Figure 2 shows the system configuration for performing the "low-pressure gradient elution method," which is one type of gradient elution method. In this example, three types of buffers with different compositions are used. There is one liquid delivery mechanism (11), and the type of buffer delivered to the column is switched in steps by opening and closing the on-off valves (6) placed in the flow path of each buffer, which is called the "low-pressure step gradient elution method." It is difficult to gradually change the buffer composition with this method, but since there is only one liquid delivery mechanism, the frequency of trouble is relatively low, and this method is often used in preparative chromatography intended for commercialization.

[0006] However, even with this system configuration, multiple on-off valves are required to switch between buffers, and there are many other components as well, so there are still many challenges to overcome before it can be commercialized. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made to solve the above problems, and provides a simplified liquid chromatograph in which the low-pressure gradient mechanism can be further simplified and the sample injection mechanism can also be simplified. [Means for solving the problem]

[0008] In a first embodiment, the plunger pump does not have a check valve and is composed of a pump chamber having three or more through holes, and a plunger which reciprocates and rotates within the pump chamber, the plunger having a groove in a horizontal direction, and the groove of the plunger stops at the position of the through hole provided in the pump chamber during the rotation of the plunger. An analytical column is connected to a first port of the through hole, a first buffer is connected to a second port of the through hole, and a second buffer is connected to a third port of the through hole. The following steps: (i) horizontally moving the plunger to a tip position of the pump chamber, and aligning the groove position of the plunger with the position of a port to which a first buffer is connected by a rotational movement; (ii) in this state, by sucking the plunger, the first buffer is sucked into the pump chamber; (iii) Rotating the plunger to align it with the port to which the analytical column is connected; (iv) discharging the plunger to push the first buffer in the pump chamber into the analytical column; By periodically repeating steps (i) to (iv), the first buffer is delivered to the analytical column; Furthermore, (v) horizontally moving the plunger to a tip position of the pump chamber, and aligning the groove position of the plunger with a port position to which a second buffer is connected by a rotational movement; (vi) In this state, the plunger is sucked to suck the second buffer into the pump chamber; (vii) aligning the plunger with the port to which the analytical column is connected by a rotating motion; (viii) expelling the plunger to push the second buffer in the pump chamber into the analytical column; By periodically repeating steps (v) to (viii), the second buffer is pumped to the analytical column; This is a method of performing a "two-liquid step gradient" in which the first buffer and the second buffer are switched in a stepwise manner by switching between the above (i) to (iv) and the above (v) to (viii) over time.

[0009] In addition, a third buffer is connected to a fourth port of the through hole, and following the above-mentioned operation, the third buffer is pumped into the analytical column, thereby performing a "three-liquid step gradient" in which the first buffer, the second buffer, and the third buffer are switched in a stepwise manner.

[0010] In addition, a fourth buffer is connected to the fifth port of the through hole, and following the above-mentioned operation, the fourth buffer is pumped into the analytical column, thereby performing a "four-liquid step gradient" in which the first buffer, second buffer, third buffer, and fourth buffer are switched in a stepwise manner.

[0011] In addition, a fifth buffer is connected to the sixth port of the through-hole, and following the above-mentioned operation, the fifth buffer is pumped into the analytical column, thereby performing a "five-liquid step gradient" in which the first buffer, second buffer, third buffer, fourth buffer, and fifth buffer are switched in a stepwise manner (configuration in Table 1, configurations in Figures 4 to 7). TIFF2025068524000002.tif86170

[0012] As a second embodiment, a plunger-type pump without a check valve is comprised of a pump chamber having five or more through holes, and a plunger which reciprocates and rotates within the pump chamber, the plunger having a groove in the horizontal direction, and the groove of the plunger stops at the position of the through hole provided in the pump chamber during the rotation of the plunger, characterized in that an analytical column is connected to a first port of the through hole, a first buffer is connected to a second port of the through hole, a second buffer is connected to a third port of the through hole, a sample to be analyzed is connected to a fourth port of the through hole, and a fifth port of the through hole is used as a waste port, The following steps: (i) moving the plunger horizontally to a tip position of the pump chamber, and aligning the groove position of the plunger with the position of a port to which a sample to be analyzed is connected by a rotational movement; (ii) In this state, the plunger is sucked to suck the sample to be analyzed into the pump chamber; (iii) rotating the plunger to align the groove of the plunger with the waste port; (iv) discharging the plunger to push the sample to be analyzed in the pump chamber to waste; (v) rotating the plunger to align the groove of the plunger with the port to which the sample to be analyzed is connected; (vi) In this state, the plunger is pulled to aspirate the sample to be analyzed; (vii) rotating the plunger to align the groove of the plunger with the port to which the analytical column is connected; (viii) discharging the plunger to push the sample to be analyzed in the pump chamber into the analytical column; Furthermore, (ix) horizontally moving the plunger to a tip position of the pump chamber, and aligning the groove position of the plunger with a port position to which a first buffer is connected by a rotational movement; (x) in this state, by sucking the plunger, a first buffer is sucked into the pump chamber; (xi) Rotating the plunger to align it with the port to which the analytical column is connected; (xii) expelling the plunger to push the first buffer in the pump chamber into the analytical column; By periodically repeating steps (ix) to (xii), the first buffer is delivered to the analytical column, Furthermore, the following steps: (xiii) horizontally moving the plunger to a tip position of the pump chamber, and aligning the groove position of the plunger with a port position to which a second buffer is connected by a rotational movement; (xiv) In this state, the plunger is sucked to suck the second buffer into the pump chamber; (xv) aligning the plunger with the port to which the analytical column is connected by a rotating motion; (xvi) expelling the plunger to push the second buffer in the pump chamber into the analytical column; By periodically repeating steps (xiii) to (xvi), the second buffer is pumped to the analytical column; By switching between the above (ix) to (xii) and the above (xiii) to (xvi) over time, sample injection in which the first buffer and the second buffer are switched in a stepwise manner and a "two-liquid step gradient" is performed (configuration in Table 2, configuration in FIG. 8). TIFF2025068524000003.tif51122

[0013] As a third embodiment, a plunger-type pump without a check valve is characterized in that it is composed of a pump chamber having five or more through holes, and a plunger that reciprocates and rotates within the pump chamber, the plunger has a groove in the reciprocating direction and in the horizontal direction, and when the plunger rotates, the groove of the plunger stops at the position of the through hole provided in the pump chamber, and the plunger pump has a check valve-free plunger-type pump including: an analytical column connected to a first port of the through hole; a first buffer connected to a second port of the through hole; a second buffer connected to a third port of the through hole; a sample to be analyzed connected to a fourth port of the through hole; and a fifth port of the through hole serving as a waste outlet; The following steps: (i) moving the plunger horizontally to a tip position of the pump chamber, and aligning the groove position of the plunger with the position of a port to which a sample to be analyzed is connected by a rotational movement; (ii) In this state, the plunger is sucked to suck the sample to be analyzed into the pump chamber; (iii) rotating the plunger to align the groove of the plunger with the waste port; (iv) discharging the plunger to push the sample to be analyzed in the pump chamber to waste; (v) rotating the plunger to align the groove of the plunger with the port to which the sample to be analyzed is connected; (vi) In this state, the plunger is pulled to aspirate the sample to be analyzed; (vii) rotating the plunger to align the groove of the plunger with the port to which the analytical column is connected; (viii) discharging the plunger to push the sample to be analyzed in the pump chamber into the analytical column; Furthermore, (ix) horizontally moving the plunger to a tip position of the pump chamber, and aligning the groove position of the plunger with a port position to which a first buffer is connected by a rotational movement; (x) In this state, the plunger is sucked to suck the first buffer into the pump chamber; (xi) Rotating the plunger to align it with the port to which the analytical column is connected; (xii) expelling the plunger to push the first buffer in the pump chamber into the analytical column; By periodically repeating steps (ix) to (xii), the first buffer is delivered to the analytical column, Furthermore, (xiii) horizontally moving the plunger to a tip position of the pump chamber, and aligning the groove position of the plunger with a port position to which a second buffer is connected by a rotational movement; (xiv) In this state, the plunger is sucked to suck the second buffer into the pump chamber; (xv) aligning the plunger with the port to which the analytical column is connected by a rotating motion; (xvi) expelling the plunger to push the second buffer in the pump chamber into the analytical column; By periodically repeating steps (xiii) to (xvi), the second buffer is pumped to the analytical column; Furthermore, (xvii) horizontally moving the plunger to a tip position of the pump chamber, and aligning a groove position of the plunger with a port position to which a third buffer is connected by a rotational movement; (xviii) in this state, sucking the plunger to suck a third buffer into the pump chamber; (xix) aligning the plunger with the port to which the analytical column is connected by a rotating motion; (xx) discharging the plunger to push the third buffer in the pump chamber into the analytical column; By periodically repeating steps (xvii) to (xx), the third buffer is delivered to the analytical column; By switching between the above (ix) to (xii), (xiii) to (xvi), and (xvii) to (xx) over time, sample injection is performed in which the first buffer, the second buffer, and the third buffer are switched in a stepwise manner, and a "three-liquid step gradient" is performed (configuration of Table 3, configuration of FIG. 9). TIFF2025068524000004.tif49113

[0014] As a fourth embodiment, a plunger pump without a check valve is provided, which is composed of a pump chamber having five or more through holes, and a plunger which reciprocates and rotates within the pump chamber, the plunger having a groove in a horizontal direction, and the groove of the plunger stops at the position of the through hole provided in the pump chamber during the rotation of the plunger, the plunger having a groove in a horizontal direction, the plunger having a groove in a horizontal direction, the plunger having a groove in a horizontal direction, the plunger having a groove in a horizontal direction, the plunger having a groove in a horizontal direction, the plunger having a groove in a horizontal direction, the plunger having a groove in a horizontal direction, the plunger having a groove in a horizontal direction, the plunger having a groove in a horizontal direction, the plunger having a groove in a horizontal direction The following steps: (i) moving the plunger horizontally to a tip position of the pump chamber, and aligning the groove position of the plunger with the position of a port to which a sample to be analyzed is connected by a rotational movement; (ii) In this state, the plunger is sucked to suck the sample to be analyzed into the pump chamber; (iii) rotating the plunger to align the groove of the plunger with the port to which the analytical column is connected; (iv) discharging the plunger to push the sample to be analyzed in the pump chamber into the analytical column; (v) rotating the plunger to align the groove of the plunger with a port to which a cleaning fluid is connected; (vi) In this state, the plunger is sucked to suck the cleaning liquid into the pump chamber; (vii) rotating the plunger to align the groove of the plunger with the port to which the sample to be analyzed is connected; (viii) ejecting the plunger to push out excess sample into a sample container; Furthermore, (ix) horizontally moving the plunger to a tip position of the pump chamber, and aligning the groove position of the plunger with a port position to which a first buffer is connected by a rotational movement; (x) in this state, by sucking the plunger, a first buffer is sucked into the pump chamber; (xi) rotating the plunger to align the groove of the plunger with the port to which the analytical column is connected; (xii) expelling the plunger to push the first buffer in the pump chamber into the analytical column; By periodically repeating steps (ix) to (xii), the first buffer is delivered to the analytical column, Furthermore, (xiii) horizontally moving the plunger to a tip position of the pump chamber, and aligning the groove position of the plunger with a port position to which a second buffer is connected by a rotational movement; (xiv) In this state, the plunger is sucked to suck the second buffer into the pump chamber; (xv) rotating the plunger to align the groove of the plunger with the port to which the analytical column is connected; (xvi) expelling the plunger to push the second buffer in the pump chamber into the analytical column; By periodically repeating steps (xiii) to (xvi), the second buffer is pumped to the analytical column; By switching between the above (ix) to (xii) and the above (xiii) to (xvi) over time, there is a method of sample injection in which the first buffer and the second buffer are switched stepwise, and a "two-liquid step gradient" is performed (the configuration in Table 4, the configuration in FIG. 10). TIFF2025068524000005.tif52108

[0015] As a fifth embodiment, a plunger-type pump without a check valve is characterized in that it is composed of a pump chamber having five or more through holes, and a plunger that reciprocates and rotates within the pump chamber, the plunger having a groove in a horizontal direction, and the groove of the plunger stops at the position of the through hole provided in the pump chamber during the rotation of the plunger, the plunger having a groove in a horizontal direction, the plunger having a groove in a horizontal direction, the plunger having a groove in a horizontal direction, the plunger having a groove in a horizontal direction, the plunger having a groove in a horizontal direction, the plunger having a groove in a horizontal direction, the plunger having a groove in a horizontal direction, the plunger having a groove in a horizontal direction, the plunger having a groove in a horizontal direction, the plunger having a groove in a horizontal direction The following steps: (i) moving the plunger horizontally to a tip position of the pump chamber, and aligning the groove position of the plunger with the position of a port to which a sample to be analyzed is connected by a rotational movement; (ii) In this state, the plunger is sucked to suck the sample to be analyzed into the pump chamber; (iii) rotating the plunger to align the groove of the plunger with the port to which the analytical column is connected; (iv) discharging the plunger to push the sample to be analyzed in the pump chamber into the analytical column; (v) rotating the plunger to align the groove of the plunger with a port to which a cleaning fluid is connected; (vi) In this state, the plunger is sucked to suck the cleaning liquid into the pump chamber; (vii) rotating the plunger to align the groove of the plunger with the port to which the sample to be analyzed is connected; (viii) ejecting the plunger to push out excess sample into a sample container; Furthermore, (ix) horizontally moving the plunger to a tip position of the pump chamber, and aligning the groove position of the plunger with a port position to which a first buffer is connected by a rotational movement; (x) in this state, by sucking the plunger, a first buffer is sucked into the pump chamber; (xi) rotating the plunger to align the groove of the plunger with the port to which the analytical column is connected; (xii) expelling the plunger to push the first buffer in the pump chamber into the analytical column; By periodically repeating steps (ix) to (xii), the first buffer is delivered to the analytical column, Furthermore, (xiii) horizontally moving the plunger to a tip position of the pump chamber, and aligning the groove position of the plunger with a port position to which a second buffer is connected by a rotational movement; (xiv) In this state, the plunger is sucked to suck the second buffer into the pump chamber; (xv) rotating the plunger to align the groove of the plunger with the port to which the analytical column is connected; (xvi) expelling the plunger to push the second buffer in the pump chamber into the analytical column; By periodically repeating steps (xiii) to (xvi), the second buffer is pumped to the analytical column; Furthermore, (xvii) horizontally moving the plunger to a tip position of the pump chamber, and aligning a groove position of the plunger with a port position to which a third buffer is connected by a rotational movement; (xviii) in this state, sucking the plunger to suck a third buffer into the pump chamber; (xix) rotating the plunger to align the groove of the plunger with the port to which the analytical column is connected; (xx) discharging the plunger to push the third buffer in the pump chamber into the analytical column; By periodically repeating steps (xvii) to (xx), the third buffer is pumped to the analytical column, By switching between the above (ix) to (xii), (xiii) to (xvi), and (xvii) to (xx) over time, sample injection is performed by switching between the first buffer, the second buffer, and the third buffer in a stepwise manner, and a "three-liquid step gradient" is performed (configuration of Table 5, configuration of FIG. 11). TIFF2025068524000006.tif53112

[0016] As a sixth embodiment, a plunger-type pump without a check valve is characterized in that it is composed of a pump chamber having six or more through holes, and a plunger that reciprocates and rotates within the pump chamber, the plunger has a groove in a horizontal direction, and when the plunger rotates, the groove of the plunger stops at the position of the through hole provided in the pump chamber, the plunger has a groove in a horizontal direction, and the groove of the plunger stops at the position of the through hole provided in the pump chamber, the plunger has a pump chamber having six or more through holes, and the pump chamber has a pump chamber having six or more through holes, the .... The following steps: (i) moving the plunger horizontally to a tip position of the pump chamber, and aligning the groove position of the plunger with the position of a port to which a sample to be analyzed is connected by a rotational movement; (ii) In this state, the plunger is sucked to suck the sample to be analyzed into the pump chamber; (iii) rotating the plunger to align the groove of the plunger with the waste port; (iv) discharging the plunger to push the sample to be analyzed in the pump chamber to waste; Furthermore, the following steps: (v) rotating the plunger to align the groove of the plunger with the port to which the sample to be analyzed is connected; (vi) In this state, the plunger is pulled to aspirate the sample to be analyzed; (vii) rotating the plunger to align the groove of the plunger with the port to which the analytical column is connected; (viii) discharging the plunger to push the sample to be analyzed in the pump chamber into the analytical column; Furthermore, (ix) rotating the plunger to align the groove of the plunger with a port to which a cleaning fluid is connected; (x) In this state, the plunger is sucked to suck the cleaning liquid into the pump chamber; (xi) rotating the plunger to align the groove of the plunger with the port to which the sample to be analyzed is connected; (xii) ejecting the plunger in this state to collect excess sample to be analyzed in a sample container; Furthermore, (xiii) horizontally moving the plunger to a tip position of the pump chamber, and aligning the groove position of the plunger with a port position to which a first buffer is connected by a rotational movement; (xiv) in this state, sucking the plunger to suck the first buffer into the pump chamber; (xv) rotating the plunger to align the groove of the plunger with the port to which the analytical column is connected; (xvi) expelling the plunger to push the first buffer in the pump chamber into the analytical column; By periodically repeating steps (xiii) to (xvi), the first buffer is delivered to the analytical column; Furthermore, (xvii) horizontally moving the plunger to a tip position of the pump chamber, and aligning the groove position of the plunger with a port position to which a second buffer is connected by a rotational movement; (xviii) in this state, sucking the plunger to suck a second buffer into the pump chamber; (xix) rotating the plunger to align the groove of the plunger with the port to which the analytical column is connected; (xx) discharging the plunger to push the second buffer in the pump chamber into the analytical column; By periodically repeating steps (xvii) to (xx), the second buffer is delivered to the analytical column; By switching between the above (xiii) to (xvi) and the above (xvii) to (xx) over time, sample pre-suction operation, loading of the sample onto the column, recovery of excess sample, and a "two-liquid step gradient" in which the first buffer and the second buffer are switched in a stepwise manner are performed (configuration in Table 6, configuration in FIG. 12). TIFF2025068524000007.tif51109

[0017] Figure 3 is a schematic diagram showing the structure of a plunger pump used in general HPLC. In general, it is composed of a pump chamber (26), a plunger (25), a suction side check valve (23), and a discharge side check valve (24). When the plunger (25) moves in the direction A, the pressure inside the pump chamber becomes negative, the suction side check valve (23) is opened, the discharge side check valve (24) is closed, and the buffer is sucked into the pump chamber. Conversely, when the plunger (25) moves in the direction B, the pressure inside the pump chamber becomes positive, the discharge side check valve (24) is opened, the suction side check valve (23) is closed, and the buffer in the pump chamber is pumped to the column side. The liquid delivery mechanism used in the present invention is a plunger type pump, but can deliver liquid without providing a check valve on the suction side or a check valve on the discharge side. In addition, since a general plunger type pump has a check valve, it is difficult to deliver a fluid with an extremely high or extremely low viscosity. On the other hand, the liquid delivery mechanism used in the present invention can deliver a fluid with an extremely high or extremely low viscosity, and can also transport gas. In addition, since the liquid delivery mechanism used in the present invention has multiple suction or discharge ports in the pump chamber, it is possible to aspirate / discharge multiple types of buffers and aspirate / discharge samples. Therefore, it is possible to integrate the liquid delivery / gradient mechanism (A) and the sample injection mechanism (B) of the general low-pressure step gradient system shown in FIG. 2, and it is possible to construct a simpler HPLC system. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a schematic diagram showing the configuration of a liquid chromatograph capable of executing a typical high-pressure step gradient. [Diagram 2] FIG. 1 is a schematic diagram showing the configuration of a liquid chromatograph capable of executing a typical low-pressure step gradient. [Diagram 3] 1 is a diagram showing a schematic diagram of the operation of a typical plunger pump. [Figure 4] FIG. 1 is a schematic diagram showing the configuration of a liquid chromatograph according to a first embodiment (configuration 1a) of the present invention. [Diagram 5] FIG. 1 is a schematic diagram showing the configuration of a liquid chromatograph according to a first embodiment (configuration 1b) of the present invention. [Figure 6] FIG. 1 is a schematic diagram showing the configuration of a liquid chromatograph according to a first embodiment (configuration 1c) of the present invention. [Figure 7] FIG. 1 is a schematic diagram showing the configuration of a liquid chromatograph according to a first embodiment (configuration 1d) of the present invention. [Figure 8] FIG. 2 is a schematic diagram showing the configuration of a liquid chromatograph according to a second embodiment of the present invention. [Figure 9]FIG. 1 is a diagram showing a schematic configuration of a liquid chromatograph according to a third embodiment of the present invention. [Figure 10] FIG. 1 is a diagram showing a schematic configuration of a liquid chromatograph according to a fourth embodiment of the present invention. [Figure 11] FIG. 13 is a diagram showing a schematic configuration of a liquid chromatograph according to a fifth embodiment of the present invention. [Figure 12] FIG. 13 is a diagram showing a schematic configuration of a liquid chromatograph according to a sixth embodiment of the present invention. [Figure 13] FIG. 1 is a schematic diagram showing one embodiment of the structure of a check-valve-less plunger pump used in the present invention. [Figure 14] 1 is a diagram showing a schematic diagram of each operation of the check valve-less plunger pump used in the present invention, which shows the pre-suction operation of a sample. [Figure 15] 1 is a diagram showing a schematic diagram of each operation of the check valve-less plunger pump used in the present invention, which shows a sample injection operation. [Figure 16] 1 is a diagram showing each operation of the check valve-less plunger pump used in the present invention, which shows the delivery of buffer A. [Figure 17] FIG. 13 is a diagram showing a schematic diagram of a connection state of each port in the third embodiment of the present invention. [Figure 18] FIG. 11 is a diagram showing a schematic flow of sample presuction in a third embodiment of the present invention. [Figure 19] FIG. 11 is a diagram showing a schematic flow of a sample injection operation in a third embodiment of the present invention. [Figure 20] 11 is a diagram showing a schematic diagram of a flow during buffer delivery in the third embodiment of the present invention, in which FIG. 11A shows the delivery of Buffer_A and FIG. 11B shows the delivery of Buffer_B. [Figure 21] 10A and 10B are diagrams showing a schematic diagram of a flow of a buffer solution during delivery in the third embodiment of the present invention, Fig. 10C showing delivery of Buffer_C. [Figure 22]FIG. 13 is a diagram showing a series of sequences including sample presuction, sample injection, and separation using a step gradient of buffers A, B, and C in the third embodiment of the present invention. [Figure 23] FIG. 13 is a diagram showing a series of sequences including sample presuction, sample injection, and separation using a step gradient of buffers A, B, and C in the third embodiment of the present invention. [Figure 24] FIG. 1 is a diagram showing a schematic diagram of the operation when performing a variable ratio low pressure gradient using the check valve-less plunger pump used in the present invention (from the initial state to the aspiration of Buffer_A). [Diagram 25] FIG. 1 is a schematic diagram showing the operation of a variable ratio low pressure gradient using the check valve-less plunger pump used in the present invention (from suction of buffer_B to discharge of buffer_A / B). [Figure 26] 1 is a schematic diagram showing the operation of a variable ratio low pressure gradient using the check valve-less plunger pump used in the present invention, where FIG. a shows the case of two-division suction and FIG. b shows the case of four-division suction. [Figure 27] FIG. 13 is a schematic diagram showing the operation of a check-valve-less plunger pump used in the present invention to produce a pseudo-linear gradient by a variable ratio low-pressure gradient. [Figure 28] FIG. 1 shows the measurement results in Example 1 (three-step gradient using buffers A, B, and C). FIG. a shows a representative example of the results, and FIG. b shows five measurement results superimposed on each other. [Figure 29] FIG. 1 shows the measurement results in Example 2 (three-step gradient with buffers A and C). FIG. 1 shows a representative example of the results, and FIG. 1 shows five measurement results superimposed on each other. [Diagram 30] FIG. 13 is a diagram showing the set values ​​in Example 3 (multi-step gradient using buffers_A and C). FIG. 13A is a diagram showing the ratio of buffer_C over time, and FIG. 13B is a diagram showing the state inside the pump chamber. [Diagram 31]FIG. 1 shows the measurement results in Example 3 (multi-step gradient with buffers A and C). FIG. a shows a representative example of the results, FIG. b shows five measurement results superimposed, and FIG. c shows the error relative to the set value. [Diagram 32] It is a figure showing the measurement results in Example 4. Figure a shows the results of injecting 10 μL, 25 μL, 50 μL, 100 μL, 150 μL, 300 μL, and 600 μL from the top, and Figure b shows the results superimposed on the same axis. [Diagram 33] 1 shows the measurement results in Example 4. The horizontal axis indicates the injection amount, and the vertical axis indicates the area value. FIG. a shows the entire region where the positioning was performed, and FIG. b shows an enlarged view of the vicinity of the origin. [Diagram 34] 1 shows the measurement results in Example 5. FIG. a is a representative example of the results, FIG. b is a graph in which five measurement results are overlapped, and FIG. c is an enlarged view of the vicinity of the injection site. [Diagram 35] FIG. 1 shows an example of the results of measurement using a glycohemoglobin analyzer manufactured by Tosoh Corporation. [Diagram 36] FIG. 13 is a diagram showing a liquid transfer sequence in Example 6. [Figure 37] 1 shows the results of measurements of actual samples in Example 6. Panel a shows the results of measurements of HbA1c control samples at level 1, panel b shows the results of measurements of the same samples at level 2, and panels c and d show the results of measurements of whole blood. [Figure 38] FIG. 13 is a diagram showing a liquid transfer sequence in Example 7. [Figure 39] 1 shows the measurement results of actual samples in Example 7. Panel a shows the measurement results of HbA1c control sample level 1, panel b shows the measurement results of the same sample level 2, and panels c and d show the measurement results of whole blood. [Diagram 40] FIG. 1 is a diagram comparing the results of Example 6 and Example 7. EXAMPLES

[0019] The effects of the present invention were verified by the following method. In this example, a DP-11 made by Flom Corporation was used as a check valve-less plunger pump. The pump serves as a liquid delivery mechanism, a low pressure gradient mechanism, and a sample injection mechanism. In addition, a UV-8020 made by Tosoh Corporation was used as an ultraviolet-visible detector. The structure of the pump is shown in FIG. The pump is composed of a pump chamber with six liquid passage ports at 60° intervals, and a plunger capable of reciprocating linear drive and rotational motion. The plunger also has a groove that allows liquid to pass in the linear drive direction. In the case of a normal plunger type pump, check valves are arranged above and below the pump chamber, and liquid flows from the bottom to the top of the pump chamber as the plunger reciprocates. In the case of this pump, the plunger groove is aligned with the groove position of the pump chamber to perform the suction / discharge operation. When the plunger is sucked in at the position of a specific liquid passage port, the fluid is sucked in from that liquid passage port. Next, the plunger is rotated to another port position and the plunger is discharged, and the fluid is discharged to the port. In other words, the port to be sucked in and the port to be discharged can be selected, making it possible to send liquid even without a check valve. In addition, the pump chamber has six through ports at 60° intervals, so the port to be sucked in and the port to be discharged can be freely selected. Example 1

[0020] In this embodiment, in order to enable switching between three types of buffer, the following flow path configuration was adopted (see FIG. 5), which corresponds to the first embodiment (configuration 1b) of the present invention. Port 1: Buffer_A suction port Port 2: Buffer B suction port Port 3: Buffer C suction port Port 4: Unused Port 5: Unused Port 6: Discharge port to the resistance tube Next, the delivery of Buffer_A will be described. The plunger (16) is moved horizontally to the tip position of the pump chamber (20), and the groove (21) is rotated to the position of port 1. In this state, the plunger (16) is sucked in. The plunger is rotated 60 degrees to align the plunger groove position with port 6. In this state, the plunger is pushed in, and Buffer_A is delivered to the resistance tube. By periodically repeating this operation, Buffer_A is delivered to the resistance tube. The delivery flow rate is determined by the plunger's suction / discharge capacity and its speed. Similarly, the delivery of Buffer_B will be described. The plunger (16) is moved horizontally to the tip position of the pump chamber (20), and the position of the groove (21) is rotated to the position of port 2. In this state, the plunger (16) is sucked in. The plunger is rotated 120° to align the position of the plunger groove with port 6. In this state, the plunger is pushed in, and Buffer_B is delivered to the resistance tube. By periodically repeating this operation, Buffer_B is delivered to the resistance tube. The delivery flow rate is determined by the suction / discharge capacity and speed of the plunger.

[0021] Similarly, the delivery of Buffer_C will be described. The plunger (16) is moved horizontally to the tip position of the pump chamber (20), and the groove (21) is rotated to the position of port 3. In this state, the plunger (16) is sucked in. The plunger is rotated 180 degrees to align the plunger groove position with port 6. In this state, the plunger is pushed in, and Buffer_C is delivered to the resistance tube. By periodically repeating this operation, Buffer_C is delivered to the resistance tube. The delivery flow rate is determined by the suction / discharge capacity and speed of the plunger.

[0022] In this example, the basic performance of the low-pressure step gradient was verified without using a column. The basic configuration was as shown in Figure 5, but instead of the analytical column (13), a coiled PEEK piping with an outer diameter of 1 / 16', an inner diameter of 0.75 mm ID, and a length of 2 m was used as the resistance tube. The solution was pumped in the order of Buffer_A → Buffer_B → Buffer_C → Buffer_A for 10 minutes each, and the pumping operation was confirmed by the fluctuation of the detector signal. Table 7 shows the analysis conditions, and Table 8 shows the pump operation. TIFF2025068524000008.tif42147

[0023] TIFF2025068524000009.tif34170

[0024] Figure 28 shows the results of five measurements. The horizontal axis is time (min) and the vertical axis is detector output (mABU). Figure a shows a typical result, and Figure b shows the results of five measurements overlaid on top of each other. Table 9 lists the detector output when it becomes constant. Note that *2 in Table 9 indicates the ratio of liquid B to the height when liquid C is present. As can be seen from the figure, the detector output changes in steps, and approximately the same profile was obtained in the five measurements. The Cv% is also an extremely good value of less than 0.1%. The liquid B% is 47.6%. It should be 50%, but this difference is due to buffer adjustment error. TIFF2025068524000010.tif58170

[0025] In this example, a step gradient with three solutions switched between buffers A, B, and C was verified, but a step gradient with up to five solutions switched can be implemented by using ports 4 and 5, which were not used this time. Conversely, if port 3 is left unused, a step gradient with two solutions switched can also be implemented, and the number of buffers is not limited. Example 2

[0026] In Example 1, a method for switching buffers stepwise was described. In this Example, a method for simply delivering a buffer with an arbitrary composition was verified. This configuration corresponds to the first embodiment (configuration 1a) of the present invention. The flow path configuration was as follows (see Figure 4). Port 1: Buffer A intake port Port 2: Buffer C aspiration port Port 3: Unused Port 4: Unused Port 5: Unused Port 6: Discharge port to the resistance tube The measurement conditions and basic operation are the same as those in the first embodiment, so a detailed explanation will be omitted here, but the details of the operation are as shown in FIGS.

[0027] By performing the plunger suction operation from multiple ports, any composition can be easily created. For example, if you want to deliver a 50% composition of Buffer A (■) and Buffer C (□), as shown in Table 10, Buffer A (■) is aspirated from Port 1 in an amount equal to 1 / 2 the plunger stroke volume (300 μL in this case), then it is rotated to Port 2, and 1 / 2 the amount of Buffer C (□) is aspirated. After that, the plunger is rotated to Port 6, and the entire amount is discharged, which essentially delivers a 50% composition of Buffer A (■) and Buffer C (□) (■□) (see Figure 26a). By performing this operation periodically, a fluid with a 50% composition of Buffer A and Buffer C flows at a constant flow rate. TIFF2025068524000011.tif45100

[0028] TIFF2025068524000012.tif40170

[0029] Figure 29 shows the results of five measurements. The horizontal axis is time (min) and the vertical axis is the detector output (mABU). Figure a shows a typical result, and Figure b shows five measurements overlaid on top of each other. Table 12 also shows the output when the detector output becomes constant. Note that *2 in Table 12 shows the ratio of the height of the solution sent at A / C:1 / 1 to the height of solution C. As can be seen from the figure, the detector output changes in a step-like manner. Also, the output in the area where Buffer_A / C is sent at a 1:1 ratio shows an almost constant value, and it can be seen that Buffer_A / C is mixed and sent at a 1:1 ratio as set. Also, almost the same profile was obtained for the five measurements. Regarding Cv%, while it is 0.014% in the area where a single composition is sent (Buffer_C), it is about 0.025% even in the area where Buffer_A / C is 1:1, which is equivalent, and is not a problem in use. In addition, the theoretical output percentage for liquid C in this region is 50%, but in this measurement it was about 49.8%, which is almost the same as the theoretical value, and good precision was obtained. TIFF2025068524000013.tif55152

[0030] Inside the plunger, Buffer_A and Buffer_C are segmented, so the fluid sent to the analytical column is inevitably in a segmented state. To solve this problem, when aspirating multiple buffers in one stroke, it is desirable to eliminate the segmented state by arranging a device (mixer) that can mix fluids dynamically or statically between the discharge port (port 6 in this case) and the analytical column, as shown in Figure 24. A static mixer has a distorted flow path structure, and increases the mixing efficiency by making the fluid flow irregular. In contrast, a dynamic mixer increases the mixing efficiency by placing a stirrer in the liquid storage space and changing the magnetic field from the outside to rotate the rotor. In any case, there are no limitations on the type as long as the fluids can be mixed.

[0031] If you want to further improve the accuracy, simply increase the number of divisions in one aspiration (see Figure 26b). When the aspiration is divided into four, it will look like Table 13. 1 / 4 of the plunger stroke volume (300 μL in this case) of Buffer_A (■) is aspirated from port 1, rotated to port 2, 1 / 4 of Buffer_C (□) is aspirated, rotated to port 1 and 1 / 4 of Buffer_A (■), rotated to port 2, 1 / 4 of Buffer_C (□) is aspirated, then rotated to port 6 and the entire volume is discharged, resulting in the delivery of a 50% composition of Buffer_A (■) and Buffer_C (□) (■□■□). TIFF2025068524000014.tif33140

[0032] In this example, the results are shown for two-part suction, but the number of parts is not limited. The more parts are divided, the more efficient the mixing, and the more accurate the measurement during analysis. However, there are also negative aspects, such as the time required for the suction operation and the complexity of the operation, so it is not good to increase the number of parts more than necessary, and two-part suction is preferable. Example 3

[0033] In this example, the performance of a pseudo linear gradient was verified. This configuration corresponds to the first embodiment (configuration 1a) of the present invention.

[0034] In the second embodiment, a fluid with a 50% composition of buffers A and C is delivered at a constant flow rate, but by changing the suction ratio of buffers A and C over time, it is also possible to perform a pseudo-linear gradient. For example, by changing the chronological order of buffers A / C: 100 / 0 → 90 / 10 → 80 / 20 → 70 / 30 → 60 / 40 → 50 / 50 → 40 / 60 → 30 / 70 → 20 / 80 → 10 / 90 → 0 / 100, the composition of buffer C can be changed in 10% steps as shown in FIG. 27. If a mixer is placed between the discharge port and the analytical column as shown in FIG. 24, the steps are relaxed, and a pseudo-linear gradient can be performed.

[0035] The flow path configuration was as follows (see Figure 4). Port 1: Buffer_A suction port Port 2: Buffer C suction port Port 3: Unused Port 4: Unused Port 5: Unused Port 6: Discharge port to the resistance tube The measurement conditions and basic operation are the same as in Example 2, so a description thereof will be omitted here. TIFF2025068524000015.tif102156 Figure 30 shows the set values ​​for this gradient. Figure a shows the time (min) on the horizontal axis and the percentage of buffer C on the vertical axis, and Figure b shows a schematic diagram of the percentage of buffer C in the pump chamber. Figure 31 shows the measurement results. The horizontal axis shows time (min) and the vertical axis shows the detector output (mABU). Figure 31a shows the results of a typical example, and Figure 31b shows five measurements taken at the same time. Figure 31c shows the C solution % (set value) on the horizontal axis and the output at each step (left axis) and the difference from the previous step (right axis) on the vertical axis. The detector output changes in a step-like manner, and the same profile is obtained even after five measurements. As can be seen from FIG. 31c, in the region where C% is small (near the start point) and the region where A% is small (near the end point), the amount of change in the detector is lower than the set value, but in the mixed region of 20 to 80%, the difference between steps shows an almost constant value, and it is understood that it is operating as set. In the method of variable amount suction in this embodiment, first Buffer_A is sucked, then Buffer_B is sucked, and it passes through the port of Buffer_A and is discharged to the column (resistance tube) connection port, so it is presumed that the accuracy will be slightly reduced when the ratio is extremely low. However, since the obtained profile has good reproducibility as described above, it is considered that the measurement accuracy is sufficient in actual analysis. Example 4

[0036] In this embodiment, the basic performance of the sample injection operation was verified. This configuration corresponds to the second embodiment of the present invention.

[0037] The flow path configuration was as follows (see FIG. 8). Port 1: Buffer_A suction port Port 2: Unused Port 3: Unused Port 4: Discharge port to drain Port 5: Sample suction port Port 6: Discharge port to the resistance tube The basic liquid delivery operation is the same as in the first embodiment, and therefore the explanation will be omitted here. Only the sample injection operation will be explained.

[0038] First, the sample pre-suction operation will be described. A sample is set in port 5, but in the initial stage, the inside of the piping of port 5 is not filled with sample. The plunger (16) is moved horizontally to the tip position of the pump chamber (20), and the position of the groove (21) is rotated to the position of port 5. In this state, the plunger (16) is sucked in until the tip of the sample reaches at least inside the pump chamber, and the inside of the piping of port 5 is filled with sample. Next, the plunger is rotated 60° counterclockwise to align the groove position of the plunger with port 4. In this state, the plunger is pushed in and the sample is discharged into the drain. If the inside of the piping is not filled with sample with one stroke of the plunger, the above process is carried out multiple times (see FIG. 14).

[0039] Next, the loading of the sample onto the column will be described.

[0040] The plunger (16) is moved horizontally to the tip of the pump chamber (20), and the groove (21) is rotated to the position of port 5. In this state, the plunger (16) is sucked in by the volume to be loaded onto the column. The plunger is rotated 60° clockwise to align the plunger groove with port 6. In this state, the plunger is pushed in to directly load the sample onto the column. If the amount of sample loaded is insufficient with one stroke of the plunger, the above process is repeated multiple times (see Figure 15). TIFF2025068524000016.tif42152

[0041] Next, the plunger (16) is moved horizontally to the tip position of the pump chamber (20), and the position of the groove (21) is rotated to the position of port 1. In this state, the plunger (16) is sucked. The plunger is rotated 60 degrees to align the position of the plunger groove with port 6. In this state, the plunger is pushed in to deliver Buffer_A to the resistance tube (see FIG. 16).

[0042] The system configuration was the same as above, except that a resistance tube was used instead of a column. The sample used was a 0.06 mM aqueous solution of p-hydroxybenzoic acid.

[0043] First, Buffer_A is pumped for a while, then the sample is aspirated and discharged into the column (resistance tube). Buffer_A is then pumped, and the injection operation is confirmed by the signal fluctuation of the detector. The sample aspirated amounts were 10μL, 25μL, 50μL, 100μL, 150μL, 300μL, and 600μL. Note that since the maximum plunger capacity is 300μL, in the case of a 600μL load, the plunger was moved back and forth twice (300μL x 2). TIFF2025068524000017.tif56148

[0044] Figure 32 shows the results when the injection amount was changed. Figure a shows the individual plots, and Figure b shows the overlapped plots. The horizontal axis shows time (min), and the vertical axis shows the detector output (mABU). It can be seen that the peaks become larger as the injection amount increases (see Table 16).

[0045] Figure 33 shows the linearity of the load. Figure a shows the whole picture, and Figure b shows an enlarged view of the area near the origin. Overall, R 2At 0.999 or more, good linearity is shown. However, the result does not pass through the origin, and there is an intercept of about 900. In the region with a small load amount, the error is large when approximating with a linear equation. This is partly due to the resolution of the pump used in this example, but another factor is that in this sample loading sequence, the liquid delivery is stopped once, the sample is loaded, and then liquid delivery is resumed, which causes some baseline fluctuation in the detector and makes it difficult to calculate the peak area correctly. For reference, when approximating with a linear equation that passes through the origin after excluding the data at 10 and 25 μL, R 2 = 0.9971, which is an improvement (see Table 17).

[0046] TIFF2025068524000018.tif46117 TIFF2025068524000019.tif48149In this embodiment, the plunger capacity is 300 μL, so the amount of sample aspirated / discharged (loaded amount onto the column) is 300 μL. As shown in this embodiment, if you want to aspirate / discharge a larger amount of sample, you can do so by repeating this process multiple times. Example 5

[0047] In this example, verification was carried out in a system close to the actual analysis flow. Sample loading and analysis using a step gradient of three liquids were assumed. This corresponds to the third embodiment of the present invention. The flow path configuration was as follows (see FIG. 9). Port 1: Buffer_A suction port Port 2: Buffer B suction port Port 3: Buffer C suction port Port 4: Sample discharge port (drain) Port 5: Sample suction port Port 6: Discharge port to the resistance tube The switching operation between buffers A, B, and C and the sample injection operation are the same as those in the previous embodiment and will not be described here, but the details of the operation are as shown in FIG.

[0048] TIFF2025068524000020.tif53150 TIFF2025068524000021.tif46146 Figure 34 shows the results of five measurements. The horizontal axis is time (min) and the vertical axis is detector output (mABU). Figure a shows a typical result, Figure b shows five measurement results superimposed, and Figure c shows an enlarged view of the sample peak region. Table 21 lists the detector output when it becomes constant, and Table 22 lists the peak heights and areas. Note that *2 in Table 21 indicates the ratio of Liquid B to the height when Liquid C is used.

[0049] The peak appearing at approximately 1 minute is the sample peak, and it can be seen that thereafter, buffers are switched in the order of Buffer_A, Buffer_B, and Buffer_C in 10 minute steps. As can be seen from the figure, the detector output changes in a step-like manner, and almost the same profile is obtained even after five measurements. The Cv% is also very good, at less than 0.1%. The B solution % is 46.7%. It should be 50%, but this difference is due to the adjustment error of the buffer (similar to Example 1).

[0050] Regarding the sample injection reproducibility Cv%, good values ​​were obtained, with 0.49% for peak height and 1.7% for peak area. TIFF2025068524000022.tif54141

[0051] TIFF2025068524000023.tif50125

[0052] This embodiment is a form simulating everything from sample injection to a three-liquid low-pressure step gradient. As shown in Example 2, a low-pressure step gradient of any composition can be performed using two types of buffers. Also, as shown in Example 3, it is possible to perform a pseudo-linear gradient by combining multiple low-pressure step gradients using two types of buffers. Example 6

[0053] In this example, the basic performance of the low-pressure step gradient was verified by using an actual column. The basic configuration was the same as in Example 5. As the analytical column, TOYOPEARL, a bulk gel for separation, manufactured by Tosoh Corporation was packed in an empty column and used. The separation conditions were as shown in Table 23, and the sequence was as shown in Table 24.

[0054] The flow path configuration was as follows (see FIG. 17). Port 1: Sample discharge port (drain) Port 2: Sample suction port Port 3: Suction port for B liquid Port 4: Suction port for liquid A Port 5: Outlet port to analytical column Port 6: Suction port for C liquid TIFF2025068524000024.tif99159

[0055] TIFF2025068524000025.tif76152

[0056] FIG. 22 is a flow chart showing the overall flow, and FIG. 36 is a diagram that expresses this sequence in an easy-to-understand manner. Also, FIGS. 18 to 21 are schematic diagrams showing the flow paths in each process. After sample injection, Buffer_A is pumped for 4 minutes, followed by Buffer_B for 16 minutes, followed by Buffer_C for 15 minutes, resulting in a step gradient with three composition switching. Since there is a single mechanism for sample injection and buffer pumping, no buffer is pumped to the column during the sample injection operation. 200 μL of sample is aspirated and pushed out and added directly to the column.

[0057] Figure 37 shows the chromatograms obtained under these conditions. Figure a shows the results for control sample level 1 (CTL_1), Figure b shows the results for control sample level 2 (CTL_2), Figure c shows the results for real blood sample #1, and Figure d shows the results for real blood sample #2. The column used here is a cation exchanger, and is filled with a gel that is also used in the analysis of glycated hemoglobin (HbA1c) in blood. Figure 35 shows an example of blood sample measurement using the Tosoh Corporation automatic glycohemoglobin analyzer HLC-723G11.

[0058] Fractions of A1A, A1B, F, L-A1c, s-A1c, and A0 were obtained, and HbA1c% was calculated from the area% of the s-A1c peak (black). The gel used in this example has a large particle size and its performance is significantly lower than that of HLC-723G11, but a separation pattern similar to that of HLC-723G11 was obtained. Table 25 shows the measurement results. CTL_1 and CTL_2 are standard samples with low and high HbA1c%, and have different s-A1c peak area%. In the results of this example, the peak designated as peak 2 is presumed to contain the s-A1c peak. The differences were 12.67% for CTL_1 and 15.5% for CTL_2, and it can be assumed that these adequately reflect the results. TIFF2025068524000026.tif108156

[0059] Table 26 shows the reproducibility (n=5) of this example. Here, the measurement results of CTL_1 are shown. Cv of 0.02-0.9% was obtained for elution time, 0.8-2.1% for peak height, and 0.6-0.9% for peak area was obtained. Between peaks 2 and 3, peak 2, which elutes earlier, tends to have worse reproducibility. One of the reasons for this is presumably that in the mechanism of the present invention, the liquid delivery is stopped during the sample injection operation, but this is considered to be at a level that does not cause any particular problems in fractionation, etc. TIFF2025068524000027.tif57156 Example 7

[0060] In this example, a three-step step gradient was performed using two types of buffers, and the following flow path configuration was used for the purpose of making it possible to load the sample onto the column (see FIG. 10). The results are from the analysis of Example 6 using two types of buffers. The flow path configuration was as follows (see FIG. 38). Port 1: Suction port for liquid A Port 2: Suction port for C liquid Port 3: Sample suction port Port 4: Sample discharge port (drain) Port 5: Sealed Port 6: Outlet port to analytical column TIFF2025068524000028.tif88149

[0061] TIFF2025068524000029.tif56147

[0062] Figure 38 is a diagram that clearly expresses this sequence. From the sample injection, Buffer_A is delivered for 4 minutes, Buffer_A and Buffer_C are delivered at a ratio of 8.82:1 for 16 minutes, and then Buffer_C is delivered for 15 minutes, resulting in a step gradient with three composition switching. Since there is a single mechanism for delivering sample injection and buffer, no buffer is delivered to the column during the sample injection operation. 200 μL of sample is aspirated and pushed out and added directly to the column.

[0063] In Example 6, a phosphate buffer containing 45 mM NaCl was used as buffer_B, but here, a phosphate buffer containing 400 mM NaCl (buffer_C) and a phosphate buffer containing no NaCl (buffer_A) are used, so that one suction operation of the plunger is divided at a ratio of buffer_A / C = 8.82 / 1, resulting in almost the same conditions as in Example 6.

[0064] Chromatograms obtained under these conditions are shown in Figure 39. Figure a shows the measurement results for control sample level 1 (CTL_1), Figure b shows the measurement results for control sample level 2 (CTL_2), Figure c shows the measurement results for real blood sample #1, and Figure d shows the measurement results for real blood sample #2. It can be seen that the same separation as in Example 6 is obtained. The measurement results are shown in Table 29. CTL_1 and CTL_2 are standard samples with low and high HbA1c%, and are samples with different s-A1c peak area%. In the results of this example, it is estimated that the peak called peak 2 contains the s-A1c peak. There was a difference of 12.0% for CTL_1 and 16.4% for CTL_2, and it can be assumed that this adequately reflects the results.

[0065] FIG. 40 is a diagram showing the measurement results of Examples 6 and 7 side by side. The difference in elution time between Peak 2 (L-A1c, s-A1c) and Peak 3 (A0) is shown. Legend: 3_Buffer shows the results of Example 6, and Legend: 2_Buffer shows the results of Example 7. As can be seen, there is almost no difference in elution time. In other words, this shows that good results can be obtained even if measurements are performed by adjusting the composition arbitrarily using two buffers, as was done in this example. TIFF2025068524000030.tif106147

[0066] Table 30 shows the reproducibility (n=5) of this example. Here, the results of CTL_2 are shown. Cv of 0.0-1.0% was obtained for elution time, 0.7-2.1% for peak height, and 0.6-1.0% for peak area was obtained. Between peaks 2 and 3, peak 2, which elutes earlier, tends to have worse reproducibility. This is presumably due in part to the fact that in the mechanism of the present invention, the liquid delivery is stopped during the sample injection operation, but this is considered to be at a level that does not cause any particular problems in fractionation, etc. Also, reproducibility at a level roughly equivalent to that of Example 6 was obtained. TIFF2025068524000031.tif56156

[0067] As explained above, by using the method of the present invention, the optimum configuration can be selected according to the purpose. In addition, one of the major features of the present invention is that a liquid chromatograph, which previously consisted of multiple functional devices, can be integrated into a single mechanism. [Explanation of symbols]

[0068] 1. First buffer 2. Second Buffer 3. Third Buffer 4. Fourth Buffer 5. The fifth buffer 6.Shut-off valve 7. Liquid delivery pump 8. Drain 9. Sample 10. Measuring Syringe 11. Sample injection valve 12. Sample injection mechanism 13. Column 14. Detectors 15. Check valve-less pump 16. Plunger (general) 17. Ball screw 18. Motor 19. Pump head 20. Pump Room 21. Groove 22. Median Strip 23. Inlet check valve 24.Outlet side check valve 25. Plunger (present invention) 26. Pump Room 27. Liquid delivery pump (missing) 28. Cleaning fluid 29. Mixer 30. Column oven 31. Deaeration device 32. Stop plug

Claims

1. a pump chamber in which a plurality of through holes are provided; A plunger that reciprocates and rotates within the pump chamber; A simplified liquid chromatograph having a liquid delivery section comprising: The polygon formed by the connection lines of the through holes is a regular polygon inscribed in the cross section of the pump chamber. and, The plunger has a groove that is aligned horizontally with the through hole. A simplified liquid chromatograph.

2. Among the plurality of through holes, one through hole is a discharge port and at least two through holes are suction ports.

2. The simplified liquid chromatograph according to claim 1.

3. different buffers are delivered through at least two through-holes serving as the suction ports; 2. The simplified liquid chromatograph according to claim 1.

4. Among the plurality of through holes, one through hole is used as a waste port.

2. The simplified liquid chromatograph according to claim 1.

5. One of the through holes is connected to a cleaning liquid.

2. The simplified liquid chromatograph according to claim 1.

6. Regarding the plunger, During reciprocating motion, the pump moves horizontally to the tip position of the pump chamber. and, During the rotational movement, the groove stops at the position of the through hole and is aligned with the through hole.

2. The simplified liquid chromatograph according to claim 1.

7. After aligning the plunger with the through hole, At least two through holes serving as suction ports are connected to different buffers; Connecting the through-hole serving as the discharge port to an analytical column; 2. The simplified liquid chromatograph according to claim 1.

8. 2. The simplified liquid chromatograph according to claim 1, wherein the number of said through-holes is six.

9. For the at least two through holes serving as the suction ports, The liquid delivery ratio between any two through holes is 100 / 0 to 0 / 100; 2. The simplified liquid chromatograph according to claim 1.

10. There are two through holes serving as suction ports, and the liquid delivery speed is constant; The liquid is delivered to one through hole in an arithmetic progression with a positive tolerance in terms of the delivery time, and The other through hole is fed in an arithmetic progression with a corresponding reciprocal tolerance for the feeding time. A step having a linear gradient property, 2. The simplified liquid chromatograph according to claim 1.

11. Switching the type of buffer delivered to the column in a stepwise manner; 2. The simplified liquid chromatograph according to claim 1.

12. The liquid delivery unit is a plunger pump.

2. The simplified liquid chromatograph according to claim 1.

13. A method for delivering a plurality of liquids in a step gradient using the simplified liquid chromatograph according to any one of claims 1 to 12.

14. (i) horizontally moving the plunger to a tip position of the pump chamber, and aligning the groove position of the plunger with the position of a port to which a first buffer is connected by a rotational movement; (ii) drawing the plunger to draw a first buffer into the pump chamber; (iii) aligning the plunger with a port to which an analytical column is connected by a rotating movement; (iv) expelling the plunger to push the first buffer in the pump chamber into an analytical column; 14. The method of claim 13, further comprising cyclically repeating steps (i) to (iv) to deliver each of the second through n buffers to the analytical column.

15. By rotating the plunger, the groove of the plunger is aligned with a port to which a cleaning liquid is connected; Suctioning the plunger to draw cleaning liquid into the pump chamber; The method of claim 14 further comprising:

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