JP2003098166A - Method for feeding liquid of liquid chromatography and gradient method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prevent irregularities in liquid feeding by combining two liquid feeding units or more each comprising a liquid feeding device and performing control between the units. SOLUTION: The at least two liquid feeding units each comprising a liquid feeding pump device for feeding a fluid or its mixture are made to communicate with each other. Each liquid feeding unit is provided with a pressure sensor for monitoring pressure during liquid feeding. The first liquid feeding unit performs discharge at a desired set value. At least one another liquid feeding unit senses the pressure of the liquid feeding unit during its discharge. Preliminary pressurization is performed according to the pressure of discharge of the first liquid feeding unit. A desired discharge pressure is obtained. Then discharge is performed at a desired set value.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a liquid feeding method and apparatus for liquid chromatography, and a gradient method.

[0002]

2. Description of the Related Art The use of high performance liquid chromatography with a small flow rate is expanding as the needs for analysis of trace substances in the environment and biological samples increase. It goes without saying that the analytical column, which is the key to separation, must be miniaturized, but in addition, high performance is required for liquid delivery as well, and it is possible to deliver a small flow rate, and further stable liquid delivery under high pressure conditions. Two things that are possible are required of the liquid delivery pump. Currently, the range of minute flow rate in liquid chromatography is several nL / min to several μL / min or several tens.
μL / min is common. There are various types of pumps, but the widely used one is a double-plunger type pump that uses two plungers.
The double plunger pump is designed so that both plungers alternately discharge and suck, and the combined flow rate is constant, and is suitable for stable continuous liquid transfer.

However, since a normal double plunger pump is driven by one motor via an interlocking cam, the movements of the two plungers are not independent, so under the condition that pressure is applied to the outlet side. The combined flow rate is not constant due to discharge delay. For example, when one plunger finishes the discharge process and enters the suction process, another plunger finishes the suction process and starts at atmospheric pressure, and the compressibility of the solvent and the elasticity of the polymer It takes time for the internal pressure of the pump chamber to rise to the system pressure (pressure at the pump outlet side) during the initial stage (compression process) of the discharge process due to deformation of the plunger seal (compression at high pressure), etc. The outlet check valve of the pump is not opened, the fluid is not immediately discharged from the pump chamber, and continuous liquid transfer is not substantially performed. 1
It is possible to design the interlocking cam so that the second plunger is started earlier before the second plunger finishes discharging and the discharging process is not delayed under a specific pressure condition. 2 if the pressure is lower than the specified pressure
The plunger of the book simultaneously delivers fluid to the system. In addition, if the actual operating pressure is higher than the specific pressure, a delay in discharge still occurs. Therefore, in the double plunger pump, stable continuous liquid transfer is practically impossible when one motor and an interlocking cam are used.

US Pat. No. 5,253,981 and JP-A-7-
Japanese Patent No. 167846 discloses that a suction step for all the steps of liquid transfer is rapidly performed to shorten the time interval during which the liquid supply is stopped during the suction step, and the plunger is rapidly advanced in the initial stage of the discharge step. To stop the pressure drop. At that time, the system pressure will overshoot from a stable pressure. Then, a high-pressure grunge system has been proposed in which a gradient system is constructed by combining the above-mentioned liquid transfer mechanisms based on such a concept.

However, since such a system does not have a mechanism capable of stable and continuous liquid feeding, a large pulsating flow is generated and reproducibility is deteriorated when performing continuous analysis. When using a mechanism such as a damper to suppress pulsating flow, the dead volume of the system increases extremely, the change over time of the mobile phase composition reaching the column inlet is significantly delayed, and it is still possible to follow the program. I can't. In order to suppress the adverse effect of the dead volume of the system, it is necessary to flow the mobile phase composition in the initial stage of the gradient at a constant flow rate faster than the set flow rate before starting the gradient program.

US Pat. No. 5,360,320 discloses a system for delivering a supercritical fluid. The above system is composed of multiple pumps, and each pump generates the liquid delivery pressure of the fast pump before the liquid is actually delivered. It has been proposed that a plurality of solvents or supercritical fluids are allowed to flow and merge to maintain a correct mixture composition without causing pressure fluctuation.

However, the disclosed control method is a combination of the constant pressure control mode and the constant flow rate control mode, and the check valve that opens and closes due to the pressure difference is controlled so as not to be disturbed by the system pressure and the system flow rate. It is impossible. However, in the case of continuous liquid transfer, such a disturbance occurs when one stroke of the plunger ends and another plunger discharges instead, which affects the reproducibility of the analysis result.

US Pat. No. 5,637,208 and International Patent Application No. In WO98 / 55199, a liquid delivery device is proposed in which a valve actuator and a control device for operating the valve actuator are used instead of the check valve, and the liquid delivery device is composed of two or more pump systems each having an independent driving unit. There is.

A system in which a pressure sensor is provided in each pump system so that the liquid in the syringe of the pump sucked in the offline state is compressed while being monitored by the pressure sensor and the operation in the online state is not disturbed And a control system is disclosed. Also, one pump system, including the first and second pistons, alternately delivers compressed liquid. The gradient chromatography system is composed of at least two pump systems and the inside of each pump system
Only the discharge of compressed liquid is disclosed (ie between two pistons).

However, what is disclosed about the control between pump systems is to refill one pump system while it is operating while the other is off-line, and then bring the refill pump system and the receiving system to the same state. It is a point to control to do. No control is disclosed between the two pump systems. However, since the valve is interposed between the pump system and the receiving system, the shock cannot be reduced to zero. Furthermore, it does not support a specific control method corresponding to a complicated configuration such as a gradient chromatography system, or control of rapid equilibration during gradient elution. Therefore, the use in a gradient system is not disclosed and cannot be carried out.

The gradient elution method in liquid chromatography has been widely applied as an extremely effective separation and analysis method. When the sample contains components with widely different distribution ratios, the components with large distribution ratios have a wide peak width,
Not only is the height low, but the time required for analysis itself is long. In such a case, at the beginning of the analysis, by weakening the solvent strength and gradually increasing the solvent strength,
The analysis time as a whole can be shortened and the peak width can be kept narrow without impairing the separation of each component. The equipment related to the gradient elution method is a low pressure (inlet) of the liquid delivery pump.
By mixing on the side or by mixing at a high pressure (outlet), there can be roughly classified into a low pressure gradient and a high pressure gradient.

[0012]

A high-pressure gradient system in liquid chromatography uses a plurality of liquid feed pumps, but in the prior art, the system flow rate (total flow rate) is several units of liquid feed pumps. Further instability is due to the synergistic or counteracting effect of the disturbance. Since each pump in the gradient system always delivers at different flow rates, it has different periods of turbulence and the phase is different depending on the position of the plunger of each pump when starting the gradient program. Even if the gradient program is used, the programmed eluent composition is not always obtained, and repeatability cannot be obtained.

When one stroke of one liquid feeding unit is completed during the analysis according to the gradient program and the liquid is fed by switching to another unit feeding the same eluent, a pressure shock is inevitably generated. . If the retention time of the analysis target peak is unfortunately overlapped with that time,
The result of analysis must be inaccurate.
Further, even if the same analysis is performed again, the time when the pressure shock occurs is determined by the position of the plunger and is difficult to predict, so that it is not reproducible.

Further, in gradient elution in liquid chromatography, when the composition of the eluent is continuously changed so that the elution power becomes weak to strong, the pump for delivering the solvent having a strong elution power is zero or extremely high. In many cases, starting from a low flow rate. When the flow rate starts from zero, the pump that delivers a solvent with a strong elution power rises from the initial pressure inside the pump chamber from zero, and since the flow rate is low at the initial stage of the gradient program, the pump outlet side pressure (system pressure) It takes a long time to rise, and the discharge is significantly delayed. Not only does the solvent with a high elution force delay delivery to the system, but the flow rates of the other pumps continuously decrease based on the gradient program, so the total flow rate of the system also decreases. Under such conditions, the eluent composition based on the gradient program is difficult and repeatability cannot be obtained.

[0015]

The present invention provides a liquid chromatography liquid feeding method and apparatus by combining two or more liquid feeding units for stable continuous liquid feeding without discharge delay time. In addition, we constructed a high-pressure gradient system that was also composed of multiple liquid transfer units, and controlled following each liquid transfer process of each of these liquid transfer units to follow change in composition programmed, gradient accuracy, and separation.・ Improved the reproducibility of analysis and enabled rapid equilibration and re-equilibration of a liquid chromatograph. Firstly, a liquid feed pump device for feeding a fluid or a mixture thereof. In the unit, at least two liquid sending units are in fluid communication with each other, and each liquid sending unit is provided with a pressure sensor for monitoring the pressure during liquid sending, and the first liquid sending unit is also provided. While the liquid discharge unit performs discharge at a desired set flow rate, the other liquid transfer units detect the discharge pressure of the liquid transfer unit during discharge, and the liquid transfer unit corresponding to the discharge pressure at that time. Secondly, a liquid feeding pump device for feeding a fluid or a mixture thereof is characterized in that the fluid is discharged at a desired set flow rate through a step of performing prepressurization in the inside and obtaining a desired discharge pressure. In the unit, at least two liquid sending units are in fluid communication with each other, each liquid sending unit is provided with a pressure sensor for monitoring the pressure during liquid sending, and a large flow rate value is set for one liquid sending unit. A small flow rate value is installed in the other liquid feeding unit, and the first liquid feeding unit performs the discharging process at the desired set flow rate value, while the other liquid feeding unit discharges the discharging pressure of the liquid feeding unit being discharged. And the sensing process that senses While the perform preheating pressure in the liquid-feeding unit in response to said discharge exit pressure, and discharges at the desired set flow rate value through obtaining a desired discharge pressure,
A gradient process is started after each discharge pressure is stabilized, and thirdly, at least three liquid feeding units each including a liquid feeding pump for feeding a fluid or a mixture thereof are provided.
The first liquid sending unit when the two liquid sending units are connected to each other and a pressure sensor is provided in each of the liquid sending units, and at least two liquid sending units of the same kind of eluent are set to switch and send the liquid. Performs a discharge process at a desired set flow rate, while the other liquid sending units detect the discharge pressure of the liquid sending unit during discharging, and the inside of the liquid sending unit corresponding to the sensing process at that time. At the same time as the gradient process is started through the process of obtaining the desired discharge pressure by performing pre-pressurization with the first unit, and the first unit is switched from the liquid feeding process to the suction process, and at least one flow rate set to the first unit is set. Fourth, it is characterized in that the liquid is fed from the liquid feeding unit at a desired set value, and fourthly, at least two liquid feeding units each including a liquid feeding pump for feeding a fluid or a mixture thereof are connected to each liquid feeding unit. A pressure sensor for monitoring the pressure during liquid feeding is provided, and the first liquid feeding unit has a discharging function at a desired set flow rate value, while the other liquid feeding units discharge the first liquid feeding unit. The feature is that it has a pressure sensing function, a pressurizing function that approaches the discharge pressure at that time, and a function that discharges at a desired set flow rate value.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described below with reference to the embodiments shown in the drawings. The liquid sending unit is configured as follows. As shown in FIGS. 1, 2, 3 and 4, a plunger seal 9 is provided in the pump head 1, and a backup ring made of a polymer that is harder than the material of the plunger seal 9 and softer than stainless steel is provided after the plunger seal 9. 10
The backup ring 10 when the pump head 1 is mounted on the pump head mounting block 16.
Gives a force to hold down the plunger seal 9.

A ball spline is adopted in order to prevent center swing of the plunger seal 9 and rotational movement of the plunger 8 when the plunger 8 reciprocates. The ball spline is a linear motion system in which the balls incorporated in the spline outer cylinder 17 are capable of transmitting torque while making smooth linear motion in the precision-ground rolling groove of the spline shaft 11. Further, the plunger 8 is hardly subjected to external vibrations and impacts by applying a pressure by the spline outer cylinder 17, so that the positioning accuracy can be improved and the structure can be simplified.

The other end of the spline shaft 11 is connected to the nut 21 of the ball screw 20 via a connecting unit 19, and a cylindrical space 25 larger than the diameter of the ball screw 20 and longer than the longest stroke of the plunger 8 is provided. The rotational movement of the motor 24 is transmitted to the ball screw 20 via the coupling 23, and the ball screw 20
The rotational movement of the screw part of the ball screw 20 causes the nut 21 of the ball screw 20 to move.
It changes into a linear motion. The nut 21 of the ball screw 20 drives the spline shaft 11 and realizes a reciprocating linear movement of the plunger.

As the spline shaft 11 moves forward and backward, the plunger 8 advances into the pump chamber 7 and moves backward. At the time of entering, the liquid sucked from the container 2 via the pipe 3 is extruded and sent to the liquid chromatography via the outlet conduit 6. Will be done. The pressure sensor 27 communicating with the pump chamber 7 is connected to the control unit 26 by the signal transmission line 28.
Connected to. The pressure sensor 27 can monitor the pressure inside the pump head. This pressure is transmitted to the control unit 26 to control the motor 24 as desired. The control unit 26 is controlled by control software 29.

Combination of Two Liquid Feeding Units FIG. 2 shows an embodiment in which two liquid feeding units are combined and alternately fed. The operation of this liquid sending unit will be described. In the normal liquid feeding state of the first liquid feeding unit 30A, the control software 29 is operated by the operation keys of the control unit 26.
Start A. The liquid transfer unit 30B having a low flow rate or starting from zero monitors the internal pressure of the pump chamber of the liquid transfer unit 30A by the pressure sensor 27 of the liquid transfer unit A via the control unit 26 in preparation for liquid transfer, The discharge pressure of the unit A is set as a target value, and pre-pressurization is performed for a short time to bring the internal pressure of the pump chamber close to the target value before the discharge step, and then the control unit 26
The motor 24 is controlled via, and the discharge is started at the set flow rate. The liquid sending unit 30A discharges at a set flow rate. Then, each discharge pressure is monitored by the pressure sensor of each liquid feeding unit to determine the stability of the discharge pressure. FIG. 5 is a flow chart for explaining these operations.

Pre-pressurization at this time will be described. In this specification, prepressurization and accelerated pressurization are defined as "pressurization that changes the flow rate in proportion to the deviation".
FIG. 11 shows an example of pre-pressurization control. The vertical axis indicates the relative speed of the pre-pressurization side plunger, that is, the relative speed (F / Fo) of the pre-pressurization side plunger speed (F) to the discharge side or the plunger speed (Fo) on the side where the set value flow rate is fast. ). Although the value of F / Fo is shown as an example in the above figure, the typical range of F / Fo is 0-10.
00. The horizontal axis is the relative pressure on the pre-pressurization side, that is, the pressure (P) on the pre-pressurization side is the relative pressure (P / Po) with respect to the discharge pressure (Po) of the discharge side or the plunger with the faster set value flow rate. is there. A typical range for P / Po is 0-1.

As shown in FIG. 11, the pre-pressurization curve has various patterns because the moving speed of the plunger on the pre-pressurization side changes depending on the system pressure. At the initial stage of pre-pressurization, the difference with the system pressure is large, so the speed of the plunger on the pre-pressurization side is fast, but the plunger speed on the pre-pressurization side should be set to zero as the lower limit value just before it becomes equal to the system pressure. It will decrease.
Specifically, the curve as shown above is programmed and monitored by each pressure sensor to automatically control the pre-pressurization process while performing high-speed processing and high-speed data processing.

These pre-pressurization curves have three typical patterns as shown in FIG. 11 by mathematical processing, and are almost expressed by the following polynomial (1). However, pre-pressurization control is possible by programming other formulas (for example, exponent, logarithm, cumulative approximation formula). Formula of pre-pressurization pattern 1 and pattern 3 (F / Fo) = a (P / Po) ⁴ + b (P / Po) ³ + c (P / Po) ² + d (P / Po) + e (1) Formula (1 ), A, b, c, d, and e are constants related to the system and the control method. When the entire system and system control are simplified, the higher order term of the above polynomial (1) can be omitted, so the pre-pressurization pattern is the following expression (2). (F / Fo) = a ′ (P / Po) + b ′ (2) a ′ and b ′ shown in the equation (2) are constants related to the system and the control method. a'is a constant smaller than 0.

Combination of Two Liquid-Feeding Units FIG. 2 shows an embodiment of the grungeent system in the case of combining two liquid-delivery units. The control software 29A is started by the operation key of the control unit 26 in the state of normal liquid feeding. The liquid supply unit 30B having a low flow rate or starting from zero monitors the internal pressure of the pump chamber of the liquid supply unit 30A via the control unit 26 by the pressure sensor 27 of the liquid supply unit A in preparation for the gradient program, Using the discharge pressure of as a target value, accelerated pressurization is performed in the pump chamber for a short time before the formal discharge process, and then discharge is started at the set flow rate. The liquid sending unit A discharges at a set flow rate. Then, each discharge pressure is monitored by the pressure sensor of each liquid feeding unit to determine the stability of the discharge pressure. When the individual requirements are met, the gradient program is started and discharge is started according to the program of each liquid transfer unit. FIG. 6 is a flow chart of these operations.

Combination of Three Liquid Feeding Units FIG. 3 shows an embodiment of a gradient system when three liquid feeding units are combined. The eluents contained in the solvent tank 31C and the solvent tank 31D are of the same kind, and the eluent in the solvent tank 31E is a solvent having a larger dissolution output. The control software 2 is operated by the operation keys of the control unit 26 in the state of normal liquid feeding
Start 9B. The liquid feeding unit 30E that starts from a low flow rate or zero monitors the internal pressure of the pump chamber of the liquid feeding unit 30C with a pressure sensor of the liquid feeding unit 30C in preparation for a gradient program, and sets the discharge pressure of the liquid feeding unit 30C as a target value. ,
Before the discharging step, the internal pressure of the pump chamber is brought closer to the liquid feeding unit 30C, so that the pressure is applied in a short time, and then the discharging is started at the set flow rate. Liquid sending unit 30
C discharges at a set flow rate.

Then, each discharge pressure is monitored by the pressure sensor of each liquid feeding unit to determine the stability of the discharge pressure. Further, the liquid feeding unit D performs pressurization for approaching the discharge pressure of the liquid feeding unit 30C, and the pressurizing pressure is monitored by the pressure sensor of the liquid feeding unit 30D to judge the stability of the pressurizing pressure. When each condition is met, the gradient program is started, and the liquid feeding unit 30D and the liquid feeding unit 30E start discharging according to the program. The liquid sending unit C starts the suction process immediately after the start of the program. After the suction process is completed, a pre-pressurization process for the liquid feeding unit 30D is performed. FIG. 7 is a flow chart of these operations. Simultaneously with the start of the gradient program, the liquid delivery unit 30D in which one stroke of the eluent is completely left in the chamber discharges, so that it is possible to minimize the occurrence of pressure shock during analysis.

In the above example, in the gradient liquid feeding of the embodiment shown in FIG. 3, as shown in FIG. 12, X indicates that the two liquid feeding units 30C and 30D are alternately switched for each analysis. Since Y is basically assumed to have a small flow rate like the liquid feeding unit 30E, the pressure of the liquid feeding units 30C and 30D should be checked regardless of how many times the analysis is performed. Send the liquid. In the above figure, the pressure fluctuation is expressed flatly, but since the ratio of the solution changes during the gradient, it follows a gradual change.

As described above, the liquid feeding units 30C and 30D alternately feed the liquid, and one analysis is performed between the switching of the liquid feeding. Therefore, no shock occurs during analysis other than switching. Of course, for that purpose, it is preferable to monitor the system pressure of the unit during liquid feeding and pre-pressurize the unit that is not during liquid feeding (eg, about twice the set flow rate) so that the pressure can be followed. .

Combination of Four or More Liquid Feeding Units FIG. 4 shows an embodiment of a gradient system in which four liquid feeding units are combined. The eluents contained in the solvent tanks 31F and 31G are of the same type, the eluents contained in the solvent tanks 31H and 31I are of the same type, and the eluents contained in the solvent tanks 31H and 31I are It is a solvent with a higher elution output. The control software 29C is started by the operation key of the control unit 26 in the state of normal liquid feeding. The liquid feeding unit 30H having a low flow rate or starting from zero sets the pressure inside the pump chamber of the liquid feeding unit 30F in preparation for the gradient program.
The pressure is monitored by the 0F pressure sensor, and the discharge pressure of the liquid feeding unit 30F is used as a target value to pressurize the inside of the pump chamber for a short time before the formal discharge step, and then discharge at a set flow rate. The liquid sending unit 30F discharges at a set flow rate.

Then, each discharge pressure is monitored by the pressure sensor of each liquid feeding unit to judge the stability of the discharge pressure. Further, the liquid sending unit 30G pressurizes the discharge pressure of the liquid sending unit 30F, and the pressurizing pressure is monitored by the pressure sensor of the liquid sending unit 30G to determine the stability of the pressurizing pressure. The liquid feeding unit 30I is the liquid feeding unit 3
Pressurization is applied to the discharge pressure of 0H, and the pressurization pressure is monitored by the pressure sensor of the liquid feeding unit 30I to determine the stability of the pressurization pressure. When each condition is met, the gradient program is started, and the liquid feeding unit 30G and the liquid feeding unit 30I start discharging according to the programs. The liquid feeding unit F and the liquid feeding unit 30H start the suction process immediately after the start of the program. After the suction process is completed, the liquid feeding unit 30H performs a pre-pressurizing process on the liquid feeding unit 30I. FIG. 8 is a flow chart of these operations.

When the amount of liquid to be delivered is relatively large, a plurality of liquid delivery units are combined to deliver one type of eluate by sequentially switching each unit. The simplest example is the case of 3 units of 2 × 1, and at the same time as the gradient starts, the liquid feeding unit that has been discharging until then turns to suction, and the other unit starts liquid feeding. By doing so, the switching shock will be forced into the beginning of the gradient and will be kept out of the analysis as much as possible. After that, when the liquid transfer unit that has turned to suction reaches a certain point, it starts prepressurizing in preparation for the next liquid transfer. One 2 × 1 independent liquid delivery unit performs the same operations as the basic pattern (pressure sensing, accelerated pre-pressurization, pressure judgment, set value flow rate liquid delivery).

Gradient followability and reproducibility FIG. 9 shows a comparison between the embodiment and the conventional gradient when the step gradient is performed using the above-mentioned four liquid feeding units. The same organic solvent (methanol) is fed to the two liquid feeding units, and the organic solvent added with a few percent of the light absorbing substance (acetone) is fed to the other liquid feeding unit at a total flow rate of 5 μL / min. The system pressure is around 10 MPa. The conventional gradient is obtained by two liquid feed pumps without using the gradient control method of the present invention. The detector is an absorptiometric detector. The program changes the flow rate of the one containing the light absorbing substance from 0% to 20,1 at regular time intervals (10 minutes).
It was changed to 0, 50, 90, 100%.
In the conventional gradient, the initial rise is slow and the value is far from the program setting value.

This delay time T is approximated by the equation T = xVo (P-Po) by the relationship among the liquid compression rate x, the volume Vo in the pump chamber, the discharge pressure or system pressure P, the pump chamber inlet pressure Po, and the flow rate F. ) / F can be theoretically calculated. In this formula, the compression ratio of methanol is 1
27 (10 ⁻⁶ / atm), Vo = 200 μL, P = 10
When calculated with MPa, Po = 0 MPa, and F = 5 μL / min, the delay time is about 30 seconds. Further, under high pressure conditions, deformation of the plunger seal and the like also cause a delay time. By adding these delay times, the conventional gradient cannot follow the program, and in the example of FIG. 8, the delay time is about 3 minutes.

FIG. 10 shows reproducibility data obtained by performing a step gradient three times using four liquid feeding units. The same organic solvent (methanol) is fed to the two liquid feeding units, and the organic solvent added with a few percent of the light absorbing substance (acetone) is fed to the other liquid feeding unit at a total flow rate of 5 μL / min. The program sets the flow rate of the light absorbing substance to 0% at regular time intervals (10 minutes).
To 10, 50, 90, 100%. Good reproducibility has been achieved with no delay in the initial rise.

In the embodiment of FIG. 10, the flow rate range is 0.1 μL / min to 50 μL / min.
We were able to confirm the operation of a similar step gradient with good followability without any problems. In this way, by further reducing the dead volume of the liquid sending unit body and piping,
Accurate gradient liquid transfer is possible up to about 0.1 μL / min.

A chromatogram obtained by actually separating the sample by setting a certain gradient program is shown in FIG.
Shown in. The chromatogram in the figure is the data obtained by repeating 6 times, but it looks like one chromatogram because of the reproducibility. The experimental conditions were as follows. Flow rate: 5 μL / min Eluent A: Acetonitrile / water = 50/50 Eluent B: Acetonitrile gradient program: Eluent B from 0% to 70% 1
Change linearly in 0 minutes. Analytical column: Inner diameter 0.3 mm Length 150 mm Inertosyl (registered trademark) ODS-3 Detection: UV254 nm Sample: para-hydroxybenzoic acid ester (C1-C)
7) Sample amount: 0.1 μL In the figure, 1 is methyl parahydroxybenzoate (C1) ester, 2 is methyl parahydroxybenzoate (C2) ester, 3 is propyl parahydroxybenzoate (C3) ester, and 4 is parahydroxyl. Butyl benzoate (C4) ester, 5 is para-hydroxybenzoic acid pentyl (C5) ester, 6 is hexyl para-hydroxybenzoate (C6)
Ester, 7 is heptyl parahydroxybenzoate (C
7) It is an ester.

[0037]

According to the first aspect of the present invention, at least two liquid feeding units are in fluid communication with each other in the liquid feeding unit including the liquid feeding pump device for feeding the fluid or the mixture thereof.
Each liquid transfer unit is provided with a pressure sensor for monitoring the pressure during liquid transfer, and the first liquid transfer unit discharges at a desired set flow rate value, while the other liquid transfer units discharge liquid during discharge. Discharge at a desired set flow rate through a sensing step of sensing the discharge pressure of the unit and a step of prepressurizing in the liquid sending unit corresponding to the discharge pressure at that time to obtain a desired discharge pressure Therefore, by reading the system pressure of the unit during liquid feeding and accelerating the pressure to the unit that is not feeding so that it can follow it, liquid feeding at the target accurate flow rate is possible. The time can be greatly reduced. This changes the flow rate ratio of multiple solvents,
This is a very effective means when examining the conditions.

According to a second aspect of the present invention, at least two liquid sending units are in fluid communication with each other and each liquid sending unit is sent by a liquid sending unit comprising a liquid sending pump device for sending a fluid or a mixture thereof. A pressure sensor for monitoring the pressure in the liquid is provided, and a large flow rate value is set for one liquid sending unit, and a small flow rate value is set for the other liquid sending unit. Performs a discharge process at a desired set flow rate, while another liquid sending unit senses the discharge pressure of the liquid sending unit being discharged, and a sensing process in the liquid sending unit corresponding to the discharge pressure at that time. In addition to the effect according to claim 1, the pre-pressurization in step (1) and the step of obtaining a desired discharge pressure are performed to discharge at a desired set flow rate value, while the gradient step is started after each discharge pressure is stabilized. , These g The discharge process is controlled by monitoring the system pressure and pre-pressurization pressure, which is included in the event program, so that the discharge process can be performed accurately without delay, and the followability to the programmed composition change, the accuracy of the gradient, and the separation / analysis Improves reproducibility. It is also effective for speeding up equilibration and re-equilibration of the liquid chromatograph.

At the start of the gradient program, the analyst or the autosampler can efficiently obtain the injection of the analysis sample by notifying the outside that the pressure is stabilized via the control unit. Thereby, analysis can be performed without wasting valuable components.

Further, according to the third aspect, after the completion of the gradient step, each liquid sending unit automatically performs the pre-pressurizing step until the pressure is stabilized, and waits in a state where the gradient is ready. In addition to the effect described in 2, the shock that enters at the start of liquid transfer can be made extremely small, and the liquid can be transferred at the correct target flow rate at any time, and the time can be greatly shortened, and the effect of improving the gradient accuracy can be achieved. Have.

According to a fourth aspect of the invention, at least three liquid feed units each including a liquid feed pump for feeding a fluid or a mixture thereof are in fluid communication with each other, and each liquid feed unit is provided with a pressure sensor. When the eluent is switched and sent by at least two liquid sending units set to the same flow rate, the first liquid sending unit performs the discharging process at the desired set flow rate value while the other liquid sending units The sensing step of sensing the discharge pressure of the liquid sending unit during discharging, and the step of performing pre-pressurization in the liquid sending unit corresponding to the discharge pressure at that time to obtain the desired discharge pressure, the gradient Simultaneously with the start of the process, the first unit is switched from the liquid feeding process to the suction process, and at least one liquid feeding unit having the same flow rate as the first unit feeds the liquid at a desired set value. In the combination of liquid units, in the control that switches the liquid feeding unit that is performing the pre-pressurization process to the discharge process immediately after the start of the gradient program, it can be analyzed by the pressure fluctuation that occurs when each liquid feeding unit switches from the discharge process to the suction process. By generating the turbulence of the baseline immediately after the start of the gradient program, it becomes possible to perform a stable ejection process in which the ejection process does not switch from the suction process for a long time thereafter. As a result, the analysis of the target component during the execution of the gradient program becomes very stable, and the reproducibility of the analysis is also improved.

According to the fifth aspect, in the trace amount gradient, since the liquid feeding unit is switched only at the start of the gradient program and the unit is not switched at the time of analysis, the liquid feeding unit is switched only at the start of the gradient program. By putting pressure shocks and controlling so that liquid transfer shocks do not enter during analysis, it is possible to prevent the occurrence of irregular shocks, prevent the elution time of the target component from overlapping the shock peak detection time, and Disturbance can be prevented, and smooth quantitative analysis can be performed accurately.

Further, according to claim 6, at least two liquid feeding units each including a liquid feeding pump for feeding a fluid or a mixture thereof are connected, and the pressure during liquid feeding is monitored in each liquid feeding unit. In addition to providing a pressure sensor, the first liquid sending unit has a discharge function at a desired set flow rate, while the other liquid sending units have a discharge pressure sensing function of the first liquid sending unit and a discharge pressure at that time. Since it has a pressurizing function to bring it closer to the discharge pressure and a function to discharge at a desired set flow rate, the discharge pressure of the first liquid sending unit is constantly monitored by other liquid sending units, and the discharge pressure of the liquid sending unit is made closer to the discharge pressure. Internal pressure is applied by the pressure function, and it is possible to discharge at a desired set value. In this way, alternate control between two or more pump systems is reliably performed, and stable continuous liquid delivery without discharge delay becomes possible. Furthermore, by constructing a gradient system with multiple liquid transfer units and controlling the process of each liquid transfer unit, the followability to programmed composition changes, high gradient accuracy, separation, and reproducibility of analysis are improved. However, it is possible to provide an apparatus capable of speeding up equilibration and re-equilibration of a liquid chromatograph.

[Brief description of drawings]

FIG. 1 is an explanatory diagram of an embodiment of a main part of the present invention

FIG. 2 is an explanatory view of an embodiment of the present invention.

FIG. 3 is an explanatory diagram of another embodiment of the present invention.

FIG. 4 is an explanatory view of another embodiment of the present invention.

FIG. 5 is an explanatory diagram of the flow of one embodiment of the present invention.

FIG. 6 is a flow chart for explaining another embodiment of the present invention.

FIG. 7 is a flowchart for explaining another embodiment of the present invention.

FIG. 8 is a flow chart for explaining another embodiment of the present invention.

FIG. 9 Conventional example gradient table

FIG. 10: Gradient table of one embodiment of the present invention

FIG. 11: Preload curve graph

FIG. 12 is an explanatory diagram of an example of gradient liquid feeding.

FIG. 13: Chromatogram in one example of gradient liquid transfer

[Explanation of symbols]

1 pump head 8 Plunger 9 Plunger seal 10 backup ring 11 spline shaft 16 Pump head mounting block 19 Connection unit 24 motor 26 Control unit

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Zhou Yasushi             2 G of 237 Sayamagahara, Iruma City, Saitama Prefecture             L Science Co., Ltd. Musashi Factory

Claims (6)

[Claims] 1. A liquid-sending unit comprising a liquid-sending pump device for sending a fluid or a mixture thereof, wherein at least two liquid-sending units are in fluid communication with each other, and each of the liquid-sending units monitors the pressure during the feeding. With a pressure sensor,
The first liquid sending unit discharges at a desired set flow rate value, while the other liquid sending units correspond to the sensing step of sensing the discharge pressure of the liquid sending unit being discharged and the discharge pressure at that time. A method for feeding liquid in liquid chromatography, comprising: prepressurizing in a liquid feeding unit to obtain a desired discharge pressure, and then discharging at a desired set flow rate value. 2. A liquid-sending unit comprising a liquid-sending pump device for sending a fluid or a mixture thereof, wherein at least two liquid-sending units are in fluid communication with each of the liquid-sending units for monitoring the pressure during the liquid feeding. With a pressure sensor,
A large flow rate value is set for one liquid sending unit, a small flow rate value is set for the other liquid sending unit, and the first liquid sending unit performs the discharge process at a desired set flow value, The other liquid sending unit performs a sensing step of sensing the discharge pressure of the liquid sending unit during discharging and a prepressurization in the liquid sending unit corresponding to the discharging pressure at that time to obtain a desired discharge pressure. A method for sending liquid in liquid chromatography, characterized in that, after discharging through a step of obtaining and discharging at a desired set flow rate, the gradient step is started after each discharge pressure is stabilized. 3. The method according to claim 2, wherein after the completion of the gradient step, each liquid sending unit automatically performs a pre-pressurization step until the pressure stabilizes and waits in a state where the gradient is ready. Liquid chromatography liquid feeding method. 4. A fluid feed unit comprising at least three fluid feed pumps for feeding a fluid or a mixture thereof, which are in fluid communication with each other, and each fluid feed unit is provided with a pressure sensor.
When at least two liquid sending units that set the same flow rate of the same type of eluent are switched and sent, the first liquid sending unit performs the discharge step at the desired set flow rate value, while the other liquid sending units The unit has a sensing step of sensing the discharge pressure of the liquid sending unit during discharging, and a step of prepressurizing in the liquid sending unit corresponding to the discharging pressure at that time to obtain a desired discharging pressure. After the start of the gradient process, the first
A liquid chromatography liquid feeding method, characterized in that a unit is switched from a liquid feeding process to a suction process, and at least one liquid feeding unit having the same flow rate as the first unit is fed at a desired set value. 5. The liquid chromatography liquid sending according to claim 2 or 4, wherein in the trace amount gradient, the liquid sending unit is switched only at the start of the gradient program, and the unit is not switched at the time of analysis. Method. 6. A liquid feeding unit comprising a liquid feeding pump for feeding a fluid or a mixture thereof, at least two liquid feeding units are connected, and each liquid feeding unit is provided with a pressure sensor for monitoring the pressure during liquid feeding. The first liquid sending unit has a discharge function at a desired set flow rate value, while the other liquid sending units have a discharge pressure sensing function of the first liquid sending unit and a pressurizing function close to the discharge pressure at that time. A liquid delivery device for liquid chromatography having a function of discharging at a set flow rate value.