JP7011921B2 - Liquid feeding method - Google Patents

Description

The present disclosure relates to a method of feeding an eluent in a low pressure linear gradient method.

In high performance liquid chromatography, gradient elution is used, which elutes while changing the eluent. A low-pressure gradient method is known in which a plurality of solenoid valves connected to each liquid on the upstream side of the pump are switched while the pump is sucking to mix the plurality of liquids. (See, for example, Patent Document 1).

Japanese Unexamined Patent Publication No. 2-238358

By the way, when a small capacity pump is used in the low pressure linear gradient method, the solenoid valve must be switched in a short time, the reproducibility of the solution mixture depends on the performance of the solenoid valve, and it is difficult to accurately adjust the concentration of the liquid. there were.

It is an object of the present disclosure to provide a liquid feeding method capable of accurately controlling the mixing of liquids without depending on the performance of the on-off valve.

The liquid feeding method of one aspect of the present disclosure includes a plurality of storage portions in which different types of liquids are stored, a pump that sucks the liquid stored in the storage portions and discharges the liquid to the downstream flow path, and the pump and the above. It is used in a liquid feeding mechanism equipped with a plurality of on-off valves provided in each of the upstream flow paths connecting the storage portions, and one of the on-off valves is opened and one of the upstream flow paths is pumped. It includes a step of sucking a liquid, a step of stopping the pump and closing the on-off valve in an open state, and a step of discharging the liquid sucked by the pump to the downstream flow path.

According to the present disclosure, it is possible to provide a liquid feeding method capable of accurately controlling the mixing of liquids without depending on the performance of the on-off valve.

It is a schematic diagram which shows the schematic structure of the measuring apparatus which used the liquid feeding method which concerns on one Embodiment of this disclosure. It is a flow figure explaining the liquid feeding sequence of the measuring apparatus shown in FIG. It is a graph which shows the opening / closing time of each solenoid valve in the liquid feeding sequence shown in FIG. 2 and the flow rate in a liquid feeding pump. It is a chromatographic pattern obtained by the liquid feeding sequence shown in FIG. The chromatographic pattern when the chromatographic pattern shown in FIG. 4 is corrected is shown. It is a flow figure explaining the liquid feeding sequence of the measuring apparatus using the liquid feeding method which concerns on other embodiment. It is a graph which shows the relationship between the set value of one eluent, the concentration of the measured value, and time when the measuring apparatus of an Example is used. It is a graph which shows the relationship between the concentration and time of the set value of one eluent and the measured value when the measuring device of the comparative example is used.

Hereinafter, a liquid feeding method according to an embodiment of the present disclosure will be described.

FIG. 1 shows a schematic configuration of a measuring device 20 using the liquid feeding method of the present embodiment.

First, the measuring device 20 will be described.
As shown in FIG. 1, the measuring device 20 is a low-pressure gradient type measuring device for measuring hemoglobins contained in a measurement sample. The measuring device 20 includes an eluent tank 23, an eluent tank 24, a solenoid valve 25, a solenoid valve 26, a liquid feed pump 28, a control unit 29, a mixer 30, a damper 32, an injection valve 34, an auto sampler 36, and a separation column. 38, a detector 40 and a waste liquid container 42 are provided.

The eluent tank 23 is a tank for storing the eluent, and the eluent is stored.

The eluent tank 24 is a tank for storing the eluent, and stores an eluent different from the eluent in the eluent tank 23.

The solenoid valve 25 is provided in the flow path between the eluent tank 23 and the liquid feed pump 28. The opening and closing of the solenoid valve 26 is controlled by the control unit 29.

The solenoid valve 26 is provided in the flow path between the eluent tank 24 and the liquid feed pump 28. The opening and closing of the solenoid valve 26 is controlled by the control unit 29.
The solenoid valve 25 and the solenoid valve 26 are examples of the on-off valves of the present disclosure.

The liquid feed pump 28 sucks each of the eluents stored in the eluent tank 23 and the eluent tank 24 and discharges them to the flow path on the downstream side (mixer 30 side). The drive and stop of the liquid feed pump 28 are controlled by the control unit 29. As the liquid feed pump 28, for example, a plunger pump may be used.

The liquid feeding mechanism 22 is composed of the eluent tank 23, the eluent tank 24, the solenoid valve 25, the solenoid valve 26, and the liquid feeding pump 28.

The control unit 29 controls opening and closing of the solenoid valve 25 and the solenoid valve 26 and driving / stopping of the liquid feeding pump 28 based on the liquid feeding sequence described later.

The mixer 30 is a device that mixes the eluent discharged from the liquid feed pump 28. The eluate mixed by the mixer 30 is sent to the damper 32 on the downstream side.

The damper 32 is a device that suppresses pressure fluctuation (pulsation) of the eluent sent from the mixer 30. For example, a diaphragm type damper or the like may be used.

The autosampler 36 is a device that sends the measurement sample to the separation column 38. The drive / stop of the autosampler 36 is controlled by the control unit 29.

The injection valve 34 is provided in the flow path between the autosampler 36 and the separation column 38. The opening and closing of the injection valve 34 is controlled by the control unit 29.

The separation column 38 is a member that internally separates hemoglobins. The eluent that has passed through the damper 32 and the measurement sample that has passed through the injection valve 34 are sent to the separation column 38. The inside of the separation column 38 is filled with a filler, and hemoglobins are separated according to the difference in the eluate output due to the sent eluate, and flow out from the separation column 38.

The hemoglobins flowing out from the separation column 38 are irradiated with light having a predetermined wavelength, and are detected by a detector 40 such as an ultraviolet / visible absorptiometer. Based on the detection result of the detector 40, a chromatogram is drawn and displayed on a display device (not shown).

After the detection by the detector 40, the waste liquid container 42 collects the liquid containing hemoglobins as a waste liquid.

Next, the first liquid feeding sequence by the control unit 29 using the liquid feeding mechanism 22 will be described.
As shown in FIG. 2, the first liquid feeding sequence includes steps S2 to S22.

First, in step S2, the type of eluent and the mixing ratio of the eluent are set based on the measurement sequence selected by using an input device (not shown) to the control unit 29.

Next, in step S4, the suction amount of each eluent by the liquid feed pump 28 is calculated from the value set in step S2.

Next, in step S6, the eluent to be sucked is selected from the plurality of eluents.

Next, in step S8, the solenoid valve corresponding to the eluent selected in step S6 is opened. For example, when the eluent in the eluent tank 23 is selected, the solenoid valve 25 is opened.

Next, in step S10, the liquid feed pump 28 is driven, and in step S8, the eluent is sucked from the eluent tank on the side where the solenoid valve is opened. For example, when the solenoid valve 25 is opened in step S8, the eluent is sucked from the eluent tank 23.

Next, in step S12, the liquid feed pump 28 is stopped. The stop of the liquid feed pump 28 is carried out after a predetermined time has elapsed.

Next, in step S14, the solenoid valve opened in step S8 is closed. For example, when the solenoid valve 25 is opened in step S8, the solenoid valve 25 is closed in step S14.

Next, in step S16, the liquid feed pump 28 is driven to discharge the eluent in the liquid feed pump 28 (inside the pump head) to the flow path on the downstream side (mixer side).

Next, in step S18, it is determined whether all the eluates set in step S2 have been fed from the liquid feed pump 28 to the mixer 30. When all the set eluents have been sent to the mixer 30, the process proceeds to step S22. On the other hand, if there is an eluent that has not yet been sent to the mixer 30, the treatment proceeds to step S20.

In step S20, the eluent that has not been subjected to the liquid feeding process is selected, and the process proceeds to step S8.

In this embodiment, steps S8 to S20 are set as one sequence.

In step S22, the amount of eluent to be sucked in the next liquid feeding sequence is calculated, and steps S8 to S20 are repeated.

Next, an example of the operation of the liquid feeding mechanism 22 when the first liquid feeding sequence is set will be specifically described with reference to FIG. In the first setting condition, the mixing ratio of the eluent is 100% of the eluent in the eluent tank 23 (that is, not mixed), and in the next setting condition, the mixing ratio of the eluent is 33% of the eluent in the eluent tank 23. , 66% of the eluent from the eluent tank 24.

First, as shown in FIG. 3, the solenoid valve 25 is opened to drive the liquid feed pump 28 to suck the eluent from the eluent tank (an example of the step of sucking the liquid (liquid suction step) in the present disclosure). .. At this time, the eluent is sucked up to the upper limit suction amount (100%) of the liquid feed pump 28. After that, the liquid feed pump 28 is stopped and the solenoid valve 25 is closed (a step of closing the on-off valve in the present disclosure (valve closing step)). Then, the liquid feed pump 28 is driven to discharge the eluent to the mixer 30 side (the step of discharging the liquid (liquid discharge step) in the present disclosure). This completes the liquid delivery sequence based on the initial setting conditions.

Next, after opening the solenoid valve 25, the liquid feed pump 28 is driven to suck the eluate from the eluent tank 23. At this time, the eluent is sucked up to 33% of the upper limit suction amount of the liquid feed pump 28. Next, the liquid feed pump 28 is stopped and the solenoid valve 25 is closed. Then, the liquid feed pump 28 is driven to discharge the eluent to the mixer 30 side. The discharged eluent is sent to the mixer 30.

Next, after opening the solenoid valve 26, the liquid feed pump 28 is driven to suck the eluate from the eluent tank 24. At this time, the eluent is sucked up to 66% of the upper limit suction amount of the liquid feed pump 28. Next, the liquid feed pump 28 is stopped and the solenoid valve 26 is closed. Then, the liquid feed pump 28 is driven to discharge the eluent to the mixer 30 side. The discharged eluent is sent to the mixer 30. This completes the liquid feeding sequence based on the following setting conditions.

Next, the action and effect of this embodiment will be described.
In the liquid feeding method of the present embodiment, the suction amount of the eluent is controlled by opening the solenoid valve 25 or the solenoid valve 26 and changing the driving time on the liquid feeding pump 28 side, so that the suction amount is controlled by the solenoid valve. It does not depend on the performance of the 25 and the solenoid valve 26. Therefore, as compared with, for example, a method in which the solenoid valve is switched to control the suction amount while the liquid feed pump 28 is driven, the suction amount of the liquid does not fluctuate and the liquid can be sucked accurately. As a result, the mixer 30 provided in the downstream flow path of the liquid feed pump 28 can accurately control the mixed concentration of a plurality of different types of eluents.

In the above-described embodiment, the discharge of the eluent to the mixer side is stopped when the solenoid valve is switched and when the liquid is sucked into the liquid feed pump from the eluent tank, so that the chromatographic pattern observed by the detector 40 is observed. As shown in FIG. 4, a step (a portion where the continuity is reduced) is formed due to the fluctuation of the flow velocity. This step can be reduced by repeating one sequence of the liquid feeding sequence in a very short time (see FIG. 5). Further, the suction amount of the liquid feed pump 28 can be set to an arbitrary value, but the total suction amount of each eluent of the liquid feed pump 28 should be equal to the upper limit amount that the liquid feed pump can feed at one time. For example, the influence of fluctuations in the flow velocity on the chromatographic pattern can be reduced, which is preferable (see FIG. 5).

Further, the timing of driving / stopping the liquid feeding pump 28 is calculated in advance, and the measured value by the detector 40 is sampled according to the timing at which the liquid feeding pump 28 is actually feeding. From this calculated data and measurement data, the continuity of the chromato pattern is ensured and the accuracy is improved by correcting the part where the continuity of the chromato pattern deteriorates and adjusting the sampling timing of the measured value. Can be made to.

In the first liquid feeding sequence in the liquid feeding method of the above-described embodiment, the eluent is sucked into the liquid feeding pump 28, and then the eluent is discharged to the mixer 30 each time, but the present disclosure is not limited to this configuration. .. For example, as in the second liquid feeding sequence in the liquid feeding method of another embodiment shown in FIG. 6, a plurality of eluents may be sucked into the liquid feeding pump 28 and then discharged to the mixer 30. The specific contents will be described below.

Steps S32 to S44 of the second liquid feeding sequence shown in FIG. 6 correspond to steps S2 to S14 of the first liquid feeding sequence shown in FIG. 2, respectively. In step S46, it is determined whether all the eluates set in step S32 have been sucked from the eluent tank into the liquid feed pump 28. When all the set eluents are sucked into the liquid feed pump 28, the process proceeds to step S50. On the other hand, if there is still an unsucked eluent in the liquid feed pump 28, the process proceeds to step S48.

In step S48, the amount of eluent to be sucked in the next sequence is calculated, and steps S38 to S48 are repeated.

In step S50, the liquid feed pump 28 is driven to discharge a plurality of eluents in the liquid feed pump 28 (inside the pump head) to the flow path on the downstream side (mixer 30 side).

In step S52, the amount of eluent to be sucked in the next liquid feeding sequence is calculated, and steps S38 to S50 are repeated.

Next, an example of the operation of the liquid feeding mechanism 22 when the second liquid feeding sequence is set will be described.
First, after opening the solenoid valve 25, the liquid feed pump 28 is driven to suck the eluent from the eluent tank 23 (liquid suction step). Next, the liquid feed pump 28 is stopped and the solenoid valve 25 is closed (valve closing step).

Next, after opening the solenoid valve 26, the liquid feed pump 28 is driven to suck the eluent from the eluent tank 24 (another step of closing the on-off valve in the present disclosure (another liquid suction step).

As a result, the eluents sucked in the liquid feed pump 28 are mixed with each other.

Then, the liquid feed pump 28 is driven to discharge the eluent to the mixer 30 side (liquid discharge step). The discharged eluent is sent to the mixer 30.

As described above, in the liquid feeding method of the other embodiment, the suction amount of the eluent is controlled by opening the solenoid valve 25 or the solenoid valve 26 and changing the driving time on the liquid feeding pump 28 side. The control does not depend on the performance of the solenoid valve 25 and the solenoid valve 26. Therefore, as compared with, for example, a method in which the solenoid valve is switched to control the suction amount while the liquid feed pump 28 is driven, the suction amount of the liquid does not fluctuate and the liquid can be sucked accurately. Further, since the liquid feed pump 28 continuously sucks one eluent and the other eluent and stores them inside, the eluents can be mixed in the liquid feed pump 28. As a result, the mixer 30 provided in the downstream flow path of the liquid feed pump 28 can more uniformly control the mixed concentration of a plurality of different types of eluents.

In the liquid feeding method of the above-described embodiment, two types of eluents provided with a solenoid valve are mixed in each eluent feeding line, but the present disclosure is not limited to this configuration. Multiple types of eluents may be mixed.

Next, examples of the present disclosure will be described. The present disclosure is not limited by the following examples.

(Example)
The concentration of one eluent was measured when the liquid feeding mechanism of the measuring device of the present disclosure was operated in the first liquid feeding sequence.

<Measuring device>
As an example, the measuring device of the above-described embodiment was prepared.
Further, the liquid feeding sequence was set to the first liquid feeding sequence, and the concentration of the eluent was measured as the change in the absorbance at the detector 40. As a comparative example, step S42 of the second liquid feeding sequence is omitted, and step S44 and step S48 (switching between opening and closing of the solenoid valve 25 and the solenoid valve 26) are performed with the liquid feeding pump 28 operating, and two elutions are performed. The liquids were mixed and the change in absorbance in the detector 40 was measured in the same manner as in the examples.
FIG. 7 shows the set value (theoretical value) of the absorbance corresponding to the concentration of the mixed eluent and the measured value in the example, and FIG. 8 shows the measured value in the comparative example.

As can be seen by comparing FIGS. 7 and 8, the actually measured values in the examples are closer to the set values than the actually measured values in the comparative examples. From this, it can be seen that the mixed concentration of different types of eluents can be accurately controlled by using the liquid feeding sequence in the present disclosure.

20 Measuring device 22 Liquid feeding mechanism 23 Eluent tank (reservoir)
24 Eluent tank (reservoir)
25 Solenoid valve (on-off valve)
26 Solenoid valve (on-off valve)
28 Liquid feed pump (pump)

Claims (3)

A plurality of reservoirs in which different types of liquids are stored, a plunger pump that sucks the liquid stored in the reservoir and discharges it to the downstream flow path, and an upstream flow path that connects the plunger pump and the reservoir. Used in a liquid feeding mechanism equipped with multiple on-off valves, each of which is provided in
A suction step of opening the on-off valve and sucking a liquid from the upstream flow path of the plunger pump.
After the suction step, the plunger pump is stopped and the on-off valve in the open state is closed .
After the closing step, a discharge step of discharging the liquid sucked by the plunger pump to the downstream flow path and a discharge step.
Liquid feeding method equipped with. Further provided between the closing step and the discharging step is another suction step in which the opening valve different from the closed opening valve is opened and the liquid is sucked from the upstream flow path by the plunger pump. The liquid feeding method according to claim 1. The liquid feeding method according to claim 2, wherein the suction amount of the plunger pump when producing a liquid having a predetermined mixed concentration is the upper limit suction amount of the plunger pump.

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