JP2023119604A - Mobile phase priming method and priming control method - Google Patents

Abstract

To shorten the action time before measurement that pertains to the priming of a mobile phase in liquid chromatographic analysis.SOLUTION: While suppressing the occurrence of an excessive decompression state and air bubbles, prompt priming is achieved, by executing a suction operation to suck in a mobile phase intermittently or in an on-and-off manner. Provided herein is a priming method which, in a liquid chromatograph equipped with a mobile phase source for storing and supplying a mobile phase to one end of an analysis flow path, causes the flow path to branch off in the middle of the analysis flow path and replaces at least an inside portion of the analysis flow path with the mobile phase, using a syringe in liquid connection with the distal end of said flow path, this method comprising a suction step for sucking in the mobile phase from the mobile phase source by causing the syringe to stroke within the range of its capacity, the suction step including a stop step in which the motion of the syringe is stopped for a given duration, and a plurality of partial suction steps which are created by being thereby divided in time.SELECTED DRAWING: Figure 1

Description

The present invention relates to a mobile phase priming method and a priming control method in a liquid chromatograph.

There is a demand for shortening the analytical measurement time per measurement in clinical laboratory measurements using a liquid chromatograph. For example, glycated hemoglobin analysis in blood can be measured in an extremely short time of less than 1 minute per measurement. Furthermore, not only the measurement time but also shortening the total throughput from preparation for measurement and activation of the device to completion of measurement is important as a contribution to the operation of clinical laboratories and shortening patient waiting times.

Liquid chromatography separates analytes in a sample according to the properties of one or more mobile phases (eluents) and stationary phases (analytical columns). Therefore, the stability of the mobile phase greatly contributes to the stability of the measurement results. An example of mobile phase instability is when mobile phases manufactured with the same composition cannot be reproduced in exactly the same state due to manual errors, instrumental errors, or manufacturing date differences. be done. Therefore, generally, when replacing the mobile phase, not only the supply source of the mobile phase is replaced, but also an operation called priming (purging or draining) is performed to replace the old mobile phase in the device with a new mobile phase. For example, Patent Literature 1 discloses a method of priming a plunger-type liquid transfer pump with a syringe pump.

In addition, the mobile phase remaining in the apparatus for a certain period of time may change the dissolved oxygen concentration, pH, etc., and an operation of discarding a certain amount may be performed before the start of measurement. Whether they are incorporated into the pre-measurement series of automated actions or performed as manual actions, they all affect the total throughput, especially when the number of mobile phases is multiplied. Increased impact. On the other hand, if the syringe suction speed is increased in order to shorten the priming time, the supply of the mobile phase cannot keep up and bubbles are generated in the syringe or the channel. These air bubbles greatly fluctuate the results of analyte measurements.

Further, conventionally, in a liquid chromatograph that switches between a plurality of mobile phases, it is common to perform a suction operation and a discharge operation related to priming for each mobile phase. In addition, some clinical testing equipment using liquid chromatographs are equipped with a preliminary mobile phase due to the high operating frequency, and the mobile phase can be automatically replaced with the preliminary mobile phase during measurement or during standby. Yes, and the frequency and duration of priming is on the rise.

Japanese Patent Application Laid-Open No. 2020-112060

An object of the present invention is to provide a mobile phase priming method and a priming control method that can shorten the operation time before measurement and contribute to the stability of measurement in liquid chromatographic analysis.

A liquid-feeding channel normally used in a liquid chromatograph has an inner diameter of 2 mm or less. In the fluid moving along the longitudinal direction in the channel, a velocity gradient is generated from the state of zero flow velocity at the fluid particles in contact with the channel wall toward the flow velocity near the center of the channel. In narrow channels, large channel resistance occurs due to velocity gradients, ie, shear stresses in the fluid. Therefore, if the moving speed of the plunger of the syringe is increased to shorten the priming time and suction is continuously performed, the movement of the mobile phase, which is a liquid, will be delayed, and the inside of the channel will be rapidly depressurized, causing air bubbles. may occur or be mixed. Focusing on this point, the present inventors achieved rapid priming while avoiding an excessively depressurized state by intermittently or intermittently performing a suction operation for sucking the mobile phase from the mobile phase source.

That is, one aspect of the present invention for solving the above problems is a liquid chromatograph having a mobile phase source for storing and supplying a mobile phase at one end of the analysis channel, in which the channel is branched from the middle of the analysis channel. A priming method for replacing at least part of the interior of an analysis channel with a mobile phase using a syringe whose distal end is fluidly connected, wherein the mobile phase source is removed by stroking the syringe within its volume range. aspiration step of aspirating the mobile phase from the be. Although the branching portion of the channel where the syringe pump is provided is not limited, it is preferably installed in the channel before the inlet of the analysis column provided in the liquid chromatograph. This is because the analysis column usually has a high flow path resistance, and thus it takes a considerable amount of time for priming. In order to shorten the priming time, it is particularly preferable to install the branch part in the inlet-side channel of the mobile phase liquid-sending pump.

The liquid chromatograph may be equipped with a plurality of mobile phase sources, and the partial aspiration step may be an operation of aspirating the mobile phase from one of the plurality of mobile phase sources with a syringe. An embodiment may include a mobile phase switching operation for switching the mobile phase to be aspirated to another mobile phase during the stop operation between the partial aspiration steps. According to this aspect, the priming operation for a plurality of mobile phases can be quickly achieved by one suction stroke operation of the syringe pump.

A syringe has a limited aspirating capacity per stroke, and it is desirable to include a step of discharging the aspirated mobile phase in order to continue liquid chromatographic analysis continuously or intermittently. A further aspect of the priming method of the present invention may further include a discharge step of discharging the mobile phase aspirated by the one-stroke operation into the drain channel or the cleaning channel with a syringe. This aspect is preferable in that the time required for ejection can be saved compared to the conventional general priming method in which the suction operation and the ejection operation are repeated for each mobile phase.

The mobile phase to be switched in the mobile phase switching operation is effective for cleaning and liquid replacement related to priming, and the mobile phase retained in the flow path after priming does not affect the measurement results. Selection can be made according to the suction order set based on the difference in at least one of the properties (pH, salt concentration, hydrophobicity, etc.) and temperature.

In the partial aspiration step, the aspiration speed and the aspiration amount for aspirating the mobile phase are not limited, but are preferably 0.5 mL/sec or more and 1.0 mL or more, respectively. Also, each suction speed and discharge speed may be different from each other, and the suction speed and discharge speed can be changed according to the type and remaining amount of the mobile phase, the order of priming, and the like. For example, when the amount of suction for priming is small, the suction speed or the ejection speed can be decreased, and when the amount of suction is large, the suction speed or the ejection speed can be increased. Alternatively, the suction speed or discharge speed can be changed according to the viscosity of the mobile phase.

Priming should be kept to a minimum, as it consumes mobile phase and delays the start of measurement. Therefore, the previous measurement end time is stored in advance, the elapsed time is calculated from the measurement start time or the device startup time, and control is performed so that priming is performed only when the elapsed time exceeds a certain time threshold. can do. The present invention also includes controlling the priming method by inputting and verifying user authentication information so that priming can be omitted at the discretion of a user who has management authority and who has grasped the operating state of the measuring device.

By intermittently or intermittently performing the aspiration operation for aspirating the mobile phase, rapid priming is achieved while suppressing excessive pressure reduction and generation of air bubbles. The priming operation time including the ejection step can be shortened by performing the suction of various mobile phases step by step including the stop operation and ejecting the entire amount of the aspirated mobile phase in one syringe operation.

1 is an example of a schematic configuration diagram of a liquid chromatograph of the present invention. FIG. It is another example of the structural schematic diagram of the liquid chromatograph of this invention. FIG. 4 is a diagram showing the influence of the priming methods of Examples and Comparative Examples on the elution time of HbA1c. FIG. 4 is a diagram showing the influence of the priming methods of Examples and Comparative Examples on the elution time of HbA0.

FIG. 1 shows an outline of the configuration of a liquid chromatograph (low pressure gradient). In this apparatus, three different types of mobile phases (hereinafter also referred to as eluents) 101 , 102 and 103 are fluidly connected to one liquid-sending pump 111 . Each eluent is sucked into the liquid feed pump 111 through the degassing unit 105 , the channel opening/closing sections 106 , 107 , 108 corresponding to each eluent, and the confluence section 109 . The channel opening/closing units 106, 107, and 108 can control opening/closing operations based on control instructions.

The sample injection part 112 has a sample loop and an injection valve, and injects the measurement sample into the channel between the liquid sending pump 111 and the column 113 . In addition, pretreatments such as dilution and hemolysis are performed as necessary before sample injection. Aspiration and ejection of the sample, aspiration and ejection of the washing liquid, dilution operation, and the like are performed by the syringe. Although one syringe can perform all these operations, a plurality of syringes can be combined in order to improve the accuracy of suction and discharge and shorten the measurement time. In FIG. 1, a small syringe 115 with a small volume and a large syringe (priming syringe) 116 with a large volume are provided. The small syringe 115 mainly aspirates the sample to be measured, and the large syringe 116 is responsible for cleaning the sampling line, cleaning the outer wall of the needle, priming, and the like. A priming syringe 116 refers to a syringe used for priming.

In the column 113, the components to be measured in the injected sample are adsorbed and desorbed. The components are eluted with a time lag according to the difference in elution power of the eluent alone or mixed eluent with a gradient. The measurement object separated by column 113 is detected by detector 114 . A plunger pump, for example, can be exemplified as the liquid transfer pump 111 . The liquid-sending pump 111 includes check valves before and after the flow path. The eluent reservoir (corresponding to the mobile phase source) can be exemplified by a pouch or bottle containing the eluent, and for example, a temporary storage tank can be provided in the channel. Electromagnetic valves and switching valves can be exemplified for the channel opening/closing units 106 , 107 , and 108 .

Priming is usually performed by aspirating and discharging the mobile phase using the priming syringe 116 . The supply destination of the mobile phase when the priming syringe 116 is sucked is not limited to the upstream or downstream portion of the liquid transfer pump, and is determined by the position of the branched channel (branching portion) that fluidly connects the priming syringe 116 . The liquid feeding operation of the pump may be stopped during priming, and particularly when the mobile phase is introduced from the upstream portion of the pump to the priming syringe 116, the liquid feeding operation of the pump may be accompanied. A channel from the branching part 110 to the switching rotary valve 117 is called a prime channel, which is a channel branched from the analysis channel. The prime flowpath may include a prime flowpath blocker 118 . The prime channel blocker 118 prevents liquid from flowing from the prime channel to the analysis channel due to the operation of the rotary valve 117 or the like during measurement. A two-way solenoid valve or the like can be exemplified as the prime flow passage blocker 118 . The branching portion 110 and the merging portion 109 can also be integrated.

An example of priming operation for the eluent 101 in FIG. 1 will be described. The channel opening/closing part 106 is opened to allow the eluent 101 to pass through. On the other hand, the channel opening/closing part 107 and the channel opening/closing part 108 are closed. If the prime channel blocker 118 is provided, it is opened. A switching rotary valve 117 connects the prime channel and the priming syringe 116 . Thereby, the eluent 101 and the priming syringe 116 are fluidly connected. In this state, the eluent 101 is led to the prime flow path by the priming syringe 116 entering a suction operation. On the other hand, since the liquid-sending pump 111 is equipped with a check valve, the inflow from the upstream side of the liquid-sending pump can be suppressed. After a predetermined amount of eluent has been sucked into the priming syringe, the priming syringe switches to the discharging process after a waiting time (stopping process) for stabilizing the liquid flow. The switching rotary valve 117 connects the priming syringe 116 and the injection valve 112 , and in this state, the priming syringe 116 is ejected to eject the sucked eluent from the needle 119 . This ejection operation also serves as a cleaning operation for the sampling line.

Regarding priming, in order to avoid contamination of the priming flow path, etc., ideally, when switching between different eluents or at the start and end of priming, operate the syringe with a solution that does not affect the measurement results. It is desirable to wash and replace the inside of the syringe. However, preparation of a liquid other than the eluent and consideration of the time required for the cleaning/replacement operation may make it difficult to implement them. In particular, as shown in Examples described later, the order of the eluents to be primed is important in a situation where the aspiration operation is performed continuously and the eluents are primed with a plurality of types of eluents. It is preferable that the last aspirated eluent be the eluent that has the least effect on the measurement. The eluent that has the least effect may be determined according to the measurement system (interaction of stationary phase, mobile phase and sample components) by differences in pH, salt concentration, hydrophobicity, and the like. In addition, in a situation where the temperature of the eluent is controlled, the order of priming can be changed according to the temperature difference. Generally, it is desirable not to select an eluent with a high elution power in the final order of priming according to each measurement system.

By the way, when it is assumed that the dissolved oxygen, pH, etc. of the eluent staying in the apparatus for a certain period of time change, the priming operation may be incorporated into a series of start-up operations before measurement. However, if batch measurements are performed continuously, or if the power is turned off and then immediately restarted, these operations can result in excessive liquid consumption. Therefore, these operations can be omitted according to the authority of the user. A user's authority can be determined by a user ID, its password, or the like. Also, the previous measurement end time is stored in advance, the elapsed time is calculated from the measurement start time or the device startup time, and control is performed so that priming is performed only when the elapsed time exceeds a certain time threshold. You can also

As a measurement device, an automatic glycohemoglobin analyzer HLC-723G11 (standard mode) (manufactured by Tosoh Corporation) was used. The configuration of this device is as shown in FIG. This device is a liquid chromatograph based on cation exchange chromatography. Hemoglobin in blood is usually divided into 6 fractions of hemoglobin A1a, hemoglobin A1b, hemoglobin F, unstable hemoglobin A1c, stable hemoglobin A1c (HbA1c) and A0 (HbA0), and each measurement takes 30 seconds. The present invention achieves the effect of shortening the total throughput by shortening the pre-measurement operation and start-up processing when the measurement time is shorter than about one minute.

In order to consider shortening the measurement time, TSKgel G11 (standard mode) (manufactured by Tosoh Corporation) was used as the analytical column, and the eluent was G11 as the first liquid and G11 as the second liquid. G11 eluent No. 3 (both manufactured by Tosoh Corporation) was used as the eluant No. 2 and No. 3, respectively. A priming syringe was driven by a pulse motor and had a stroke volume of 5 mL. The suction/discharge speed was controlled by a syringe speed parameter, and theoretically, the syringe speed parameter and the operation time were set to be inversely proportional. As actual values, it is about 13.3 seconds (0.375 mL/sec) when aspirating 5 mL with a syringe speed parameter of 500, and about 10 seconds (0.5 mL/sec) when aspirating 5 mL with a syringe speed parameter of 750. and about 6.7 seconds (0.75 mL/sec) when aspirating 5 mL with a syringe speed parameter of 1000.

This liquid chromatograph uses a salt concentration gradient due to the difference in salt concentration of the eluent, and the third eluent serves both as elution of the A0 peak and washing. Therefore, the order of priming was the third eluent with a high salt concentration, the second eluent with a medium salt concentration, and finally the first eluent with a low eluent concentration. It should be noted that, unless otherwise specified, the device configuration of the following embodiments or comparative examples is the same as the device configuration described above.

(Example 1)
In Example 1, a priming method was carried out based on Table 1 in which the type of eluent was switched at the time of stopping the suction, and finally discharged all at once. The aspiration speed was 0.5 mL/sec. The liquid passage state of the channel opening/closing part from the eluent source is represented by the open state (white circle ◯) and the closed state (black circle ●) for each eluent (same below). First, the third eluent was sucked (partial suction step 1/3 in Table 1). The channel opening/closing valve (reference numeral 108 in FIG. 1) for the third liquid 103 was opened, and 1.5 mL of the third liquid was sucked over a required time of 3 seconds. A waiting time (stopping step) of 15 seconds was provided because the follow-up of the liquid was delayed with respect to the suction operation of the syringe. In the meantime, the solenoid valve 3 was switched to the closed state and the solenoid valve 2 (reference numeral 107) was switched to the open state to prepare for the subsequent suction of the eluent.

Next, the second liquid of the eluent was sucked (partial suction step 2/3 in Table 1). 1.5 mL of the second liquid was sucked over a required time of 3 seconds. A standby time (stopping step) of 15 seconds was provided, during which the solenoid valve 2 was switched to the closed state and the solenoid valve 1 (reference numeral 106) was switched to the open state to prepare for the subsequent suction of the eluent. Next, the first liquid of the eluent was aspirated (partial aspiration step 3/3 in Table 1). 1.5 mL of the second liquid was sucked over a required time of 3 seconds. A standby time (stopping step) of 15 seconds was provided, during which the solenoid valve 1 was switched to the closed state.

At this point, the syringe contains the three eluents as a mixture. After the 15-second stop process, the entire amount was discharged into the drain passage by one discharge operation over a required time of 9 seconds (discharge process (total amount)). Before the discharge step (total amount), all channels to the eluent source were shut off. The time required to complete the entire priming process was approximately 75 seconds including the final stop process of 12 seconds.

(Comparative example 1)
In Comparative Example 1, a conventional priming method in which a suction step and a discharge step are performed as a set for each type of eluent was performed based on Table 2. The device configuration used is the same as in Example 1. The syringe operating liquid volume of 1.5 mL, the suction speed of 0.5 mL/sec, and the required time of 15 seconds for the stopping step were also the same as in Example 1 in each aspiration step. The time required to complete the entire priming process was approximately 105 seconds.

Compared with Comparative Example 1, Example 1 was able to shorten the total priming time to about 74% by omitting the ejection operation of each eluent and finally ejecting the entire amount.

(Example 2)
In order to investigate the influence of the aspiration speed in advance, the aspiration speed during the aspiration operation of the priming syringe was set to 0.375 mL/sec, 0.50 mL/sec, 0.63 mL/sec and 0.75 mL/sec, respectively, and the syringe operation was performed. A 4.5 mL volume aspiration/dispense motion (no stop motion during the aspiration stroke) was attempted. Among these suction speed conditions, generation of air bubbles was confirmed at 0.63 mL/sec, and significant generation of air bubbles was confirmed at 0.75 mL/sec.

In order to verify the effect of the present invention in which the aspiration operation is performed intermittently and intermittently at this relatively high aspiration speed of 0.75 mL/sec, priming of the third eluent alone was performed as shown in Table 3. . First, in the partial suction step 1/3, the flow state of the third liquid was set to the open state, and 1.5 mL was sucked over a required time of 2 seconds. The waiting time (stop process) was 5 seconds. A partial suction step 2/3 similar to the partial suction step 1/3 was performed without changing the liquid feeding state, and after a waiting time (stopping step), a similar partial suction step 3/3 was further performed. Then, a 15-second stop step was performed, during which all the flow path opening/closing parts from the eluent source were closed, and the entire amount was discharged to the drain flow path over a required time of 6 seconds in one discharge operation (discharge step (whole amount)). The time required to complete the entire priming step was very short at 49 seconds including the final stop step of 12 seconds.

Immediately after the priming shown in Table 3, five samples containing hemoglobin were continuously measured, and after the same priming was performed again, five samples were continuously measured. FIG. 3 shows changes in HbA1c elution time for a total of 10 samples (black square plots). Also, FIG. 4 shows changes in the HbA0 elution time of a total of 10 specimens (black square plots). In both FIG. 3 and FIG. 4, the peak elution time after the first priming and after priming after continuous measurement of 5 samples was stable and hardly changed. As described above, it was confirmed that the three-stage suction operation has an excellent effect of keeping the elution time stable. No further improvement was seen with more than 3 stages of aspiration. Thus, it was confirmed that stepwise priming contributed to the stability of the measurement when the syringe suction speed was increased in the priming of the same type of mobile phase.

(Comparative example 2)
In Comparative Example 2, priming of the eluent 3rd solution was performed at the same aspiration speed of 0.75 mL/sec as in Example 2, but as shown in Table 4, a method that did not include a stop step during the aspiration stroke. The total amount of mobile phase in priming is the same as in Example 2. That is, the suction step 1 was performed over a required time of 6 seconds so that the operating volume of the syringe was 4.5 mL, and the stop step was 15 seconds. After that, 4.5 mL was discharged into the drain channel over a required time of 6 seconds in one discharging operation (discharging step 1). The total priming time was extremely short at 39 seconds including a 12 second stop step after dispensing step 1.

Immediately after the above priming, 5 samples containing hemoglobin were continuously measured, and after the same priming was performed again, 5 samples were continuously measured. Changes in HbA1c elution time for a total of 10 specimens are shown in FIG. 3 (white diamond plot), and changes in HbA0 elution time are shown in FIG. 4 (white diamond plot). Although the total priming time is shorter than in Example 2 because there is no stopping step in the middle, it can be confirmed that the HbA1c and HbA0 elution times are not stable in the measurement immediately after priming. In particular, the discontinuity of the HbA0 elution time before and after priming in FIG. 4 was remarkable. Therefore, it was shown that priming at a rate (0.75 mL/sec) at which air bubbles are generated naturally shortens the priming time, but has an unfavorable effect on the measurement.

(Comparative Example 3)
In Comparative Example 3, similarly to Comparative Example 2, the aspiration step did not include the stopping step during the stroke, and the aspiration rate was set as low as 0.375 mL/sec. was carried out as follows. The liquid volume of the mobile phase in priming is the same as in Comparative Example 2. That is, the suction step 1 was performed over a required time of 12 seconds so that the operating volume of the syringe was 4.5 mL, and the stop step was 15 seconds. After that, 4.5 mL was discharged into the drain channel over a required time of 12 seconds in one discharging operation (discharging step 1). Total priming time was 51 seconds including a 12 second stop step after dispensing step 1.

Immediately after performing the above priming, continuous measurement of 5 samples was performed, and after performing priming again in the same manner, continuous measurement of 5 samples was performed. Changes in HbA1c elution time for a total of 10 specimens are shown in FIG. 3 (filled circle plot), and changes in HbA0 elution time are shown in FIG. 4 (filled circle plot). The elution time of HbA0 was stable even when measured immediately after priming, but the elution time of HbA1c varied. The required time is 51 seconds, which is equivalent to 49 seconds in Example 2, but if the mobile phase species or the number of mobile phases are increased, each required time is accumulated and may become longer.

(Example 3)
The apparatus configuration of Example 3 is shown in FIG. A difference from the apparatus configuration (FIG. 1) of the first embodiment will be described. This configuration has two series of reservoirs, reservoir A (201A, 202A and 203A) and reservoir B (201B, 202B and 203B) for each of the three different types of eluents (mobile phases). . Reservoir A and reservoir B of the same type of mobile phase are connected via mobile phase switching units (204, 205, 206) present upstream of respective channel opening/closing units (106, 107, 108). there is

Examples of the mobile phase switching unit include a three-way solenoid valve and a switching valve, which can be controlled by the control unit. In Example 3, a three-way solenoid valve was used. Also, the merging portion of the eluent and the branching portion of the analysis channel and the prime channel were integrated into a merging/branching portion 207 . During measurement or during standby, the amount of mobile phase used, weight, or mobile phase status is monitored by a liquid level sensor, etc., and when the consumption or remaining amount of liquid reaches a certain standard, the flow switching means is controlled. Then, the mobile phase source can be changed (liquid switching) between the reservoirs A and B. Although the mobile phase of the same type has the same liquid composition, there may be slight differences between the liquids due to changes in temperature and pH during use.

In order to avoid the influence of the liquid-to-liquid difference on the measurement results, priming of a new mobile phase to be used may be automatically performed at the time of liquid switching. Here, the consumption amounts of various mobile phases per measurement are often not the same, and the timing of switching various liquids differs. The control of liquid switching may be performed at the timing when the consumption of each mobile phase is detected, or the liquid exchange of all mobile phases may be performed according to the mobile phase with the largest consumption. The former has the disadvantage of increasing the total throughput by increasing the number of times of priming, and the latter has the disadvantage of increasing the amount of unconsumed liquid that is discarded.

In this example, it was assumed that the total throughput was required to be shortened and the first liquid of the eluent was consumed the most. As an initial state, the eluent 1st liquid is in reservoir A (mobile phase 1A), the eluent 2nd liquid is in reservoir B (mobile phase 2B), and the eluent 3rd liquid is in reservoir A (mobile phase 3A), Due to the consumption of the mobile phase 1A, the eluent 1st liquid enters the reservoir B (mobile phase 1B), the eluent 2nd liquid enters the reservoir A (mobile phase 2A), and the eluent 3rd liquid enters the reservoir B ( Table 6 shows the priming method when switching to mobile phase 3B). The order of priming was the third eluent (3B) with a higher salt concentration, followed by the second eluent (2A), and finally the first eluent (1B) with a lower eluent concentration.

First, after all the channel opening/closing parts are closed, the mobile phase 3A is switched to 3B, and then only the channel opening/closing part for the third liquid is opened (3A→3B/white circle). In this state, partial aspiration step 1/3, ie, 1.5 mL aspiration of mobile phase 3B was performed for 2 seconds. Immediately after that, all the channel openings and closing parts are closed during the stopping step for 5 seconds, and after switching the mobile phase 2B to 2A, only the channel opening and closing part of the second liquid is opened (2B → 2A/ white circle). In this state, partial aspiration step 2/3, ie, 1.5 mL aspiration of mobile phase 2A was performed for 2 seconds. Immediately after that, all the channel openings and closing parts are closed during the stop step for 5 seconds, and after switching the mobile phase 1A to 1B, only the channel opening and closing part of the first liquid is opened (1A → 1B/ white circle). In this state, partial aspiration step 3/3, ie, 1.5 mL aspiration of mobile phase 1B was performed for 2 seconds. Then, a 15-second stop step, during which all the flow channel openings and closing parts from the eluent source are closed, and the entire amount (4.5 mL) is poured into the drain flow channel over a required time of 6 seconds in one discharge operation. Discharged (discharge step (total amount)). The time required to complete the entire priming step was very short at 49 seconds including the final stop step of 12 seconds.

By this priming method, it is possible to shorten the priming time of the present invention even in a complicated configuration in which a plurality of reservoirs are provided for the same mobile phase species.

101, 201A, 201B Eluent first liquid 102, 202A, 202B Eluent second liquid 103, 203A, 203B Eluent third liquid 104 Hemolytic washing liquid 105 Degassing device 106 Flow path opening and closing part (for eluent first liquid)
107 Channel opening/closing part (for eluent second liquid)
108 Channel opening/closing part (for eluent third solution)
109 Confluence section 110 Branch section 111 Liquid feed pump 112 Injection valve 113 Column 114 Detector 115 Small syringe 116 Large syringe (priming syringe)
117 Rotary valve 118 Prime channel blocking unit 119 Needle 204 Mobile phase switching unit (for eluent first liquid)
205 Mobile phase switching unit (for eluent second liquid)
206 Mobile phase switching unit (for eluent third solution)
207 confluence branch

Claims (7)

In a liquid chromatograph equipped with a mobile phase source that stores and supplies a mobile phase at one end of an analysis channel, the channel is branched from the middle of the analysis channel and a syringe is liquid-connected to the distal end thereof, A priming method for liquid replacement of at least part of the inside of the analysis channel with the mobile phase, wherein the aspiration step of aspirating the mobile phase from the mobile phase source by stroke-moving the syringe within its volume range. including
The priming method, wherein the suction step comprises a stopping step of stopping the movement of the syringe for a predetermined period of time, and a plurality of partial suction steps divided by the stopping step. The mobile phase source consists of a plurality of mobile phase sources,
The partial aspiration step is an operation of aspirating the mobile phase from one of the plurality of mobile phase sources with the syringe,
Further comprising a mobile phase switching operation to switch the mobile phase from one mobile phase source aspirated by the syringe to the mobile phase from another mobile phase source during the execution of the stopping step, The priming method of claim 1. 3. The priming method according to claim 1, further comprising a discharge step of discharging the mobile phase sucked in said suction step into a drain channel or a cleaning channel with said syringe. 2. The mobile phase to be switched in the mobile phase switching operation is selected according to a suction order set based on at least one condition of pH, salt concentration, hydrophobicity and temperature of the mobile phase. Or the priming method according to 3. The priming according to any one of claims 1 to 4, wherein in the partial aspiration step, the mobile phase is aspirated at an aspiration speed of 0.5 mL/sec or more and an aspiration volume of 1.0 mL or more. Method. 6. The method according to any one of claims 1 to 5, wherein at least one of the suction speeds in the plurality of partial suction steps and the discharge speed in the discharge step is different from the other speed. Priming method as described. When the end time of the measurement operation of the liquid chromatograph is monitored, and the elapsed time from the end time to the next measurement start time exceeds a preset time interval,
Alternatively, if the authentication information related to the user is input to the liquid chromatograph at any time from power-on to the start of measurement, and the input authentication information does not meet the authentication criteria,
A priming control method, comprising executing the priming method according to any one of claims 1 to 6.