Method for priming a mobile phase and method for controlling priming.

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

Technical Field

[0001] The present invention relates to a method for priming a mobile phase in a liquid chromatograph and a method for controlling priming.

Background Art

[0002] For clinical test measurements using a liquid chromatograph, there is a demand to shorten the analysis measurement time per measurement. For example, in the analysis of glycated hemoglobin in blood, it is possible to perform measurements in an extremely short time of within 1 minute per measurement. Furthermore, not only limited to the measurement time, shortening the total throughput from the preparation for measurement and the startup of the apparatus to the completion of measurement is also important as a contribution to the operation of the clinical laboratory and the shortening of the patient's waiting time.

[0003] A liquid chromatograph separates a measurement target substance in a sample according to the characteristics of one or more mobile phases (eluents) and a stationary phase (analysis column). Therefore, the stability of the state of the mobile phase greatly contributes to the stability of the measurement results. As an example of the state of the mobile phase becoming unstable, even if the mobile phase is manufactured with the same composition, it is conceivable that the same state cannot be strictly reproduced due to errors in the manufacturing method, instrument errors, differences in the manufacturing date, etc. Therefore, generally when replacing the mobile phase, not only the supply source of the mobile phase is exchanged, but also an operation called priming (purging or liquid draining) for replacing the old mobile phase in the apparatus with a new mobile phase is performed. For example, Patent Document 1 discloses a method for priming a plunger type liquid delivery pump with a syringe pump.

[0004] Furthermore, mobile phase that remains in the device for a certain period of time may experience changes in dissolved oxygen concentration, pH, etc., and a certain amount may be discarded before measurement begins. Whether these operations are incorporated into a series of automated operations before measurement or performed manually, each operation affects the total throughput, and this effect is amplified when there are multiple mobile phases. On the other hand, if the syringe aspiration speed is increased to shorten the priming time, the supply of mobile phase may not keep up, causing air bubbles to form in the syringe or flow path. These air bubbles can significantly alter the results of sample measurement.

[0005] Furthermore, in conventional liquid chromatographs that use multiple mobile phases, it is common practice to perform priming aspiration and dispensing operations for each mobile phase. In addition, some clinical testing equipment using liquid chromatographs have a reserve mobile phase due to their high operating frequency, and the mobile phase is automatically switched to the reserve mobile phase during measurement or standby, which tends to increase the frequency and time required for priming. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2020-112060 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The object of the present invention is to provide a mobile phase priming method and a priming control method that can shorten the pre-measurement operation time and contribute to the stability of the measurement in liquid chromatography analysis. [Means for solving the problem]

[0008] The fluid delivery channels typically used in liquid chromatography have an inner diameter of 2 mm or less. For fluids moving longitudinally through a channel, a velocity gradient is generated from zero velocity at the fluid particles in contact with the channel wall to the velocity near the center of the channel. In narrow channels, this velocity gradient, i.e., shear stress within the fluid, generates significant channel resistance. Therefore, if the syringe plunger is moved at a high speed to continuously aspirate the fluid in order to shorten the priming time, a delay occurs in the movement of the liquid mobile phase, leading to a rapid decrease in pressure within the channel and potentially causing or introducing bubbles. The inventors focused on this point and achieved rapid priming while avoiding excessive pressure reduction by performing the suction operation to draw the mobile phase from the mobile phase source intermittently or inconsistently.

[0009] In other words, one aspect of the present invention that solves the above problems is a priming method for a liquid chromatograph equipped with a mobile phase source that stores and supplies a mobile phase at one end of an analytical channel, wherein the analytical channel is branched from the middle of the analytical channel and a syringe connected to the distal end of the branch is used to replace at least a part of the inside of the analytical channel with the mobile phase, and the method includes an aspiration step of aspirating the mobile phase from the mobile phase source by stroking the syringe within the range of its capacity, and the aspiration step is characterized by comprising a stop step of stopping the movement of the syringe for a certain period of time and a plurality of partial aspiration steps that are divided by the stop step. The branch of the channel where the syringe pump is installed is not limited, but it is preferable to install it in the channel before the inlet of the analytical column provided in the liquid chromatograph. This is because the flow resistance of the analytical column is usually large, so priming takes a considerable amount of time. In order to shorten the priming time, it is particularly preferable to install the branch in the channel on the inlet side of the mobile phase delivery pump.

[0010] A liquid chromatograph may be equipped with multiple mobile phase sources, and the partial aspiration step may be an operation in which a mobile phase is aspirated from one of the multiple mobile phase sources using a syringe. The configuration may include a mobile phase switching operation during the pause operation between the partial aspiration steps, in which the mobile phase being aspirated is switched to another mobile phase. In this configuration, priming operations for multiple mobile phases can be quickly achieved with a single aspiration stroke of the syringe pump.

[0011] Syringes have a limited aspiration volume per stroke, and in order to continue liquid chromatographic analysis continuously or intermittently, it is desirable to incorporate a step to discharge the aspirationed mobile phase. In a further embodiment of the priming method of the present invention, a discharge step may be further included in which the mobile phase aspirationed in a single stroke is discharged by the syringe into a drain channel or a washing channel. This embodiment is preferable in that it saves the time required for discharge compared to conventional priming methods in which aspiration and discharge operations are repeated for each mobile phase.

[0012] In the mobile phase switching operation, the mobile phase to be switched can be selected according to an aspiration sequence set based on differences in at least one of the following conditions: chemical properties (pH, salt concentration, hydrophobicity, etc.) and temperature, so as to be effective for cleaning and liquid displacement related to priming, and so as not to affect the measurement results if the mobile phase stored in the flow path after priming is affected.

[0013] In the partial aspiration process, the aspiration rate and aspiration volume of the mobile phase are not limited, but are preferably 0.5 mL / second or more and 1.0 mL or more, respectively. Furthermore, the aspiration rate and discharge rate may differ from each other, and the aspiration rate and discharge rate can be changed depending on the type and amount of mobile phase, or the priming order. For example, if the amount of priming aspiration is small, the aspiration rate or discharge rate can be reduced, and if the amount of aspiration is large, the aspiration rate or discharge rate can be increased. Alternatively, the aspiration rate or discharge rate can be changed according to the viscosity of the mobile phase.

[0014] Priming should be kept to a minimum as it leads to mobile phase consumption and delays in the start of measurement. Therefore, the previous measurement end time can be stored in advance, the elapsed time can be calculated from the measurement start time or device startup time, and priming can be controlled to be performed only if the elapsed time exceeds a certain time threshold. Furthermore, the present invention also includes controlling the priming method by inputting and verifying authentication information related to the user, so that priming can be omitted at the discretion of a user with management authority who is aware of the operating status of the measuring device. [Effects of the Invention]

[0015] By performing the suction operation to aspirate the mobile phase intermittently or inconsistently, rapid priming can be achieved while suppressing excessive pressure and bubble generation. If the suction of various mobile phases is performed in stages, including stopping operations, and the entire aspirated volume is discharged in a single syringe operation, the priming operation time, including the discharge process, can be shortened. [Brief explanation of the drawing]

[0016] [Figure 1] This is an example of a schematic diagram of the liquid chromatograph configuration of the present invention. [Figure 2] This is another example of a schematic diagram of the liquid chromatograph configuration of the present invention. [Figure 3] This figure shows the effect of the priming methods of the examples and comparative examples on the HbA1c elution time. [Figure 4] This figure shows the effect of the priming methods of the examples and comparative examples on the elution time of HbA0. [Modes for carrying out the invention]

[0017] Figure 1 shows a schematic configuration of a liquid chromatograph apparatus (low-pressure gradient). In this apparatus, three different types of mobile phases (hereinafter also called eluents) 101, 102, and 103 are liquid-connected to a single liquid delivery pump 111. Each eluent is drawn into the liquid delivery pump 111 via a degassing unit 105, flow path opening / closing units 106, 107, and 108 corresponding to each eluent, and a confluence unit 109. The flow path opening / closing units 106, 107, and 108 can be controlled to open and close based on control instructions.

[0018] The sample injection unit 112 has a sample loop and an injection valve, and injects the sample to be measured into the flow path between the liquid delivery pump 111 and the column 113. Pre-treatment such as dilution or hemolysis is performed as needed before sample injection. Sample aspiration and discharge, washing solution aspiration and discharge, and dilution operations are performed using syringes. While a single syringe can perform all these operations, multiple syringes can be combined to improve the accuracy of aspiration and discharge and shorten the measurement time. Figure 1 shows a small-capacity syringe 115 and a large-capacity syringe (priming syringe) 116. The small syringe 115 is mainly used for aspiration of the sample to be measured, while the large syringe 116 is responsible for cleaning the sampling line, cleaning the needle wall, and priming. The priming syringe 116 refers to the syringe used for priming.

[0019] In column 113, the components to be measured are adsorbed and desorbed from the injected sample. The components are eluted with a time difference depending on the difference in eluent strength between the eluent alone or a gradient mixed eluent. The substances to be measured separated in column 113 are detected by detector 114. The liquid delivery pump 111 can be exemplified by a plunger pump. The liquid delivery pump 111 is equipped with check valves at both ends of the flow path. As the eluent storage section (corresponding to the mobile phase source), examples include pouches and bottles containing the eluent, and a temporary storage tank can also be provided in the flow path, for example. The flow path opening and closing sections 106, 107, and 108 can be exemplified by solenoid valves and switching valves.

[0020] Priming is usually performed by the suction and discharge operations of the mobile phase using the priming syringe 116. The supply destination of the mobile phase during suction by the priming syringe 116 is not restricted with respect to the upstream or downstream of the liquid delivery pump, and is determined by the position (branch portion) of the branch flow path that liquid-connects the priming syringe 116. During priming, the liquid delivery operation of the pump may be stopped, and particularly when introducing the mobile phase from the upstream of the pump to the priming syringe 116, it may be accompanied by the liquid delivery operation of the pump. The flow path from the branch portion 110 to the switching rotary valve 117 is called the prime flow path and is a flow path branched from the analysis flow path. The prime flow path may include a prime flow path blocking portion 118. The prime flow path blocking portion 118 prevents liquid from flowing from the prime flow path into the analysis flow path due to the operation of the rotary valve 117 or the like during measurement. Examples of the prime flow path blocking portion 118 include a two-way solenoid valve. The branch portion 110 and the confluence portion 109 can also be integrated.

[0021] An example of an operation of priming with respect to the eluent 101 in FIG. 1 will be described. The flow path opening / closing portion 106 is opened to allow the eluent 101 to pass through. On the other hand, the flow path opening / closing portions 107 and 108 are closed. When the prime flow path blocking portion 118 is provided, it is opened. The switching rotary valve 117 connects the prime flow path and the priming syringe 116. As a result, the eluent 101 and the priming syringe 116 are fluid-connected. In this state, when the priming syringe 116 starts the suction operation, the eluent 101 is guided into the prime flow path. On the other hand, since the liquid delivery pump 111 is equipped with a check valve, the inflow from the upstream of the liquid delivery pump can be suppressed. The priming syringe that has suctioned a predetermined amount of the eluent switches to the discharge process after going through a waiting time (stop 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 discharge operation of the priming syringe 116 is performed to discharge the suctioned eluent from the needle 119. This discharge operation also serves as a cleaning operation for the sampling line.

[0022] Regarding priming, in order to avoid contamination of the priming flow path or the like, ideally, when switching between different eluents or at the start and end of priming, the syringe should be operated with a liquid that does not affect the measurement results, and cleaning and replacement within the syringe should be performed. However, considering the preparation of liquids other than the eluent and the time required for the cleaning and replacement operations, it may be difficult to carry them out. In particular, as shown in the examples described later, in a situation where suction operations are continuously performed and multiple types of eluents are primed, the order of the eluents to be primed becomes important. It is preferable that the eluent to be suctioned last is the one that has the least impact on the measurement. The eluent with the least impact may be determined according to the measurement system (the interaction between the stationary phase, the mobile phase, and the sample components) due to differences in pH, salt concentration, hydrophobicity, etc. Also, in a situation where the installation temperature of the eluent is controlled, the priming order can be changed due to the temperature difference. Generally, it is desirable not to select the eluent with a high elution output in the final order of priming according to each measurement system.

[0023] By the way, when it is assumed that the dissolved oxygen, pH, etc. of the eluent staying in the device for a certain period of time change, the priming operation may be incorporated into a series of startup operations before measurement. However, when performing batch measurements continuously or restarting immediately after turning off the power, excessive liquid consumption may occur through these operations. Therefore, according to the user's authority, these operations can also be omitted. The user's authority can be determined by the user ID, its password, etc. Also, the end time of the previous measurement can be stored in advance, the elapsed time can be calculated from the measurement start time or the device startup time, and the priming can be controlled to be performed only when the elapsed time exceeds a certain time threshold.

Example

[0024] The HLC-723G11 automated glycated hemoglobin analyzer (standard mode) (manufactured by Tosoh Corporation) was used as the measuring device. The configuration of this device is shown in Figure 1. This device is a liquid chromatograph that operates on the principle of cation exchange chromatography. Hemoglobin in the blood is fractionated into six types: hemoglobin A1a, hemoglobin A1b, hemoglobin F, unstable hemoglobin A1c, stable hemoglobin A1c (HbA1c), and A0 (HbA0), and each measurement takes 30 seconds. This invention shortens the total throughput by reducing the time required for pre-measurement operations and startup processes when the measurement time is shorter than about 1 minute.

[0025] To investigate how to shorten the measurement time, a TSKgel G11 (standard mode) analytical column (manufactured by Tosoh Corporation) was used, and the eluents used were G11 Eluent Solution 1 as the first solution, G11 Eluent Solution 2 as the second solution, and G11 Eluent Solution 3 as the third solution (all manufactured by Tosoh Corporation). A priming syringe with a pulse motor drive and a stroke capacity of 5 mL was used. The aspiration and dispensing speed was controlled by the syringe speed parameter, and the syringe speed parameter and operating time were set to be theoretically inversely proportional. The measured values ​​were approximately 13.3 seconds (0.375 mL / sec) for aspiration of 5 mL with a syringe speed parameter of 500, approximately 10 seconds (0.5 mL / sec) for aspiration of 5 mL with a syringe speed parameter of 750, and approximately 6.7 seconds (0.75 mL / sec) for aspiration of 5 mL with a syringe speed parameter of 1000.

[0026] This liquid chromatograph apparatus uses a salt concentration gradient based on the difference in salt concentration of the eluents, with the third eluent serving both to elute the A0 peak and to perform washing. Therefore, the priming order was the third eluent with a high salt concentration, followed by the second eluent with a medium salt concentration, and finally the first eluent with a low salt concentration. Unless otherwise specified, the apparatus configurations of the following embodiments or comparative forms are the same as those described above.

[0027] (Example 1) In Example 1, a priming method was implemented based on Table 1, in which the eluent type was switched during the aspiration stop process and the eluent was discharged all at once at the end. The aspiration rate was 0.5 mL / second. The flow state of the channel opening / closing section from the eluent source was indicated as open (white circle ○) or closed (black circle ●) for each eluent (the same applies below). First, the third eluent was aspirated (partial aspiration process 1 / 3 in Table 1). The channel opening / closing valve for the third eluent 103 (reference numeral 108 in Figure 1) was opened, and 1.5 mL of the third eluent was aspirated over a required time of 3 seconds. Because the liquid lags behind the aspiration action of the syringe, a waiting time (stopping process) of 15 seconds was provided. During this time, solenoid valve 3 was switched to the closed state and solenoid valve 2 (reference numeral 107) was switched to the open state in preparation for the subsequent aspiration of the eluent.

[0028] Next, the second eluent solution was aspirated (partial aspiration process 2 / 3 in Table 1). 1.5 mL of the second solution was aspirated over a period of 3 seconds. A waiting period (stopping process) of 15 seconds was provided, during which solenoid valve 2 was switched to the closed position and solenoid valve 1 (reference numeral 106) was switched to the open position in preparation for the subsequent aspiration of the eluent solution. Next, the first eluent solution was aspirated (partial aspiration process 3 / 3 in Table 1). 1.5 mL of the second solution was aspirated over a period of 3 seconds. A waiting period (stopping process) of 15 seconds was provided, during which solenoid valve 1 was switched to the closed position.

[0029] At this point, the syringe contains a mixture of the three eluents. After a 15-second stop, the entire volume is discharged into the drain channel in a single dispensing operation, taking 9 seconds (dispensing process (total volume)). Prior to the dispensing process (total volume), all channels to the eluent sources were shut off. The total time required for the priming process, including the final 12-second stop, was approximately 75 seconds.

[0030] [Table 1]

[0031] (Comparative Example 1) In Comparative Example 1, a conventional priming method was performed, consisting of a suction step and a dispensing step for each type of eluent, as shown in Table 2. The apparatus configuration used was the same as in Example 1. The syringe operating fluid volume of 1.5 mL, the suction speed of 0.5 mL / second, and the time required for the stop step of 15 seconds were also the same as in Example 1. The total time required to complete the priming process was approximately 105 seconds.

[0032] Compared to Comparative Example 1, Example 1 reduced the total priming time by approximately 74% by omitting the dispensing operation of each eluent and dispensing the entire amount at the end.

[0033] [Table 2]

[0034] (Example 2) To investigate the effect of suction speed beforehand, the suction speed during the priming syringe suction operation was set to 0.375 mL / sec, 0.50 mL / sec, 0.63 mL / sec, and 0.75 mL / sec, respectively, and a suction and dispensing operation with a syringe operating volume of 4.5 mL (without stopping during the suction stroke) was attempted. Of these suction speed conditions, the generation of air bubbles was observed at 0.63 mL / sec, and the generation of air bubbles was significantly observed at 0.75 mL / sec.

[0035] To verify the effectiveness of the present invention in performing the suction operation intermittently at a relatively high suction speed of 0.75 mL / second, priming of the third eluent alone was performed as shown in Table 3. First, in partial suction step 1 / 3, the flow state of the third eluent was opened, and 1.5 mL was aspirated over a required time of 2 seconds. The waiting time (stopping step) was set to 5 seconds. Without changing the flow state, partial suction step 2 / 3 was performed in the same manner as partial suction step 1 / 3, followed by a waiting time (stopping step), and then the same partial suction step 3 / 3 was performed again. Then, after a 15-second stopping step, during which all flow channel openings from the eluent source were closed, the entire amount was discharged into the drain channel in a single discharge operation over a required time of 6 seconds (discharge step (total amount)). The total time required to complete the entire priming process was very short, at 49 seconds, including the final 12-second stopping step.

[0036] Immediately after performing the priming shown in Table 3, five samples containing hemoglobin were measured consecutively, and the same priming was performed again, followed by consecutive measurements of the same five samples. The changes in HbA1c elution time for a total of 10 samples are shown in Figure 3 (black square plot). Similarly, the changes in HbA0 elution time for a total of 10 samples are shown in Figure 4 (black square plot). In both Figure 3 and Figure 4, the peak elution time after the initial priming and after priming following the consecutive measurement of five samples was stable with almost no fluctuation. As described above, the excellent effect of maintaining stability of elution time by performing the aspiration operation in three stages was confirmed. No further improvement was observed with aspiration operations beyond three stages. Thus, in priming of the same type of mobile phase, it was confirmed that performing priming in stages contributes to measurement stability when the syringe aspiration speed is increased.

[0037] [Table 3]

[0038] (Comparative Example 2) In Comparative Example 2, priming of the third eluent was performed at the same suction speed of 0.75 mL / second as in Example 2, but without any stopping steps during the suction stroke, as shown in Table 4. The total volume of the mobile phase during priming was the same as in Example 2. Specifically, suction step 1 was performed over a period of 6 seconds to ensure the syringe's operating volume was 4.5 mL, followed by a 15-second stopping step during which all flow path openings from the eluent source were closed. Then, in a single dispensing operation, 4.5 mL was discharged into the drain channel over a period of 6 seconds (dispensing step 1). The total priming time was extremely short, at 39 seconds, including the 12-second stopping step after dispensing step 1.

[0039] Immediately after performing the priming described above, five samples containing hemoglobin were measured consecutively. The same priming procedure was then repeated, followed by consecutive measurements of the same five samples. Figure 3 shows the changes in HbA1c elution time for a total of 10 samples (outlined diamond plot), and Figure 4 shows the changes in HbA0 elution time (outlined diamond plot). The total priming time was shorter compared to Example 2 because there were no intermediate stopping steps, but it was confirmed that the HbA1c and HbA0 elution times were not stable in the measurements taken immediately after priming. In particular, the discontinuity in HbA0 elution time before and after priming in Figure 4 was significant. Therefore, it was shown that while priming at a rate that generates bubbles (0.75 mL / sec) can shorten the priming time, it also has an undesirable effect on the measurement.

[0040] [Table 4]

[0041] (Comparative Example 3) In Comparative Example 3, similar to Comparative Example 2, the priming of the third eluent was performed as shown in Table 5, with no stop step during the stroke in the aspiration process and the aspiration rate set low at 0.375 mL / second. The amount of mobile phase during priming was the same as in Comparative Example 2. Specifically, aspiration step 1 was performed over a required time of 12 seconds to achieve a syringe operating volume of 4.5 mL, followed by a 15-second stop step during which all flow channel openings from the eluent source were closed. Then, in a single discharge operation, 4.5 mL was discharged into the drain channel over a required time of 12 seconds (discharge step 1). The total priming time, including the 12-second stop step after discharge step 1, was 51 seconds.

[0042] Immediately after performing the priming described above, five samples were measured consecutively, and then the same priming process was repeated before measuring five more samples consecutively. The changes in HbA1c elution time for a total of 10 samples are shown in Figure 3 (black circle plot), and the changes in HbA0 elution time are shown in Figure 4 (black circle plot). While the elution time for HbA0 remained stable even when measured immediately after priming, fluctuations were observed in the elution time for HbA1c. The required time was 51 seconds, which is equivalent to 49 seconds in Example 2, but if the mobile phase type or the number of mobile phases increases, the required time for each can accumulate and become longer.

[0043] [Table 5]

[0044] (Example 3) The apparatus configuration of Example 3 is shown in Figure 2. The differences from the apparatus configuration of Example 1 (Figure 1) will be explained. In this configuration, there are two series of storage sections, storage section A (201A, 202A, and 203A) and storage section B (201B, 202B, and 203B), for each of the three different types of eluents (mobile phases). Storage sections A and B for the same type of mobile phase are connected via mobile phase switching sections (204, 205, 206) located upstream of each flow path opening / closing section (106, 107, 108).

[0045] Examples of mobile phase switching units include three-way solenoid valves and switching valves, which can be controlled from the control unit. In this embodiment 3, a three-way solenoid valve was used. The eluent confluence and the branching point between the analysis channel and the prime channel are integrated into a single confluence / branching section 207. During measurement or standby, the mobile phase status is monitored by the amount used, weight, or liquid level sensor, and when the liquid consumption or remaining amount reaches a certain standard, the channel switching means is controlled to change the mobile phase source (liquid switching) between storage sections A and B. Although the liquid composition is the same for the same type of mobile phase, slight differences between liquids may be observed due to changes in temperature and pH during use.

[0046] To avoid the influence of differences between liquids on measurement results, priming of the new mobile phase to be used may be performed automatically when switching liquids. Here, the consumption of each mobile phase per measurement is often not the same, and the timing of switching between each liquid differs. Liquid switching control may be performed when the consumption of each mobile phase is detected, or all mobile phase liquids may be changed in accordance with the mobile phase with the highest consumption. The former has the disadvantage of increasing the total throughput due to the increased number of priming cycles, while the latter has the disadvantage of a larger amount of liquid being discarded without being consumed.

[0047] In this embodiment, it was required to shorten the total throughput, and the scenario in which the first eluent is consumed the most was assumed. Initially, the first eluent is in storage unit A (mobile phase 1A), the second eluent is in storage unit B (mobile phase 2B), and the third eluent is in storage unit A (mobile phase 3A). Table 6 shows the priming method when switching the first eluent to storage unit B (mobile phase 1B), the second eluent to storage unit A (mobile phase 2A), and the third eluent to storage unit B (mobile phase 3B) due to the consumption of liquid in mobile phase 1A. The priming order was the third eluent (3B) with the higher salt concentration, followed by the second eluent (2A), and finally the first eluent (1B) with the lower eluent concentration.

[0048] First, all flow channel openings were closed, and then mobile phase 3A was switched to 3B, after which only the flow channel opening for the third liquid was opened (3A→3B / white circle). In this state, partial aspiration process 1 / 3, i.e., 1.5 mL of mobile phase 3B was aspirated for 2 seconds. Immediately afterward, during a 5-second stop, all flow channel openings were closed, and then mobile phase 2B was switched to 2A, after which only the flow channel opening for the second liquid was opened (2B→2A / white circle). In this state, partial aspiration process 2 / 3, i.e., 1.5 mL of mobile phase 2A was aspirated for 2 seconds. Immediately afterward, during a 5-second stop, all flow channel openings were closed, and then mobile phase 1A was switched to 1B, after which only the flow channel opening for the first liquid was opened (1A→1B / white circle). In this state, partial aspiration process 3 / 3, i.e., 1.5 mL of mobile phase 1B was aspirated for 2 seconds. Then, a 15-second stoppage was performed, during which all channels from the eluent source were closed. After that, the entire volume (4.5 mL) was discharged into the drain channel in a single discharge operation over a period of 6 seconds (discharge process (total volume)). The total time required to complete the priming process, including the final 12-second stoppage, was a very short 49 seconds.

[0049] This priming method makes it possible to shorten the priming time in the present invention, even in complex configurations where each of the same mobile phase types has multiple storage units.

[0050] [Table 6] [Explanation of Symbols]

[0051] 101, 201A, 201B Eluent 1st liquid 102, 202A, 202B Eluent 2nd solution 103, 203A, 203B Eluent 3rd solution 104 Hemolytic wash solution 105 Degassing device 106 Flow channel opening / closing section (for eluent solution 1) 107 Flow channel opening / closing section (for eluent second solution) 108 Flow channel opening / closing section (for eluent third solution) 109 Confluence 110 Branching point 111 Liquid transfer pump 112 Injection Valve 113 columns 114 detectors 115 Small Syringe 116 Large syringe (priming syringe) 117 Rotary Valve 118 Prime flow blockage section 119 Needle 204 Mobile phase switching unit (for eluent solution 1) 205 Mobile phase switching unit (for eluent second solution) 206 Mobile phase switching unit (for eluent third solution) 207 Merging / Diverging Section

Claims

1. A liquid chromatograph equipped with a mobile phase source for storing and supplying a mobile phase at one end of an analytical channel, comprising a priming method for liquid-replacing at least a portion of the interior of the analytical channel with the mobile phase using a syringe connected to a liquid at the distal end of a branch channel of the analytical channel, the method including a suction step of aspirating the mobile phase from the mobile phase source by stroking the syringe within the range of its capacity, The priming method is characterized in that the aspiration step comprises a stopping step of stopping the movement of the syringe for a certain period of time, and a plurality of partial aspiration steps that are divided over time by the stopping step.

2. The aforementioned mobile phase source consists of multiple mobile phase sources. The partial aspiration step is an operation in which the syringe is used to aspirate the mobile phase from one of the plurality of mobile phase sources. The priming method according to claim 1, further comprising a mobile phase switching operation during the execution of the stopping step, which switches the mobile phase from one mobile phase source aspirated by the syringe to a mobile phase from another mobile phase source.

3. The priming method according to claim 1 or 2, further comprising a discharge step of discharging the mobile phase aspirated in the suction step into a drain channel or a cleaning channel using the syringe.

4. The priming method according to claim 2, characterized in that the mobile phase to be switched in the mobile phase switching operation is selected according to an aspiration sequence set based on at least one of the conditions of the mobile phase: pH, salt concentration, hydrophobicity, and temperature.

5. The priming method according to any one of claims 1, 2, or 4, characterized in that, in the partial aspiration step, the aspiration rate for aspirating the mobile phase is 0.5 mL / second or more and the aspiration volume is 1.0 mL or more.

6. The priming method according to any one of claims 1, 2, 4, or 5, characterized in that at least one of the suction speeds in the plurality of partial suction steps is different from another speed.

7. The priming method according to claim 3, characterized in that the mobile phase to be switched in the mobile phase switching operation is selected according to an aspiration sequence set based on at least one of the conditions of the mobile phase: pH, salt concentration, hydrophobicity, and temperature.

8. The priming method according to claim 3 or 7, characterized in that, in the partial aspiration step, the aspiration rate for aspirating the mobile phase is 0.5 mL / second or more and the aspiration volume is 1.0 mL or more.

9. The priming method according to any one of claims 3, 7, or 8, characterized in that 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 one of the other speeds.

10. The liquid chromatograph monitors the end time of the measurement operation, and if the elapsed time from the end time to the start time of the next measurement exceeds a predetermined time interval, A priming control method characterized by performing the priming method described in any one of claims 1 to 9.

11. For a liquid chromatograph, user authentication information is entered at any point between power-on and the start of measurement, and if the entered authentication information does not meet the authentication criteria, A priming control method characterized by performing the priming method described in any one of claims 1 to 9.

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