Liquid chromatograph and method for cleaning analysis column

Abstract

A liquid chromatograph (100) comprises: an analysis column (20) that separates a component to be measured from a sample and that is provided with an inlet (8) which is provided with a filter (8a) and into which the sample flows, a packing material (9) which comes in contact with the sample, and an outlet (10) from which the sample after separation is discharged; a liquid feed pump (3, 4) that feeds a cleaning liquid and a mobile phase to the analysis column (20); and a control device (14) that controls the liquid feed pump (3, 4), wherein the control device (14) controls the feed pump (3, 4) to feed, at a first flow rate, the cleaning liquid mainly to only the filter (8a) from among the filter (8a) and the packing material (9), holds the filter (8a) in the cleaning liquid at a second flow rate smaller than the average flow rate of the first flow rate, for a prescribed period of time, and discharges, from the analysis column (20), the cleaning liquid in which the filter (8a) was held, after the predetermined period of time has elapsed.

Description

Liquid chromatograph and analytical column cleaning method

[0001] The present disclosure relates to a method for cleaning a liquid chromatograph and an analytical column.

[0002] Liquid chromatographs are widely used for separating and analyzing components contained in biological samples. The analytical column provided in the liquid chromatograph can separate components to be separated (measured components) in a biological sample after pretreatment, for example, based on differences in physical properties such as molecular weight, polarity, and chemical structure. Patent Document 1 discloses a technique related to separation using an analytical column in a liquid chromatograph.

[0003] The abstract of Patent Document 1 describes a liquid chromatograph apparatus comprising: a liquid delivery means 11 configured to deliver at least one of a plurality of eluents in response to an input signal; an analytical column 13 connected to the liquid delivery means 11 so that the eluent is supplied from the liquid delivery means 11; a detection means 14 that detects chromatographic information based on the eluate eluted from the analytical column; and a control means 12 that outputs an analytical column cleaning command signal to the liquid delivery means 11 when the number of measurements exceeds a predetermined reference number, thereby enabling automatic determination of deterioration of the analytical column and automatic cleaning of the analytical column, and a column cleaning solution used therein.

[0004] Japanese Patent Application Laid-Open No. 2001-133446

[0005] Samples, such as biological samples, may contain impurities (contaminants), such as proteins and lipids, derived from the biological sample. Therefore, when a sample is passed through a filter attached to the inlet (flow port) of an analytical column, the filter is likely to become clogged. Therefore, the technology described in Patent Document 1 involves performing a cleaning process on the analytical column every predetermined number of times (paragraph 0075). However, although the flow rate and flow rate of the cleaning solution used for cleaning are unclear, prolonged contact time between the cleaning solution and the packing material is likely to accelerate packing material deterioration. The problem addressed by the present disclosure is to provide a method for cleaning a liquid chromatograph and an analytical column that can suppress packing material deterioration due to contact with the cleaning solution.

[0006] The liquid chromatograph of the present disclosure comprises an analytical column that separates a component to be measured from the sample and includes an inlet with a filter through which the sample flows, a packing material that comes into contact with the sample, and an outlet through which the separated sample is discharged, a liquid delivery mechanism that delivers a cleaning solution and a mobile phase to the analytical column, and a control device that controls the liquid delivery mechanism, wherein the control device controls the liquid delivery mechanism to perform the following steps: a liquid delivery step of delivering the cleaning solution at a first flow rate to the filter and the packing material, mainly to only the filter; a holding step of holding the filter in the cleaning solution at a second flow rate that is smaller than the average flow rate of the first flow rate, for a predetermined time; and a discharge step of discharging the cleaning solution that has been holding the filter from the analytical column after the predetermined time has elapsed in the holding step. Other solutions will be described later in the detailed description of the invention.

[0007] According to the present disclosure, it is possible to provide a method for cleaning a liquid chromatograph and an analytical column that can suppress deterioration of the packing material due to contact with a cleaning liquid.

[0008] 10 is a block diagram showing a liquid chromatograph of one embodiment; FIG. 11 is a block diagram showing the hardware configuration of a control device; FIG. 12 is a flowchart showing a cleaning method for an analytical column of the present disclosure; FIG. 13 is a graph showing changes over time in the flow rate of a cleaning liquid during cleaning processing in a conventional cleaning method; FIG. 14 is a graph showing changes over time in the flow rate of a cleaning liquid during cleaning processing in the cleaning method of the present disclosure; FIG. 15 is a block diagram showing a liquid chromatograph of another embodiment; FIG. 16 is a block diagram showing a liquid chromatograph of another embodiment; FIG. 17 is a block diagram showing a liquid chromatograph of another embodiment; FIG. 18 is a time chart showing the timing of separation and cleaning in each analytical column in the liquid chromatograph shown in FIG.

[0009] Hereinafter, modes for carrying out the present disclosure (hereinafter referred to as "embodiments") will be described with reference to the drawings. In the following description of one embodiment, other embodiments applicable to the one embodiment will also be described as appropriate. The present disclosure is not limited to the one embodiment described below, and different embodiments can be combined with each other or modified as desired without significantly impairing the effects of the present disclosure. Furthermore, the same components will be given the same reference numerals, and duplicate descriptions will be omitted. Furthermore, components having the same functions will be given the same names. The contents shown are merely schematic, and for convenience of illustration, changes may be made from the actual configuration within the scope of not significantly impairing the effects of the present disclosure, and some components may be omitted or modified between drawings. Furthermore, the same embodiment does not necessarily have to include all of the configurations.

[0010] 1 is a block diagram showing a liquid chromatograph 100 according to one embodiment. The liquid chromatograph 100 is an apparatus for separating components from a sample containing multiple components. Separation can be performed by passing a liquid sample through an analytical column 20 (separation column).

[0011] The liquid chromatograph 100 includes a reagent bottle 1 (cleaning liquid storage container), a reagent bottle 2 (mobile phase storage container), a liquid delivery pump 3 (liquid delivery mechanism), a liquid delivery pump 4 (liquid delivery mechanism), a confluence 5, a flow path switching valve 6, a holding mechanism 7, an analytical column 20, a sample introduction mechanism 11, a detection device 12, a waste liquid tank 13, and a control device 14.

[0012] The reagent bottle 1 (or the reagent bottle 2) is a container (tank) that stores a cleaning liquid to be sent to the analytical column 20. The reagent bottle 1 stores, for example, a cleaning liquid made of a single or mixed reagent.

[0013] The cleaning liquid is a liquid used to clean the analytical column 20, the flow path switching valve 6, the flow paths 25, 26, etc. Examples of cleaning liquids that can be used include reagents for dissolving proteins, lipids, etc., or for removing proteins, lipids, etc., from objects to which proteins, lipids, etc. are attached. Specific examples of cleaning liquids include acidic cleaning liquids such as aqueous phosphoric acid solutions, aqueous trifluoroacetic acid solutions, and aqueous acetic acid solutions; alkaline cleaning liquids such as aqueous sodium hydroxide solutions, aqueous sodium carbonate solutions, and aqueous sodium bicarbonate solutions; enzyme cleaning liquids containing protease enzymes, etc.; oxidizing agent cleaning liquids such as aqueous sodium hypochlorite solutions; organic solvent cleaning liquids such as methanol, acetonitrile, and tetrahydrofuran; and combinations of at least two of these.

[0014] The reagent bottle 2 (or reagent bottle 1) is a container (tank) that stores the mobile phase to be supplied to the analytical column 20. The reagent bottle 2 stores, for example, a mobile phase consisting of a single or mixed reagent. The mobile phase is a medium that flows the liquid sample introduced by the sample introduction mechanism 11 (described later) into the analytical column 20. As will be described in detail later, the mobile phase is also used to flow (push) a predetermined amount of cleaning solution into the analytical column 20. The mobile phase is also called an eluent. The mobile phase is, for example, an aqueous solvent, an aqueous solution, an organic solvent, or a mixed solution of an aqueous solution and an organic solvent. Specific examples of the aqueous solvent include ultrapure water, and examples of the aqueous solution include an aqueous acetic acid solution, an aqueous formic acid solution, an aqueous ammonia solution, an aqueous ammonium carbonate solution, an aqueous ammonium bicarbonate solution, and an aqueous phosphoric acid solution. Specific examples of the organic solvent include methanol, ethanol, acetonitrile, acetone, chloroform, and tetrahydrofuran.

[0015] In this way, by using either reagent bottle 1 or reagent bottle 2 as a cleaning liquid bottle, the analytical column 20 (e.g., filter 8a, filler 9, etc.) can be cleaned with cleaning liquid without being restricted by the upper limit of the sample volume in the holding mechanism 7.

[0016] The reagent bottle 1 and the flow path switching valve 6 are connected by flow paths 21, 23, such as tubes or piping. Hereinafter, in this disclosure, a "flow path" is composed of, for example, tubes, piping, or the like. The flow path 21 is provided with a liquid delivery pump 3 that delivers a cleaning solution. The flow path 21 connects the reagent bottle 1 to a junction 5. The junction 5 is a junction (connection) of the flow paths 21 and 22. The flow path 23 connects the junction 5 to the flow path switching valve 6. The reagent bottle 2 and the flow path switching valve 6 are also connected by flow paths 22, 23. The flow path 22 is provided with a liquid delivery pump 4 that delivers a mobile phase. The flow path 22 connects the reagent bottle 2 to the junction 5. The liquid delivery pumps 3, 4 are devices that deliver the cleaning solution and the mobile phase to the analytical column 20.

[0017] The sample introduction mechanism 11 is a structure for introducing a sample into, for example, the analytical column 20 of the liquid chromatograph 100. The sample introduction mechanism 11 includes an opening (not shown; port) through which the sample is introduced, and a liquid delivery pump 11a that delivers the sample introduced through the opening to the analytical column 20. The sample introduction mechanism 11 and the flow path switching valve 6 are connected by a flow path 24. The control device 14 controls the flow path switching valve 6 to connect either the flow path 23 or the flow path 24 to a flow path 25. The flow path 25 connects the flow path switching valve 6 and the analytical column 20. Therefore, by switching the flow path using the flow path switching valve 6, it is possible to switch between a cleaning solution, a mobile phase, or a sample and deliver it to the analytical column 20.

[0018] The sample may be, for example, a biological sample such as tissue fragments, cell fragments, blood, urine, other liquid components, etc. The biological sample to be used may be subjected to, for example, appropriate pretreatment as required.

[0019] The flow path switching valve 6 includes, for example, a six-way valve (not shown) and a holding mechanism 7 such as a sample loop. The sample, washing solution, and mobile phase supplied to the flow path switching valve 6 are first held in the holding mechanism 7. Then, the sample, washing solution, and mobile phase held in the holding mechanism 7 are sent to the subsequent analytical column 20.

[0020] For example, the cleaning liquid stored in the reagent bottle 1 is sent to the analytical column 20 via the flow path 21, the junction 5, the flow path 23, the flow path switching valve 6, and the flow path 25 by driving the liquid sending pump 3. Therefore, the liquid sending pump 3 (liquid sending mechanism) sends the cleaning liquid stored in the reagent bottle 1 (cleaning liquid storage container) to the analytical column 20. This allows the analytical column 20 and the like to be washed using the cleaning liquid stored in the reagent bottle 1.

[0021] In another embodiment, the liquid delivery pump 11a (liquid delivery mechanism) delivers the cleaning liquid introduced through the sample introduction mechanism 11 to the analytical column 20. This allows only the amount of liquid required for cleaning to be supplied from the sample introduction mechanism 11, and space can be saved by not using the reagent bottle 1.

[0022] The cleaning solution is subjected to removal of foreign matter, degassing, etc., as appropriate, for example, by a filter and degasser (neither of which are shown) provided between the reagent bottle 1 and the solution delivery pump 3. The cleaning solution is delivered under desired flow rate conditions by the control device 14. For example, a mobile phase may be mixed with the cleaning solution. After cleaning the analytical column 20, the cleaning solution is delivered to the waste tank 13 via a flow path 26 connected to the analytical column 20. The flow path 26 is equipped with a detection device 12, which will be described later.

[0023] Furthermore, for example, the mobile phase stored in the reagent bottle 2 is delivered to the analytical column 20 by driving the liquid delivery pump 4 via the flow path 22, the junction 5, the flow path 23, the flow path switching valve 6, and the flow path 25. Of these, the flow paths 22, 23, and 25 are the first flow paths connecting the reagent bottle 2 (mobile phase storage container) and the inlet 8 of the analytical column 20. The mobile phase is delivered under desired flow rate conditions by the control device 14. For example, a sample may be mixed with the mobile phase. The mobile phase may be subjected to removal of foreign matter, degassing, etc., as appropriate, for example, by a filter and a degasser (neither of which are shown) provided between the reagent bottle 2 and the liquid delivery pump 4. The mobile phase that has flowed through the analytical column 20 is delivered to the waste tank 13 via the flow path 26 connected to the analytical column 20.

[0024] Furthermore, the sample introduced from the sample introduction mechanism 11 is sent to the analytical column 20 via the flow path 24, the flow path switching valve 6, and the flow path 25 by driving the liquid sending pump 11a. The sample that has flowed through the analytical column 20 is detected by the detection device 12, and then sent to the waste liquid tank 13 via the flow path 26.

[0025] The analytical column 20 is a structure that separates components to be measured in a sample from the sample. The analytical column 20 separates components in the sample that have been delivered thereto, for example, based on differences in physical properties such as molecular weight, polarity, and chemical structure. The analytical column 20 also includes an inlet 8, a packing material 9, and an outlet 10. The inlet 8 is an inlet (opening) through which a cleaning solution, a mobile phase, and a sample flow into the analytical column 20. The inlet 8 includes a filter 8a, such as a sintered filter. The inclusion of the filter 8a can prevent foreign matter (impurities, contaminants, etc.) large enough to affect the packing material 9 from flowing into the analytical column 20.

[0026] The packing material 9 is, for example, a material called a stationary phase, and comes into contact with the mobile phase, washing solution, and sample that flow into the analytical column 20 through the inlet 8. It is preferable to use a material that is effective for separating components in the sample as the packing material 9. The packing material 9 may be any material, such as octadecylsilylated silica gel.

[0027] The outlet 10 is an outlet (opening) through which the sample after separation, the washing liquid after washing, and the mobile phase are discharged. The outlet 10 is provided with a filter 10a such as a sintered filter.

[0028] The detector 12 detects the components to be measured and impurities contained in the delivered sample or mobile phase and evaluates them using indices such as intensity. Examples of the detector 12 include an absorptiometry device, a fluorescence intensity measurement device, and a mass spectrometer. The waste tank 13 is a tank (an example of a container) that stores the mobile phase, sample, and cleaning solution discharged from the detector 12.

[0029] 2 is a block diagram showing the hardware configuration of the control device 14. The control device 14 is configured to include, for example, a CPU (Central Processing Unit) 1001, a RAM (Random Access Memory) 1002, a ROM (Read Only Memory) 1003, an I / F (Interface) 1004, and a bus 1005. The CPU 1001, RAM 1002, ROM 1003, and I / F 1004 are connected via, for example, the bus 1005. The control device 14 is realized by loading a predetermined control program (e.g., a method for cleaning the analytical column 20 of the present disclosure, a method for operating the liquid chromatograph 100, etc.) stored in the ROM 1003 into the RAM 1002 and executing the program by the CPU 1001. In terms of hardware, signals and information are exchanged between the control device 14 and various devices (such as servers), external networks, etc. via an I / F 1004 .

[0030] 1 , the control device 14 is a device that controls the liquid delivery pumps 3, 4 (liquid delivery mechanisms). In the example of the present disclosure, the control device 14 controls the entire liquid chromatograph 100, including each part, device, and mechanism. Specifically, for example, the control device 14 controls the liquid delivery pumps 3, 4, and 11a, the flow path switching valve 6, and the detection device 12. Furthermore, the control device 14 stores and outputs information such as the pressures of the sample, cleaning solution, and mobile phase that can be measured by the liquid delivery pumps 3, 4, and 11a, and the detection results of the detection device 12.

[0031] The control device 14 adjusts the predetermined time in the retention step S2 (described below) based on the pressure in the analytical column 20 when a fluid (a sample, a cleaning solution, or a mobile phase) is delivered during a first period and the pressure in the analytical column 20 when the fluid is delivered during a second period after the first period. The first period is, for example, the period when a sample is first passed through a brand new analytical column 20. The second period is, for example, the period after a predetermined period has elapsed since the first period (which may be a predetermined number of analyses, a predetermined number of sample flows, or the like).

[0032] As the analysis is repeated and the sample is repeatedly passed through, the filter 8a becomes more susceptible to clogging. Therefore, the pressure in the analytical column 20 increases over time, placing a greater load on the liquid delivery pumps 3, 4, 11a, etc. Therefore, by using the pressure at a first time point and the pressure at a second time point after the first time point, it is possible to understand how the pressure is changing. It is believed that the greater the pressure difference, the more advanced the clogging. Therefore, by changing the cleaning time (the predetermined time in the retention step S2) depending on the degree of clogging, cleaning can be performed for an appropriate time, reducing the risk of excessive or insufficient cleaning.

[0033] The higher the pressure in the second period, the more clogged the filter 8a is considered to be, and therefore the predetermined time can be made longer.On the other hand, the lower the pressure in the second period, the less clogged the filter 8a is considered to be, and therefore the predetermined time can be made shorter, or cleaning can be omitted altogether.

[0034] For example, the difference between the pressure at the first time point and the pressure calculated by the second equation is calculated, and if the difference is equal to or greater than a predetermined threshold value that serves as an index for performing cleaning, cleaning can be performed. Furthermore, if the difference is equal to or greater than a predetermined percentage (e.g., 10% or more) of the threshold value, the cleaning time can be extended by a predetermined percentage (e.g., 10% or more) from the predetermined reference time. This allows the cleaning time to be changed according to the pressure difference that changes due to the degree of clogging.

[0035] If the pressure is measured at predetermined intervals or each time a sample is introduced, the second time period can be the time when the pressure is measured next after the first time period (the time when the next sample is introduced). The pressure may be measured using a pressure gauge, or it may be calculated, for example, from the load on the liquid delivery pumps 3, 4, and 11a. Furthermore, clogging is usually caused by impurities in the sample, but not by the cleaning solution or mobile phase. However, a pressure increase is not limited to when a sample is passed through a clogged filter 8a, but can also be detected when the cleaning solution and mobile phase are passed through the clogged filter 8a. Therefore, the pressure during the flow of the sample is not limited to the pressure during the flow of the sample, and the pressure during the flow of at least one of the cleaning solution and the mobile phase may be used.

[0036] FIG. 3 is a flowchart showing a cleaning method for an analytical column 20 according to the present disclosure. Hereinafter, the cleaning method for an analytical column 20 according to the present disclosure will be appropriately abbreviated as the "cleaning method according to the present disclosure." The cleaning method according to the present disclosure is a method for separating a target component in a sample from the sample, and cleaning an analytical column 20 having an inlet 8 equipped with a filter 8a through which the sample flows, a packing material 9 that contacts the sample flowing in through the inlet 8, and an outlet 10 through which the separated sample is discharged. The cleaning method according to the present disclosure can be performed, for example, by the control device 14 described above. Therefore, the description of FIG. 3 will be made with reference to FIG. 1 described above as appropriate. For example, the control device 14 controls the liquid delivery pumps 3, 4, and 11a to perform the liquid delivery step S1, the retention step S2, and the discharge step S3 shown in FIG. 3.

[0037] The liquid sending step S1 is a step of sending the cleaning liquid at a first flow rate mainly to the filter 8a out of the filter 8a and the packing material 9 (i.e., sending the cleaning liquid mainly to the filter 8a out of the filter 8a and the packing material 9). The cleaning liquid may be, for example, a cleaning liquid stored in the reagent bottle 1 or a cleaning liquid introduced through the sample introduction mechanism 11. "Sending the cleaning liquid mainly to the filter 8a only" means that the cleaning liquid is sent so that the filter 8a is preferably (ideally) impregnated (submerged or immersed) in the cleaning liquid but the packing material 9 does not come into contact with the cleaning liquid. This can suppress deterioration of the packing material 9 due to contact of the cleaning liquid with the packing material 9 and extend the life of the packing material 9. For example, based on design conditions such as the capacity of the piping, the cleaning liquid can be sent mainly to the filter 8a by controlling the liquid sending conditions, such as the amount and time of the cleaning liquid (and an appropriate mobile phase) sent, so that the liquid level of the cleaning liquid is located between the filter 8a and the packing material 9.

[0038] However, it is usually difficult to precisely position the liquid surface of the delivered cleaning liquid between the filter 8a and the packing material 9 (i.e., the cleaning liquid does not come into contact with the packing material 9 at all). Therefore, it is preferable that the delivered cleaning liquid does not come into contact with the packing material 9 as much as possible, but it is acceptable for the cleaning liquid to come into contact with the packing material 9 to an extent that it is unlikely to affect the deterioration of the packing material 9. In this way, the degree of deterioration of the packing material 9 as a whole can be suppressed.

[0039] Furthermore, it is preferable that the surface of the cleaning solution is located between the filter 8a and the filler 9, i.e., it is preferable that the filter 8a is completely impregnated (submerged, immersed, dipped, or covered) with the cleaning solution. However, the cleaning solution may come into contact with only a portion of the filter 8a. Specifically, clogging of the filter 8a is likely to occur on the inlet side surface of the filter 8a that is on the sample introduction mechanism 11 side. Therefore, it is preferable that the cleaning solution come into contact with at least the inlet side surface of the filter 8a that is on the sample introduction mechanism 11 side.

[0040] The cleaning agent is delivered at a first flow rate. The first flow rate can be determined, for example, according to the cross-sectional areas of the flow paths 21, 23, 24, and 25, the capacity of the delivery pumps 3 and 11a, etc. The first flow rate may be constant or may vary (e.g., a combination of increasing, constant, and decreasing flow rates).

[0041] For example, in the liquid sending step S1, the control device 14 sends the cleaning liquid while decreasing the flow rate from the maximum flow rate as the first flow rate by controlling the liquid sending pump 3. This makes it easier to position the liquid surface of the cleaning liquid, which gradually slows down, at a desired position (for example, between the filter 8a and the filler 9), and prevents the cleaning liquid from flowing too far toward the filler 9.

[0042] Furthermore, in the liquid sending step S1, the control device 14 sends the cleaning liquid at a constant flow rate as the first flow rate by controlling the liquid sending pump 3. This simplifies the control of the liquid sending pump 3 and enables the cleaning liquid to be sent stably.

[0043] Furthermore, in the liquid sending step S1, the control device 14 sends the cleaning liquid while increasing the flow rate from the minimum flow rate as the first flow rate by controlling the liquid sending pump 3. This allows the cleaning liquid to be sent quickly to the analytical column 20, and the time required to wash the analytical column 20 can be shortened.

[0044] The liquid delivery time at the first flow rate is preferably set after detailed prior verification of the area reached by the cleaning liquid relative to the liquid delivery time. This reduces the area in contact with the packing material 9, suppresses deterioration of the packing material 9, and further suppresses deterioration of the separation performance of the analytical column 20. Furthermore, when changing the first flow rate over time, it is preferable to avoid a flow rate that results in a sudden high flow rate and pressure fluctuation or a high pressure that would impair the separation performance of the analytical column 20. Therefore, it is preferable to set the first flow rate so that the flow rate changes and flow rate are appropriate. However, since a higher flow rate can be expected to have a higher cleaning effect, it is desirable to set appropriate cleaning conditions after considering them in advance.

[0045] The holding step S2 is a step of holding the filter 8a in the cleaning liquid at a second flow rate different from the first flow rate for a predetermined time. In the holding step S2, the filter 8a is mainly cleaned using the cleaning liquid. This allows the cleaning liquid to break down foreign matter that may be causing clogging in the filter 8a, thereby eliminating the clogging. Furthermore, as described above, the cleaning liquid mainly contacts only the filter 8a out of the filter 8a and the filler 9. Therefore, in the holding step S2, the time the filler 9 is in contact with the liquid can be shortened, and deterioration of the filler 9 can be suppressed.

[0046] The second flow rate is a flow rate condition for retaining the cleaning liquid in the filter 8a. The second flow rate is a flow rate smaller than the average flow rate of the first flow rate in the liquid sending step S1. Therefore, if the retention step S2 were performed while flowing the cleaning liquid at the same first flow rate as in the liquid sending step S1, the time required for the cleaning liquid to pass through not only the filter 8a but also the entire filler 9 would be extended. Therefore, by flowing the cleaning liquid at a second flow rate smaller (less than) the first flow rate in the liquid sending step S1 (which may be stopped as described below), the time required for the cleaning liquid to pass through the entire filler 9 can be shortened. This reduces the overall degree of deterioration of the filler 9.

[0047] When the second flow rate is not zero (i.e., when the feeding of the cleaning liquid is not stopped), a small amount of the cleaning liquid comes into contact with the filler 9. However, the amount of cleaning liquid that comes into contact with the filler is less than when the cleaning liquid is fed at the first flow rate, and therefore, when the entire filler 9 is considered, the portion that does not come into contact with the liquid increases. Therefore, even when the second flow rate is not zero, it can be considered that the entire filler 9 has a reduced contact time with the liquid.

[0048] During the time when the cleaning liquid is sent at the second flow rate (including a flow rate of 0), contact between the cleaning liquid and the filter 8 a is maintained. Therefore, as described above, it is desirable to set the second flow rate to at least the average flow rate (average value) of the first flow rate or less.

[0049] The holding step S2 may be performed in a state where the supply of the cleaning liquid is stopped, or may be performed in a state where the supply of the cleaning liquid is continued. When the supply of the cleaning liquid is stopped, the second flow rate is 0. In this manner, the cleaning liquid that did not come into contact with the filler 9 in the supplying step S1 can be prevented from coming into contact with the filler 9 during the holding step S2, and deterioration of the filler 9 can be prevented.

[0050] On the other hand, while the feeding of the cleaning liquid is continued, the second flow rate in the holding step S2 is preferably a flow rate lower than the lower limit of the first flow rate in the feeding step S1, thereby shortening the time during which the cleaning liquid comes into contact with the filler 9 in the holding step S2.

[0051] The discharge step S3 is a step of discharging the cleaning solution that has retained the filter 8a from the analytical column 20 after a predetermined time has elapsed in the retention step S2. The third flow rate when discharging the cleaning solution in the discharge step S3 is not particularly limited. The third flow rate is a flow rate for discharging the cleaning solution from the analytical column 20 via the packing 9 and the outlet 10. At the start of the discharge step S3, the flow path 22 is usually filled with the mobile phase. As will be described in detail later, when the mobile phase is introduced from the inlet 8 to push the cleaning solution out of the outlet 10, the third flow rate can also be considered as the flow rate of the mobile phase.

[0052] The third flow rate may be, for example, the same as the first flow rate or the second flow rate, or may be greater than the first flow rate and the second flow rate. However, from the viewpoint of quickly completing the cleaning, the third flow rate is preferably greater than the first flow rate and the second flow rate. Discharge can be performed, for example, by driving the liquid delivery pump 4 to cause the mobile phase to flow in from the inlet 8. The third flow rate may be constant or may vary (e.g., a combination of increasing, constant, and decreasing flow rates). However, when the third flow rate is changed over time, it is desirable to set the change in flow rate per unit time so as not to result in a flow rate change accompanied by a sudden pressure fluctuation that would impair the separation performance of the analytical column 20, as with the first flow rate.

[0053] Here, in order to confirm the effect of the cleaning method of the present disclosure compared to the conventional cleaning method, the flow chart shown in FIG. 3 was carried out using the liquid chromatograph 100 shown in FIG.

[0054] First, as test conditions, in the liquid chromatograph 100 of the present disclosure and the conventional cleaning method, the flow path 21 was filled with a cleaning solution, and the other flow paths 22, 23, 24, 25, and 26 were all filled with a mobile phase. Note that in the conventional cleaning method (comparative example), the second flow rate in the retention step S2 was equal to the first flow rate in the liquid delivery step S1.

[0055] The total volume (internal volume) of the flow path 23, through which the cleaning solution and mobile phase flow, the interior of the flow path switching valve 6, the flow path 25, and the filter 8a, which exist between the confluence 5 and the outlet side of the inlet 8 (filter 8a), was 10.9 μL. Furthermore, the volume from the packing 9 to the outlet 10 was 18.5 μL, and the total volume from the outlet 10 to the waste tank 13 was 10.1 μL. Therefore, the total volume of the flow path (piping and device) connecting the confluence 5 to the waste tank 13 was the sum of these, 39.5 μL.

[0056] In addition, in the present disclosure and the conventional cleaning method, the cleaning solution was assumed to be present in reagent bottle 1. The mobile phase was assumed to be present in reagent bottle 2. The cleaning solution was a mixed reagent prepared by mixing 2.5% by mass aqueous ammonia solution and acetonitrile in a volume ratio of 4:6. The mobile phase was a mixed reagent prepared by mixing ultrapure water and acetonitrile in a volume ratio of 1:9. The sample was the supernatant obtained after adding methanol as a precipitant to serum. The analytical column 20 used was an octadecylsilyl (ODS) column.

[0057] <Explanation of Conventional Cleaning Method (FIG. 4 and Table 1)> FIG. 4 is a graph showing the change over time in the flow rate of the cleaning solution during the cleaning process in the conventional cleaning method. The cleaning process is the same as the above-mentioned liquid supplying step S1, holding step S2, and discharge step S3 except that the second flow rate is equal to the first flow rate.

[0058] 4 and FIG. 5, which will be described later, are graphs showing the change in flow rate (vertical axis) versus the time (horizontal axis) elapsed since the cleaning liquid was sent from the liquid sending pump 3 in the liquid chromatograph 100. The cleaning time (the total time for each of the liquid sending step S1, the holding step S2, and the discharge step S3) is 90 seconds in both FIGS. 4 and 5. Table 1 below lists the time elapsed since the cleaning liquid was sent from the liquid sending pump 3, the operating status of the liquid sending pumps 3 and 4 at that time, and the flow rate during the cleaning process of the analytical column 20. Table 1 corresponds to FIG. 4. The times listed in Table 1 correspond to the times listed on the horizontal axis of FIG. 4. The flow rates listed in Table 1 correspond to the flow rates listed on the vertical axis of FIG. 4.

[0059]

[0060] 4 and Table 1, the conventional cleaning method involves pumping the cleaning solution at 75 μL / min for the first 49.76 seconds of the 90-second cleaning time, and pumping the mobile phase at 75 μL / min for the remaining 40.24 seconds. Therefore, the former 49.76 seconds corresponds to the solution pumping step S1 and holding step S2 in the cleaning method of the present disclosure, and the latter 40.24 seconds corresponds to the discharge step S3 in the cleaning method of the present disclosure.

[0061] Here, t1 and t2 are defined by the following equations (1) and (2).

[0062] t1=V / (2×Q)...Equation (1) t2=V / Q...Equation (2)

[0063] In equations (1) and (2), V is the volume of the flow path (μL), and Q is the flow rate (μL / min) when feeding a liquid into a flow path with a volume V. t1 in equation (1) is the time (min) required for a cleaning liquid or mobile phase to pass through a flow path with a volume V in the shortest time when feeding at a flow rate Q. t2 in equation (2) is an estimate of the time (s) required for a cleaning liquid or mobile phase to pass through a flow path with a volume V when feeding at a flow rate Q. In flow paths such as tubes and piping, due to the influence of the wall (inner wall) that divides the flow path, the flow rate of the liquid near the inner wall is slower than the flow rate of the liquid near the center of the flow path. Therefore, time t1 in equation (1) is a time (shortest time) that ignores the influence of the inner wall. On the other hand, time t2 in equation (2) is a time (slowest time) that takes into account the influence of the inner wall. Therefore, in actual fluid phenomena, it is considered that an actual time exists between time t1 and time t2.

[0064] From the above formulas (1) and (2) and the volume of 10.9 μL from the confluence 5 to the filter 8 a, it is considered that the cleaning liquid reaches the area between the inlet 8 and the filler 9 (for example, the contact area between the filter 8 a and the filler 9; the interface between them) between t1 = 4.36 seconds and t2 = 8.72 seconds after the start of liquid supply.

[0065] The difference between the cleaning liquid transfer time (elapsed time 0 to 49.76 seconds) and t1 (= 4.36 seconds) calculated from the above formula (1) is 45.4 seconds. Also, the difference between the cleaning liquid transfer time (elapsed time 0 to 49.76 seconds) and t2 (= 8.72 seconds) calculated from the above formula (2) is 41.04 seconds. Therefore, it is considered that the cleaning liquid and the filler 9 are in contact for a period of 41.04 seconds or more and 45.4 seconds or less.

[0066] Furthermore, when the mobile phase starts to be pumped at 75 μL / min, the cleaning solution remains downstream of the confluence 5 (including the analytical column 20). Therefore, it takes longer than the above-mentioned "time of 41.04 seconds or more and 45.4 seconds or less" until the remaining cleaning solution is discharged from the analytical column 20 by the pumping of the mobile phase. In other words, it takes longer than expected for the contact between the cleaning solution and the packing 9 to be eliminated.

[0067] The contact time between the cleaning liquid and the packing material 9 can be considered to be the time required for the cleaning liquid from the confluence 5 to the inlet 8 to move outside the contact area between the packing material 9 and the outlet 10. Therefore, the contact time can be calculated from the volume 10.9 μL from the confluence 5 to the inlet 8, the volume 17.5 μL of the packing material 9, and the above formulas (1) and (2). As a result of the calculation, the contact time is 11.36 seconds or more and 22.72 seconds or less from the start of the delivery of the mobile phase.

[0068] From the above calculation of the contact time, the contact time of the cleaning liquid with the filler 9 in the conventional cleaning method is "52.4 seconds or more and 68.12 seconds or less", which is the sum of the contact time of the cleaning liquid with the filler 9 (41.04 seconds or more and 45.4 seconds or less) and the time required to move the cleaning liquid from the junction 5 to the inlet 8 (11.36 seconds or more and 22.72 seconds or less).

[0069] The amount of cleaning liquid used in the conventional cleaning method can be calculated as follows: The amount of cleaning liquid used can be considered to be the amount of cleaning liquid present downstream of the confluence 5. Therefore, the amount of cleaning liquid used is calculated as 62.2 μL from the product of 75 μL / min and 49.76 seconds.

[0070] <Explanation of the cleaning method of the present disclosure (FIG. 5, Table 2)> FIG. 5 is a graph showing the change in the flow rate of the cleaning solution over time during the cleaning process in the cleaning method of the present disclosure. Furthermore, the following Table 2 is a table listing the time elapsed since the cleaning solution was sent from the solution sending pump 3, the operating status of the solution sending pumps 3 and 4 at that time, and the flow rate during the cleaning process of the analytical column 20. Table 2 also lists the first flow rate in the solution sending step S1, the second flow rate in the retention step S2, and the third flow rate in the discharge step S3. Table 2 corresponds to FIG. 5. The times listed in Table 2 correspond to the times listed on the horizontal axis of FIG. 5. The flow rates listed in Table 2 correspond to the flow rates listed on the vertical axis of FIG. 5.

[0071]

[0072] The first flow rate (flow rate in the liquid sending step S1) is a flow rate condition for sending the cleaning liquid from the confluence 5 to the contact portion (between the filter 8a and the filler 9) between the filter 8a (inlet 8) and the filler 9. The first flow rate was set to a constant flow rate of 60 μL / min. The liquid sending time at the first flow rate was set to 10 seconds.

[0073] The reason for setting the liquid transfer time to 10 seconds will be explained. It is preferable to set the liquid transfer time by calculating the time required for the cleaning liquid to travel from the confluence 5 to the contact point between the filter 8a and the packing material 9. Therefore, based on the first flow rate of 60 μL / min, the volume of 10.9 μL from the confluence 5 to the filter 8a, and the above formulas (1) and (2), the liquid transfer time was calculated to be from t1 = 5.45 seconds to t2 = 10.9 seconds. Therefore, the liquid transfer time was set to 10 seconds as an example, falling within the range of t1 = 5.45 seconds to t2 = 10.9 seconds.

[0074] The second flow rate (flow rate in the holding step S2) and time were set to 0 μL / min and 39.74 seconds (between 10.01 seconds and 49.75 seconds), respectively. Therefore, the filter 8a was held in the cleaning solution for 39.74 seconds while the supply of the cleaning solution was stopped.

[0075] The third flow rate (flow rate in the discharge step S3) was constant at 75 μL / min. The duration of the discharge step S3 was 40.24 seconds, which is the same as the delivery time of the mobile phase in the conventional cleaning method.

[0076] Under the above conditions, the contact times of the cleaning liquid with the filler 9 in the liquid sending step S1 at the first flow rate, the holding step S2 at the second flow rate, and the discharge step S3 at the third flow rate were calculated as follows, similarly to the conventional cleaning method described above.

[0077] First, at the first flow rate and the second flow rate, the cleaning liquid reaches and is retained at the contact portion between the filter 8a and the packing material 9. At this time, for the sake of simplicity of calculation, it is considered that the cleaning liquid does not come into contact with the packing material 9. However, strictly speaking, it is assumed that the cleaning liquid comes into contact with the packing material 9 near the contact portion due to the effect of the cleaning liquid being delivered to the contact portion between the filter 8a and the packing material 9 and the effect of diffusion within the flow path.

[0078] However, the liquid contact portion is a part of the filler 9 on the filter 8a side. When the cleaning liquid diffuses in the flow path, the cleaning liquid in a reduced concentration state comes into contact with the filler 9. For this reason, it is considered that the effect on separation performance due to contact with the cleaning liquid of reduced concentration is small and can be ignored. From the above, it is considered that the effect of contact at the first flow rate and the second flow rate is negligible, and the liquid contact time was set to 0 seconds.

[0079] Next, regarding the third flow rate, in the process in which the cleaning solution present from the confluence 5 to the filter 8a is discharged out of the analytical column 20 by the delivery of the mobile phase, the cleaning solution comes into contact with the packing material 9. The method for calculating the contact time at this time is the same as that for calculating the contact time when the mobile phase is delivered in the conventional cleaning method. That is, based on the volume of the cleaning solution present from the confluence 5 to the filter 8a (10.0 μL), the volume of the packing material 9 (17.5 μL), and the above (1) and (2), the contact time is calculated to be 11.0 seconds or more and 22.0 seconds or less.

[0080] From the above, in the present disclosure, the contact time of the cleaning solution with the packing material 9 is 11.0 seconds or more and 22.0 seconds or less. On the other hand, the contact time in the conventional cleaning method is 52.4 seconds or more and 68.12 seconds or less. Therefore, with the liquid chromatograph 100 of the present disclosure, a reduction of 30.4 seconds or more is expected. This value corresponds to approximately 45% to approximately 58% of the contact time in the conventional cleaning method, and indicates that the present disclosure can significantly suppress performance degradation of the analytical column 20 due to the influence of the cleaning solution.

[0081] Furthermore, the amount of cleaning solution used in the present disclosure can be calculated as 10.0 μL from the product of the first flow rate of 60 μL / min and the solution delivery time of 10 seconds. Therefore, the amount of cleaning solution used in the present disclosure is approximately 84% less than the amount used in conventional cleaning methods. Therefore, according to the present disclosure, the amount of cleaning solution used can also be reduced.

[0082] As described above, according to the present disclosure, the contact time between the packing material 9 and the cleaning solution is shortened, and the reaction between the base material or functional groups of the packing material 9 and the cleaning solution is suppressed, and a decrease in the separation performance of the analytical column 20 is suppressed, while clogging substances in the filter 8 a are removed and clogging is suppressed.

[0083] Fig. 6 is a block diagram showing a liquid chromatograph 100 according to another embodiment. The liquid chromatograph 100 shown in Fig. 6 has the same configuration as the liquid chromatograph 100 shown in Fig. 1. For convenience, however, Fig. 6 shows a liquid delivery section 15 that collectively includes the reagent bottle 1, the reagent bottle 2, the liquid delivery pump 3, the liquid delivery pump 4, the flow path switching valve 6, the sample introduction mechanism 11, etc.

[0084] 6, in the liquid supply step S1, the control device 14 supplies the cleaning liquid to the filter 8a in an amount corresponding to the volume of the filter 8a (an amount sufficient to completely immerse the filter 8a in the analytical column 20). This reduces the amount of cleaning liquid used. Furthermore, in the discharge step S3, when the cleaning liquid is directed toward the outlet 10, the amount of cleaning liquid that comes into contact with the packing material 9 is reduced, thereby suppressing deterioration of the packing material 9.

[0085] The control device 14 controls the pumps 3 and 4 to send the cleaning liquid to the filter 8a in an amount corresponding to the volume of the filter 8a, followed by the mobile phase. This allows the cleaning liquid to be sent to the filter 8a, and makes it easier to control the delivery of the cleaning liquid to the desired position compared to when the cleaning liquid is delivered using gas. This also makes it possible to further shorten the contact time between the cleaning liquid and the filler 9, and to reduce the amount of cleaning liquid used.

[0086] The tests described above with reference to Tables 1 and 2 were also carried out in the embodiment shown in Fig. 6. Table 3 below shows the conditions for the cleaning test using the liquid chromatograph 100 shown in Fig. 6. The meanings of the items are the same as those in Table 2 above.

[0087]

[0088] In the embodiment shown in FIG. 6 , the flow path of the liquid chromatograph 100 before cleaning is in a state in which the flow path 21 ( FIG. 1 ) is filled with cleaning liquid. All other portions are filled with mobile phase. Similar to the embodiment shown in Table 2 above, the embodiment shown in FIG. 6 also cleans the filter 8 a using cleaning liquid at first to third flow rates. However, unlike the embodiment shown in Table 2 above, in the liquid delivery step S1 at the first flow rate, the liquid delivery is switched from the liquid delivery pump 3 to the liquid delivery pump 4. That is, as shown in Table 3, during the 10 seconds of liquid delivery at the first flow rate, the liquid delivery pump 3 delivers cleaning liquid at 60 μL / min for 2 seconds, and then the liquid delivery pump 4 delivers mobile phase at 60 μL / min for 8 seconds.

[0089] The contact time is estimated by the following calculation. Assuming the longest contact time, the first to third flow rates are used to hold the clogged area, i.e., the cleaning liquid, from the contact point between the filter 8a and the filler 9 toward the outlet 10. The contact time is considered to correspond to the time it takes for the cleaning liquid to completely pass through the filler 9 when being delivered at the third flow rate. In addition, the amount of cleaning liquid delivered in the embodiment shown in FIG. 6 is 2.0 μL, calculated from the product of a flow rate of 60 μL / min and 2 seconds.

[0090] Therefore, from the amount of cleaning liquid of 2.0 μL, the volume of packing 9 of 17.5 μL, the flow rate of the mobile phase at the third flow rate of 75 μL / min, and the above formulas (1) and (2), the liquid contact time can be calculated to be 7.8 seconds or more and 15.6 seconds or less. From the above, the liquid contact time can be further shortened compared to the liquid contact time of 11.0 seconds or more and 22.0 seconds or less in the embodiment shown in FIG. 1 and Table 2 above.

[0091] Furthermore, the amount of cleaning solution used in the embodiment of Fig. 6, 2.0 µL, is 80% less than the amount of cleaning solution used in the embodiment of Fig. 1, 10.0 µL. However, since the time that the filter 8a is immersed in the cleaning solution due to the suspension of the solution supply is the same, it can be expected that the same cleaning effect will be achieved.

[0092] 7 is a block diagram showing a liquid chromatograph 100 according to another embodiment. In the embodiment shown in FIG. 7, the control device 14 controls the liquid delivery pumps 3, 4, and 18 (liquid delivery mechanisms) to discharge the cleaning liquid retained in the filter 8a to the side opposite the side containing the packing material 9, as viewed from the filter 8a, in the discharge step S3. In this manner, the cleaning liquid used to wash the filter 8a can be discharged from the analytical column 20 via the inlet 8 without coming into contact with the packing material 9.

[0093] The term "without contact" as used herein includes "a form in which there is no contact at all" in which the interface of the cleaning liquid is positioned strictly between the filter 8a and the packing material 9. In this case, the time of contact with the liquid is zero. However, since this form may be difficult in practice, the present invention is not limited to this form, and as described above, the cleaning liquid may come into contact with the packing material 9 to some extent. Furthermore, by circulating the cleaning liquid in the direction opposite to the flow direction of the cleaning liquid in the liquid sending step S1, so-called "backwashing" can be performed, which makes it easier to unclog the filter 8a.

[0094] 7 further includes a reagent bottle 17 (mobile phase reservoir) for storing a mobile phase, located on the side of the filter 8a that contains the packing material 9 and downstream of the packing material 9. In the illustrated example, the reagent bottle 17 is located outside the analytical column 20 and connected downstream of the flow path switching valve 16. The flow path switching valve 16 switches between a flow path 26 that connects the analytical column 20 and the waste tank 13 and a flow path 27 that connects the analytical column 20 and the reagent bottle 17. The flow path 26 includes a detection device 12. The flow path 27 includes a liquid delivery pump 18. The flow path 26 and the flow path 27 are shared between the analytical column 20 and the flow path switching valve 16.

[0095] The liquid chromatograph 100 further includes a flow path switching valve 19 in a flow path 25 between the flow path switching valve 6 and the analytical column 20. The flow path switching valve 19 switches between the flow path 25 connecting the analytical column 20 and the flow path switching valve 6 and a flow path 28 connecting the analytical column 20 and the waste tank 30. The flow path 25 and the flow path 28 are common between the analytical column 20 and the flow path switching valve 19.

[0096] In the discharge step S3, the control device 14 controls the flow path switching valve 16 to connect the reagent bottle 17 and the analytical column 20 via the flow path 27. The control device 14 also controls the flow path switching valve 19 to connect the waste tank 30 and the analytical column 20 via the flow path 28. In this state, the liquid delivery pump 18 (liquid delivery mechanism) causes the mobile phase stored in the reagent bottle 17 to flow from the outlet 10 into the analytical column 20, thereby discharging the cleaning liquid retained in the filter 8a from the analytical column 20. In this manner, the pushing force of the mobile phase from the outlet 10 side causes the cleaning liquid to be discharged from the analytical column 20. This prevents the cleaning liquid from coming into contact with the packing 9. The discharged cleaning liquid is discarded into the waste tank 30 via the flow path 28.

[0097] FIG. 8 is a block diagram showing a liquid chromatograph 100 according to another embodiment. Unlike the liquid chromatograph 100 shown in FIG. 7 , the liquid chromatograph 100 shown in FIG. 8 does not include the reagent bottle 17, the liquid delivery pump 18, and the flow path 27. Instead, the liquid chromatograph 100 of FIG. 8 includes a flow path switching valve 31 between the liquid delivery pump 4 and the junction 5. Furthermore, as described above, the liquid chromatograph 100 of FIG. 8 includes flow paths 22, 23, and 25 (each of which is a first flow path) connecting the reagent bottle 2 (mobile phase reservoir) and the inlet 8 of the analytical column 20. Furthermore, the liquid chromatograph 100 of FIG. 8 includes a flow path 32 (a second flow path) connecting the reagent bottle 2 and the outlet 10 of the analytical column 20.

[0098] The flow paths 22 and 32 are common between the liquid delivery pump 4 and the flow path switching valve 31. The flow path 26 and the flow path 32 are also common between the analytical column 20 and the flow path switching valve 16.

[0099] The flow path switching valve 31 (switching mechanism) is a valve that is controlled by the control device 14 and switches between the flow paths 22, 23, 25, and flow path 32. The control device 14 uses flow path 32 during the discharge step S3 to discharge the cleaning solution that has been retaining the filter 8a. In this way, the mobile phase stored in the reagent bottle 2 can flow into the analytical column 20 from the outlet 10 side via the flow path 32. This allows the cleaning solution that has been retaining the filter 8a to be discharged from the analytical column 20. Furthermore, since the additional reagent bottle 17 and liquid delivery pump 18 shown in FIG. 7 can be eliminated, space can be saved.

[0100] For example, during the liquid transfer step S1, the flow path switching valve 31 switches the flow paths so that the flow paths 22, 23, and 25 are connected. This allows the mobile phase stored in the reagent bottle 2 to be used in the liquid transfer step S1. The flow path switching valve 31 is preferably installed somewhere between the liquid transfer pump 4 and the flow path switching valve 19. In this way, the mobile phase flows both before and after switching by the flow path switching valve 31.

[0101] The tests described with reference to Tables 1 and 2 above were also carried out in the embodiment shown in Fig. 8. Table 4 below shows the conditions for the cleaning test using the liquid chromatograph 100 shown in Fig. 8. The items are the same as those in Table 2 above.

[0102]

[0103] Under the conditions shown in Table 4, the state of each flow path of the liquid chromatograph 100 before cleaning was such that flow path 21 was filled with cleaning solution and all other parts were filled with mobile phase. The cleaning solution was present in reagent bottle 1, and the mobile phase was present in reagent bottle 2. The cleaning solution, mobile phase, sample, and analytical column 20 were the same as those described above with reference to Tables 1 and 2.

[0104] When the filter 8a was backwashed under these conditions for the analytical column 20 in which the pressure had increased, and the sample was again passed through the analytical column 20, a decrease in the pressure in the analytical column 20 was observed.

[0105] FIG. 9 is a block diagram illustrating a liquid chromatograph 100 according to another embodiment. The liquid chromatograph 100 illustrated in FIG. 9 includes multiple (two, three, or more) analytical columns 20. Specifically, the analytical columns 20 include an analytical column 40 (a first analytical column; one analytical column 20) and an analytical column 41 (a second analytical column; the other analytical column 20) connected in parallel to the analytical column 40. The analytical columns 40, 41 are connected in parallel along flow paths 25, 26 through which the sample, cleaning solution, and mobile phase flow. For example, the analytical columns 40, 41 are installed by preparing multiple (e.g., two) analytical columns 20 of the same type and connecting them in parallel for the flow of the sample, cleaning solution, and mobile phase. The liquid chromatograph 100 illustrated in FIG. 9 further includes a flow path switching valve 42, a waste tank 43, and a flow path switching valve 46.

[0106] The flow path switching valve 42 is a valve that switches between the flow path 26, the flow path 50, the flow path 51, and the flow path 52. The switching is performed by the control device 14. The flow path 50 is a flow path that connects the analytical column 40 and the flow path switching valve 42. The flow path 51 is a flow path that connects the analytical column 41 and the flow path switching valve 42. The flow paths 50 and 51 are shared with a part of the flow path 26. The flow path 52 is a flow path that connects the flow path switching valve 42 and the waste liquid tank 43.

[0107] The flow path switching valve 46 (switching mechanism) is a valve that switches the destination of the sample, cleaning solution, and mobile phase to either the analytical column 40 or the analytical column 41. The switching is performed by the control device 14. The flow path switching valve 46 switches between a flow path 54 and a flow path 55. The flow path 54 is a flow path that connects the flow path switching valve 46 and the analytical column 40. The flow path 55 is a flow path that connects the flow path switching valve 46 and the analytical column 41. The flow paths 54 and 55 are shared with a portion of the flow path 25.

[0108] 9, the filter 8a in the analytical column 41 is washed while the analytical column 40 is analyzing a sample. After the analysis in the analytical column 40 is completed, the filter 8a in the analytical column 40 is washed, and at the same time, the analytical column 41 is analyzing a sample. The method for washing the filter 8a in the analytical columns 40 and 41 is the same as the method described above with reference to FIG. 1.

[0109] The control for cleaning the filter 8 a in the analytical column 41 during sample analysis by the analytical column 40 is as follows: First, the flow path switching valve 46 connects the sample introduction mechanism 11 to the analytical column 40. This allows the sample to be delivered to the analytical column 40. Meanwhile, the flow path switching valve 46 connects the analytical column 41 to the reagent bottle 1 that stores the cleaning solution. This allows the cleaning solution to be delivered to the filter 8 a of the analytical column 41.

[0110] Furthermore, the flow path switching valve 42 connects the analytical column 40 to the detection device 12. As a result, the reagent flowing through the analytical column 40 flows through the flow path 26 to the detection device 12 and the waste liquid tank 13. At the same time, the flow path switching valve 42 connects the analytical column 41 to the waste liquid tank 43. As a result, the cleaning solution that has flowed through the analytical column 41 is discarded in the waste liquid tank 43.

[0111] Meanwhile, when the filter 8a in the analytical column 40 is washed and the analytical column 41 analyzes the sample, the control is as follows: First, the flow path switching valve 46 connects the sample introduction mechanism 11 to the analytical column 41. This allows the sample to be sent to the analytical column 41. Meanwhile, the flow path switching valve 46 connects the reagent bottle 1, which stores the washing liquid, to the analytical column 40. This allows the washing liquid to be sent to the filter 8a of the analytical column 41.

[0112] Furthermore, the flow path switching valve 42 connects the analytical column 41 to the detection device 12. As a result, the reagent flowing through the analytical column 41 flows through the flow path 26 to the detection device 12 and the waste liquid tank 13. At the same time, the flow path switching valve 42 connects the analytical column 40 to the waste liquid tank 43. As a result, the cleaning solution that has flowed through the analytical column 40 is discarded in the waste liquid tank 43.

[0113] According to this liquid chromatograph 100, while an analysis is being performed in at least one analytical column 20, the filter 8a in at least one other analytical column 20 can be washed, thereby improving the efficiency of analysis and washing.

[0114] 10 is a time chart showing the timing of separation and cleaning in each analytical column 40, 41 in the liquid chromatograph 100 shown in FIG. 9. The cleaning time for one analytical column 20 (e.g., analytical column 40) can be made the same as the analysis time for another analytical column 20 (e.g., analytical column 41). This allows for continuous analysis using the two analytical columns 20, and allows the analytical columns 20 to be used for a longer period of time due to cleaning.

[0115] It is not necessary for the analysis and cleaning to be performed at the same time. However, considering that the sample can flow more smoothly by clearing the clogging of the filter 8a, it is preferable to give priority to the time required to clean the filter 8a and set the analysis time in accordance with the time required to clean the filter 8a.

[0116] The present disclosure is not limited to the above-described embodiments and includes various modifications. For example, the above-described embodiments have been described in detail to clearly explain the present disclosure, and the present disclosure is not necessarily limited to those including all of the described configurations. Furthermore, it is possible to replace part of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add the configuration of another embodiment to the configuration of one embodiment.

[0117] 1 Reagent bottle (cleaning liquid storage container) 10 Outlet 100 Liquid chromatograph 10a Filter 11 Sample introduction mechanism 11a Liquid delivery pump (liquid delivery mechanism) 12 Detection device 13 Waste liquid tank 14 Control device 15 Liquid delivery section 16 Flow path switching valve (switching mechanism) 17 Reagent bottle (mobile phase storage container) 18 Liquid delivery pump (liquid delivery mechanism) 19 Flow path switching valve (switching mechanism) 2 Reagent bottle (mobile phase storage container) 20 Analytical column 21 Flow path 22 Flow path 23 Flow path 24 Flow path 25 Flow path 26 Flow path 27 Flow path 28 Flow path 3 Liquid delivery pump (liquid delivery mechanism) 30 Waste liquid tank 31 Flow path switching valve 32 Flow path 4 Liquid delivery pump (liquid delivery mechanism) 40 Analytical column 41 Analytical column 42 Flow path switching valve (switching mechanism) 43 Waste liquid tank 46 Flow path switching valve (switching mechanism) 5 Confluence 50 Flow path 51 Flow path 52 Flow path 54 Flow path 55 Flow path 6 Flow path switching valve 7 Holding mechanism 8 Inlet 8a Filter 9 Filler S1 Liquid transfer process S2 Holding process S3 Discharge process

Claims

1. A liquid chromatograph comprising: an analytical column that separates components to be measured in a sample from the sample, the analytical column comprising an inlet equipped with a filter and into which the sample flows, a packing that comes into contact with the sample that flows in through the inlet, and an outlet from which the separated sample is discharged; a liquid delivery mechanism that delivers a cleaning solution and a mobile phase to the analytical column; and a control device that controls the liquid delivery mechanism, wherein the control device controls the liquid delivery mechanism to carry out the following steps: a liquid delivery step in which the cleaning solution is delivered at a first flow rate to the filter and the packing, mainly to only the filter; a holding step in which the filter is held in the cleaning solution at a second flow rate that is smaller than the average flow rate of the first flow rate, for a predetermined time; and a discharge step in which the cleaning solution that has been holding the filter is discharged from the analytical column after the predetermined time has elapsed in the holding step.

2. A liquid chromatograph as claimed in claim 1, further comprising a cleaning liquid storage container for storing cleaning liquid to be sent to the analytical column, and wherein the liquid sending mechanism sends the cleaning liquid stored in the cleaning liquid storage container to the analytical column.

3. A liquid chromatograph as claimed in claim 1, comprising a sample introduction mechanism for introducing the sample into the analytical column, and wherein the liquid delivery mechanism delivers the cleaning liquid introduced through the sample introduction mechanism to the analytical column.

4. A liquid chromatograph as described in claim 1, characterized in that the control device controls the liquid delivery mechanism to discharge the cleaning liquid that has been holding the filter to the side opposite the side containing the filler when viewed from the filter during the discharge process.

5. A liquid chromatograph as claimed in claim 4, further comprising a mobile phase reservoir for storing a mobile phase, on the side of the packing material as viewed from the filter and downstream of the packing material, and wherein the liquid delivery mechanism discharges the cleaning liquid that has been holding the filter by causing the mobile phase stored in the mobile phase reservoir to flow from the outlet into the analytical column.

6. A liquid chromatograph as claimed in claim 4, further comprising: a mobile phase reservoir container for storing the mobile phase to be supplied to the analytical column; a first flow path connecting the mobile phase reservoir container to the inlet of the analytical column; a second flow path connecting the mobile phase reservoir container to the outlet of the analytical column; and a switching mechanism for switching between the first flow path and the second flow path, wherein the control device uses the second flow path during the discharge step to discharge the cleaning liquid that has been holding the filter.

7. A liquid chromatograph as claimed in claim 1, characterized in that the analytical columns comprise a first analytical column and a second analytical column connected in parallel to the first analytical column, and further comprising a switching mechanism for switching the destination of the sample, the cleaning solution and the mobile phase to either the first analytical column or the second analytical column.

8. A liquid chromatograph as described in claim 1, characterized in that the control device adjusts the predetermined time during the retention step based on the pressure in the analytical column when any one of the sample, the cleaning solution or the mobile phase is delivered at a first time period and the pressure in the analytical column when the fluid is delivered at a second time period after the first time period.

9. A liquid chromatograph according to claim 8, wherein the second flow rate is a flow rate that is lower than the lower limit of the first flow rate.

10. A liquid chromatograph according to claim 1, wherein the second flow rate is zero.

11. A liquid chromatograph according to claim 1, wherein the control device sends the cleaning liquid to the filter in an amount corresponding to the volume of the filter during the liquid sending step.

12. A liquid chromatograph as claimed in claim 11, characterized in that the control device controls the liquid delivery mechanism to deliver the cleaning liquid to the filter in an amount corresponding to the volume of the filter, followed by the mobile phase.

13. A liquid chromatograph according to claim 1, wherein the control device, in the liquid sending step, sends the cleaning liquid while decreasing the flow rate from the maximum flow rate as the first flow rate.

14. A liquid chromatograph according to claim 1, wherein the control device sends the cleaning liquid at a constant flow rate as the first flow rate in the liquid sending step.

15. A liquid chromatograph according to claim 1, wherein the control device, in the liquid sending step, sends the cleaning liquid while increasing the flow rate from a minimum flow rate as the first flow rate.

16. A method for cleaning an analytical column that separates components to be measured in a sample from the sample and that has an inlet equipped with a filter and through which the sample flows, a packing that comes into contact with the sample, and an outlet through which the separated sample is discharged, the method comprising: a liquid supply step of supplying a cleaning liquid at a first flow rate to the filter and the packing, mainly to the filter alone; a holding step of holding the filter in the cleaning liquid at a second flow rate that is smaller than the average flow rate of the first flow rate, for a predetermined time; and a discharge step of discharging the cleaning liquid that has been holding the filter from the analytical column after the predetermined time has elapsed in the holding step.

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