LIQUID CHROMATOGRAPHY AND METHOD FOR ANALYZING LIQUID SAMPLES

Abstract

To provide a liquid chromatograph and a liquid sample analysis method by which the separation and resolution of each component as well as the measurement precision can be improved while realizing cost reduction and miniaturization of a device.SOLUTION: Provided is a liquid sample analysis device (HPLC device) that comprises one pump, a selector valve, a column (HPLC column), a first flow channel, a second flow channel, an air-introduction passage, a detector, and a control unit. The control unit controls the pump and the selector valve, can switch between introduction of an eluate and introduction of a liquid sample into the first flow channel, introduces air bubbles between the eluate and the liquid sample from the air-introduction passage, and generates an air-segmented liquid sample in a first fluid. Furthermore, the control unit controls the pump and the selector valve to cause the air-segmented liquid sample to flow into the second flow channel from the first flow channel and dissolve, in the eluate and / or the liquid sample, the air bubbles passing through the second flow channel.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a liquid chromatograph and a method for analyzing a liquid sample. [Background technology]

[0002] Conventionally, chromatographs have been known as sample analyzers that analyze the components of samples. A chromatograph is an analyzer that pressurizes a mobile phase medium with a pump or the like to pass it through a column, and detects analytes with high performance by utilizing the difference in the interactions between the stationary phase and the mobile phase. Gas chromatographs, which use a gas called a carrier gas as the mobile phase medium, are used for the analysis of gas samples. On the other hand, liquid chromatographs, which use a liquid called an eluent as the mobile phase medium, are used for the analysis of liquid samples. High performance liquid chromatography (HPLC) is known as a method for analyzing the components of a liquid sample, and is capable of easily analyzing a liquid sample containing multiple components, such as a culture solution for hydroponic culture.

[0003] For example, Non-Patent Document 1 discloses a sample introduction device for HPLC. This sample introduction device is equipped with a switching valve capable of switching the connection / separation between a mobile phase flow path connected to a column and a sample loop into which a sample is introduced, and first, the mobile phase (eluent) pressurized by a first pump is made to flow through the column via the switching valve. A sample containing a component to be analyzed is introduced into the sample loop by a second pump in a state in which the sample loop is separated from the mobile phase flow path. Thereafter, the mobile phase flow path and the sample loop are connected to introduce the sample into the mobile phase flow path, and the mobile phase (eluent) containing the sample is introduced into the column. As described above, conventional HPLC devices include a first pump for introducing a mobile phase into the column, a second pump for introducing a sample into the sample loop, and a switching valve that can switch between connection and disconnection between the mobile phase flow path and the sample loop.

[0004] Furthermore, in such HPLC devices, for example, Patent Document 1 discloses a technique for placing an air bubble in the sample loop to reduce pressure and flow disturbance in the sample loop and separate the sample from the preceding or following sample. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 5114471 [Non-patent literature]

[0006] [Non-Patent Document 1] Hirohisa Mikami et al., "Sample introduction device for liquid chromatography," CHROMATOGRAPHY, 2011, vol. 32, No. 1, pp. 17-21 Summary of the Invention [Problem to be solved by the invention]

[0007] The above-mentioned conventional HPLC device has two pumps and a function for separating and connecting the mobile phase flow path and the sample loop, which increases the size of the device and the cost. Therefore, it is conceivable to configure the HPLC device to use only one pump and to eliminate the function for separating and connecting the mobile phase flow path and the sample loop. In this case, it is possible to realize a smaller device and lower costs. However, in the case of an HPLC device using only one pump, a separation type (flow path switching type) sample loop is not provided, and the mobile phase (eluent) and the sample are introduced by switching them into one flow path connected to the HPLC column, and the mobile phase (eluent) and the sample are flowed simultaneously. At this time, the pump's suction and discharge operations are repeated. Therefore, undesired diffusion of the sample in the flow path is likely to occur. If undesired diffusion of the sample occurs, even if the sample is introduced into the HPLC column, the separation of the sample in the HPLC column will be insufficient. In this way, although the device configuration is simplified, problems may occur that could not occur in conventional separation type (flow path switching type) HPLC devices.

[0008] The above-mentioned Patent Document 1 discloses placing air bubbles in a sample loop to reduce pressure and flow disturbance in the sample loop. However, when air bubbles are introduced into an HPLC column or a detection section, separation of the components to be detected in the HPLC column becomes insufficient, and noise due to air (air bubbles) occurs in the detector. Therefore, in HPLC devices, it is common to avoid introducing air (air bubbles) into the flow path. The technology described in the above-mentioned Patent Document 1 can reduce the effects of pressure and flow disturbance, but the above-mentioned problems due to air bubbles introduced into the flow path may occur.

[0009] Therefore, an object of the present invention is to provide a liquid chromatograph and a liquid sample analyzing method that can improve the separation resolution and measurement accuracy of each component while realizing a smaller size and lower cost of the device. [Means for solving the problem]

[0010] In order to solve the above problems, one embodiment of a liquid chromatograph according to the present invention includes a pump that draws in and discharges a fluid, a switching valve having ports on multiple sides, a column to which a liquid containing an eluent and a liquid sample is supplied and which separates components of the liquid sample by interaction with a stationary phase held therein, a first flow path that connects between the pump and the switching valve and through which the eluent and the liquid sample flow simultaneously, a second flow path that connects between the switching valve and the column and through which the eluent and the liquid sample flow simultaneously, an air introduction path having one end connected to the switching valve, a detector that detects components separated in the column, and a flow path that connects between the pump and the switching valve and through which the eluent and the liquid sample flow simultaneously. and a control unit that controls the switching valve, wherein the control unit is capable of switching between introducing the eluent and the liquid sample into the first flow path by controlling the pump and the switching valve, introducing air bubbles from the air introduction path between the eluent and the liquid sample to generate an air-segmented liquid sample in which the air bubbles, the liquid sample, and the air bubbles are arranged in this order in the first flow path having the eluent therein, and controlling the pump and the switching valve to flow the eluent and the air-segmented liquid sample from the first flow path to the second flow path, and dissolving the air bubbles passing through the second flow path in at least one of the eluent and the liquid sample.

[0011] In this way, in the flow path through which the eluent and the liquid sample flow simultaneously, the eluent and the liquid sample are separated by the air bubbles, so that undesired diffusion of the liquid sample can be suppressed even when the pump performs a suction or discharge operation. Furthermore, the air bubbles introduced between the eluent and the liquid sample are dissolved in the eluent or the liquid sample in the second flow path, so that the air bubbles are not introduced into the column or the detector. In other words, the liquid containing the eluent and the liquid sample can be introduced into the column in a state in which the diffusion of the liquid sample is suppressed and the air bubbles have disappeared. Therefore, the components to be detected in the column can be appropriately separated, and the generation of noise caused by air (air bubbles) in the detector can be suppressed.

[0012] In the above liquid chromatograph, the control unit may dissolve the air bubbles in at least one of the eluent and the liquid sample immediately before a liquid inlet of the column in the second flow path. In this case, the eluent and the liquid sample can be properly separated up to just before the liquid inlet of the column, and undesirable diffusion of the liquid sample can be suppressed, so that a decrease in the separation resolution in the column can be more appropriately suppressed.

[0013] Furthermore, in the above liquid chromatograph, the control unit may control the volume of the air bubbles introduced from the air introduction path between the eluent and the liquid sample, thereby dissolving the air bubbles passing through the second flow path in at least one of the eluent and the liquid sample. In this case, it is possible to introduce a volume of air bubbles that will dissolve in the eluent or liquid sample in the second flow path, thereby appropriately preventing air bubbles from being introduced into the column or detector.

[0014] In addition, in the above-mentioned liquid chromatograph, the volume of the air bubbles introduced between the eluent and the liquid sample may be set based on the discharge volume per unit time of the pump when the air-segmented liquid sample flows from the first flow path to the second flow path, the conductance of the column, the diameters of the first flow path and the second flow path, and the lengths of the first flow path and the second flow path. In this case, an appropriate volume of bubbles can be introduced taking into account the pressure value inside the second flow path that is expected to be generated by the operation of the pump, and the bubbles can be reliably dissolved in the eluent or liquid sample in the second flow path.

[0015] Furthermore, in the above liquid chromatograph, the volume of the air bubbles introduced between the eluent and the liquid sample may be set to be equal to or greater than 1 microliter and equal to or less than 45 microliters. In this case, the air bubbles can be appropriately dissolved in the eluent or liquid sample in the second flow path.

[0016] In addition, in the above-mentioned liquid chromatograph, the control unit may control the discharge amount per unit time of the pump when the air-segmented liquid sample is flowing from the first flow path through the second flow path into the column, thereby dissolving the air bubbles passing through the second flow path into at least one of the eluent and the liquid sample. In this case, the pressure inside the second flow path can be controlled to a pressure value at which the bubbles are dissolved in the eluent or liquid sample, thereby appropriately preventing the introduction of bubbles into the column or detector.

[0017] Furthermore, in the above liquid chromatograph, the discharge volume per unit time of the pump when the air-segmented liquid sample flows from the first flow path through the second flow path into the column may be set based on the volume of the air bubbles introduced from the air inlet path, the conductance of the column, the diameters of the first flow path and the second flow path, and the lengths of the first flow path and the second flow path. In this case, the pump's discharge volume per unit time can be appropriately controlled taking into account the pressure value (target pressure value) required to dissolve the introduced bubbles, and the bubbles can be reliably dissolved in the eluent or liquid sample in the second flow path.

[0018] The liquid chromatograph may further include a pressure relief unit disposed downstream of the detector. In this case, the pressure in the flow path downstream of the column can be maintained by the exhaust pressure unit, and the pressure of the fluid discharged from the column can be released to prevent air bubbles from being generated again, thereby appropriately preventing air bubbles from being introduced into the detector.

[0019] Furthermore, in the above liquid chromatograph, the backpressure section may have a flow path provided on the flow path downstream of the detector, the flow path having a cross-sectional area smaller than a cross-sectional area of ​​a third flow path connecting between the column and the detector. In this case, the pressure inside the flow channel downstream of the column can be appropriately maintained with a relatively simple configuration.

[0020] The above liquid chromatograph may further include an eluent flow path for collecting the eluent stored in the eluent tank, and a liquid sample flow path for collecting the liquid sample stored in the liquid sample tank, and one end of the eluent flow path and one end of the liquid sample flow path may each be connected to the switching valve. In this case, by controlling one pump and switching valve, it is possible to appropriately inject the eluent and the liquid sample into the first flow path and send the eluent and the liquid sample to the second flow path.

[0021] Furthermore, one aspect of the liquid sample analysis method of the present invention is a liquid sample analysis method in a liquid chromatograph comprising: one pump that draws in and discharges a fluid; a switching valve having ports on multiple sides; a column to which a liquid containing an eluent and a liquid sample is supplied and which separates components of the liquid sample by interaction with a stationary phase held therein; a first flow path connecting the pump and the switching valve and through which the eluent and the liquid sample flow simultaneously; a second flow path connecting the switching valve and the column and through which the eluent and the liquid sample flow simultaneously; an air introduction path having one end connected to the switching valve; and a detector that detects components separated in the column, The method includes a first step of introducing the eluent into a column and filling the inside of the column with the eluent; a second step of controlling the pump and the switching valve to introduce the liquid sample and air bubbles from the air introduction path into the first flow path having the eluent therein, thereby generating an air-segmented liquid sample in which the air bubbles, the liquid sample, and the air bubbles are arranged in this order in the first flow path having the eluent therein; a third step of controlling the pump and the switching valve to flow the eluent and the air-segmented liquid sample from the first flow path to the second flow path; a fourth step of dissolving the air bubbles passing through the second flow path in at least one of the eluent and the liquid sample; and a fifth step of separating a liquid containing the eluent and the liquid sample in the column and detecting it with the detector.

[0022] In this way, in the flow path through which the eluent and the liquid sample flow simultaneously, the eluent and the liquid sample are separated by the air bubbles, so that undesired diffusion of the liquid sample can be suppressed even when the pump performs a suction or discharge operation. Furthermore, the air bubbles introduced between the eluent and the liquid sample are dissolved in the eluent or the liquid sample in the second flow path, so that the air bubbles are not introduced into the column or the detector. In other words, the liquid containing the eluent and the liquid sample can be introduced into the column in a state in which the diffusion of the liquid sample is suppressed and the air bubbles have disappeared. Therefore, the components to be detected in the column can be appropriately separated, and the generation of noise caused by air (air bubbles) in the detector can be suppressed.

[0023] Furthermore, in the above liquid sample analysis method, in the second step, the pump and the switching valve may be controlled to introduce air bubbles from the air introduction path via the switching valve into the first flow path having the eluent therein, then the pump and the switching valve may be controlled to introduce the liquid sample from a liquid sample flow path, one end of which is connected to the switching valve and which collects the liquid sample stored in a liquid sample tank, into the first flow path via the switching valve, and then the pump and the switching valve may be controlled to introduce air bubbles from the air introduction path via the switching valve into the first flow path. In this case, in the first flow path having the eluent therein, an air-segmented liquid sample consisting of air bubbles, liquid sample, and air bubbles arranged in that order can be properly generated, and the eluent and the liquid sample can be properly separated by the air bubbles.

[0024] Furthermore, in the above liquid sample analysis method, after the first step, operation of the pump may be stopped, and after a certain waiting time has elapsed, the second step may be carried out. In this case, the pressure in the first and second flow paths, which has risen due to the discharge operation of the pump in the first step, can be reduced during the standby time when the operation of the pump is stopped. In this way, by performing the second step after reducing the pressure in the flow paths, it is possible to prevent the eluent in the first and second flow paths from flowing back into the air introduction path when the switching valve is switched to introduce air bubbles from the air introduction path in the second step. As a result, it is possible to introduce a desired amount of air bubbles into the first flow path.

[0025] Furthermore, in the above liquid sample analysis method, in the first step, the pump and the switching valve may be controlled to introduce the eluent from an eluent flow path, one end of which is connected to the switching valve and which collects the eluent stored in an eluent tank, into the first flow path via the switching valve, and then the pump and the switching valve may be controlled to introduce a portion of the eluent from the first flow path via the second flow path into the column, thereby filling the inside of the column with the eluent. In this case, the inside of the flow channel including the first flow channel, the second flow channel and the column can be appropriately replaced with the eluent.

[0026] Furthermore, in the above liquid sample analysis method, in the second step, the volume of the bubbles introduced may be set based on the discharge amount per unit time of the pump in the third step, the conductance of the column, the diameters of the first flow path and the second flow path, and the lengths of the first flow path and the second flow path. In this case, an appropriate volume of bubbles can be introduced taking into account the pressure value inside the second flow path that is expected to be generated by the operation of the pump, and the bubbles can be reliably dissolved in the eluent or liquid sample in the second flow path.

[0027] Furthermore, in the above liquid sample analysis method, in the second step, the air bubbles may be introduced between the eluent and the liquid sample with a volume of not less than 1 microliter and not more than 45 microliters. In this case, the air bubbles can be appropriately dissolved in the eluent or liquid sample in the second flow path.

[0028] Furthermore, in the above liquid sample analysis method, in the fourth step, the discharge volume per unit time of the pump may be controlled based on the volume of the air bubbles introduced from the air inlet path in the second step, the conductance of the column, the diameters of the first flow path and the second flow path, and the lengths of the first flow path and the second flow path. In this case, the pump's discharge volume per unit time can be appropriately controlled taking into account the pressure value (target pressure value) required to dissolve the introduced bubbles, and the bubbles can be reliably dissolved in the eluent or liquid sample in the second flow path. Effect of the Invention

[0029] According to the present invention, it is possible to realize a compact and inexpensive device while improving the separation resolution and measurement accuracy of each component. [Brief description of the drawings]

[0030] [Figure 1] 1 is a diagram showing an example of the structure of a liquid sample analyzer according to the present embodiment; [Diagram 2] 5A to 5C are diagrams illustrating an example of the operation of the liquid sample analyzer. [Diagram 3] 5A to 5C are diagrams illustrating an example of the operation of the liquid sample analyzer. [Figure 4] 5A to 5C are diagrams illustrating an example of the operation of the liquid sample analyzer. [Diagram 5] 5A to 5C are diagrams illustrating an example of the operation of the liquid sample analyzer. [Figure 6] 5A to 5C are diagrams illustrating an example of the operation of the liquid sample analyzer. [Figure 7] 5A to 5C are diagrams illustrating an example of the operation of the liquid sample analyzer. [Figure 8] 5A to 5C are diagrams illustrating an example of the operation of the liquid sample analyzer. [Figure 9] FIG. 13 is an image of an air segmentation sample. [Figure 10]5A to 5C are diagrams illustrating an example of the operation of the liquid sample analyzer. [Figure 11] FIG. 13 is an image of the compressed state of an air segment sample. [Figure 12] 5A to 5C are diagrams illustrating an example of the operation of the liquid sample analyzer. [Figure 13] FIG. 1 is an image of an air-segmented sample immediately prior to introduction into the column. [Figure 14] FIG. 1 is a diagram for explaining an example of a basic configuration of a liquid analysis system. [Figure 15] FIG. 1 is a diagram for explaining an example of a basic configuration of a liquid analysis system. [Figure 16] FIG. 1 shows an example of the structure of a conventional liquid sample analyzer. [Figure 17] 2 is an example of a signal obtained from a detector. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Analytical systems that employ liquid chromatography are used, for example, as detection systems for the earlier detection of targets such as infectious diseases as a countermeasure against terrorism and pandemics, and as situation assessment systems based on analytical information collected at multiple locations in order to prevent the spread of such targets. In infectious disease testing, the RNA or DNA of the target pathogen or virus can be analyzed directly by liquid chromatography, or it can be analyzed by liquid chromatography via a sensitization method such as PCR, LAMP, hybridization, or intercalator. Examples of such infectious diseases include anthrax, avian influenza, Crimean-Congo hemorrhagic fever, dengue fever, Ebola hemorrhagic fever, Hendra virus infection, hepatitis, influenza, Lassa fever, Marburg fever, meningococcal disease, Nipah virus infection, plague, Rift Valley fever, Severe Acute Respiratory Syndrome (SARS), smallpox, tularemia, and yellow fever.

[0032] In addition, analytical systems that employ liquid chromatography are also used as systems for monitoring the quality of drinking water in drinking water manufacturing plants, and as systems for analyzing the components of nutrient solution in which nutrients necessary for plants (vegetables and other crops) are dissolved in water in plant factories. FIG. 14 is a diagram showing an example of a liquid analysis system in a manufacturing plant for drinking water or the like. 14, in a manufacturing plant for drinking water or the like, in order to monitor the quality of the drinking water or the like being produced, a portion of liquid 82 to be analyzed, such as drinking water, is collected from a liquid tank 81 that stores the drinking water, etc. Then, the collected liquid 82 is sent to a liquid analyzer 84 via a liquid sample collection flow path 83, and the components are analyzed by the liquid analyzer 84.

[0033] FIG. 15 is a diagram illustrating an example of a liquid analyzing system in a plant factory. Generally, in a plant factory, hydroponic cultivation is carried out using a nutrient solution in which nutrients necessary for plants (crops such as vegetables) are dissolved in water. Fig. 15 shows an example of hydroponic cultivation using a circulation type in which a nutrient solution 91 is circulated as the nutrient solution cultivation. 15 , a nutrient solution 91 supplied to a cultivation tank 93 in which a crop 92 is grown is circulated through a circulation flow path 95 by a liquid delivery pump 94. A portion of the circulating nutrient solution 91 is collected, for example automatically, through a liquid sample collection flow path 96 branched off from a part of the circulation flow path 95, and the collected nutrient solution 91 is subjected to component analysis by a liquid analyzer 97.

[0034] In the case of recirculating hydroponics, the composition of the nutrient solution held in the cultivation tank changes as the nutrient solution circulates. Since plant growth is affected by the nutrient solution components, the nutrient solution is adjusted as necessary based on the results of nutrient solution analysis. As the liquid analyzers 84 and 97, a high performance liquid chromatography (HPLC) device capable of easily analyzing multiple components contained in a liquid sample can be used. The analytical devices in these systems are desirably small, low-cost, and can be installed anywhere.

[0035] Hereinafter, an embodiment of the present invention will be described with reference to the drawings. First Embodiment First, an example of a conventional small-sized, low-cost HPLC device will be described with reference to FIG. FIG. 16 is a diagram showing an example of the configuration of a conventional HPLC apparatus 10A. The HPLC device 10A includes a pump 11, a switching valve 12, and an HPLC column 13. A control unit 32 controls the operation of the pump 11 and the switching valve 12 to introduce an eluent 41a, which is a mobile phase medium stored in an eluent tank 41, and a liquid sample (sample) 42a stored in a liquid sample tank (sample tank) 42 into the HPLC column 13 via an eluent flow path 21 and a liquid sample flow path (sample flow path) 22, respectively.

[0036] The liquid sample 42a introduced into the HPLC column 13 is separated into its constituent components by the HPLC column 13. The separated components are then detected by the detector 31, and the detection data from the detector 31 is analyzed in the data processing unit 33 to identify and quantify the separated components. The analysis results from the data processing unit 33 are sent to, for example, the control unit 32. The eluent 41a and liquid sample 42a that have passed through the HPLC column 13 and the detector 31 are passed through the drainage flow path 25 and are retained in a drainage tank 43 as a drainage liquid 43a.

[0037] When liquid sample 42a flows through HPLC column 13 together with eluent 41a, each component constituting liquid sample 42a moves while interacting with HPLC column 13, which is the stationary phase. The time at which each component is eluted from HPLC column 13 is determined by the difference in the strength of the interaction between each component and HPLC column 13. In other words, the components contained in liquid sample 42a are separated by utilizing the difference in the elution time of each component passing through HPLC column 13.

[0038] The separated components are detected by the detector 31. At this time, the detector 31 detects signals corresponding to the respective components according to the retention times of the respective components on the HPLC column 13. That is, the signals corresponding to the respective components have time dependence. Here, the retention time is the time from when it flows into the HPLC column 13 until it elutes from the HPLC column 13 and is detected by the detector 31. For example, when the liquid sample 42a contains substance A, substance B, and substance C, and the retention times of substance A, substance B, and substance C passing through the HPLC column 13 are t1, t2, and t3 (t1 < t2 < t3), signals corresponding to substance A, substance B, and substance C are obtained from the detector 31 in order as shown in FIG. 17.

[0039] The HPLC apparatus 10A shown in FIG. 16 operates roughly as follows. First, by the suction operation of the pump 11, the eluent 41a is introduced from the eluent tank 41 into the first flow path 23 through the eluent flow path 21 and the switching valve 12 (step 1). Next, by the discharge operation of the pump 11, the eluent 41a in the first flow path 23 is introduced into the HPLC column 13 through the first flow path 23, the switching valve 12, and the second flow path 24 (step 2). At this time, at least a part of the eluent 41a introduced into the HPLC column 13 is stored as the drained liquid 43a in the drain tank 43 through the detector 31 and the waste liquid flow path 25.

[0040] Next, by the suction operation of the pump 11, the liquid sample (sample) 42a is introduced from the sample tank 42 into the first flow path 23 through the sample flow path 22 and the switching valve 12 (step 3). At this time, the sample 42a is mixed with the eluent 41a remaining in the first flow path 23 in the first flow path 23. Next, by the discharge operation of the pump 11, the eluent 41a and the sample 42a in the first flow path 23 are introduced into the HPLC column 13 through the switching valve 12 and the second flow path 24 (step 4). Then, each component constituting the sample 42a in the eluent 41a is separated by the HPLC column 13, and the separated components are introduced into the detector 31 in the order of separation. Then, the detector 31 detects the sample 42a introduced for each component (step 5). After detection, the eluent 41a and the sample (each component) 42a are stored as a drainage 43a in the drainage tank 43 via the drainage flow path 25 (step 6).

[0041] The HPLC device 10A shown in FIG. 16 is equipped with only one pump and does not require two pumps for respectively introducing the eluent and the sample, as in a separation-type (flow path switching) HPLC device in which the eluent flow path and the sample flow path are separate, thereby enabling the device to be made smaller and less expensive. However, pump 11, which is, for example, a syringe pump, replaces the liquid in the flow path by repeating suction and discharge operations. In order to introduce sample 41a into HPLC column 13, steps 1 to 4 must be performed as described above, and pump 11 also performs suction and discharge operations multiple times. When the pump 11 performs the suction and discharge operations as described above, the sample 42a may be undesirably diffused in the flow path. If the sample 42a is undesirably diffused, separation of the sample in the HPLC column may be insufficient even if a liquid containing the eluent 41a and the sample 42a is introduced into the HPLC column.

[0042] The HPLC device in this embodiment is a compact and low-cost HPLC device equipped with a single pump, and suppresses problems such as undesired diffusion of the sample (liquid sample) in the flow path. Specifically, the HPLC device in this embodiment uses a configuration in which air bubbles are provided before and after the sample (liquid sample) in the flow path in the flow path (hereinafter, also referred to as "air-segmented sample") to suppress undesired diffusion of the sample (liquid sample) in the flow path.

[0043] However, when an air-segmented sample (liquid air analysis sample) is introduced into an HPLC column or detector, the components to be detected may not be separated sufficiently within the HPLC column, and noise due to air (air bubbles) may occur in the detector. Therefore, the HPLC device of this embodiment uses an air-segmented sample to suppress problems such as undesirable diffusion of the sample (liquid specimen) within the flow path, while ensuring that the air bubbles that make up the air-segmented sample disappear before being introduced into the HPLC column, thereby avoiding problems in the column and detector caused by air.

[0044] FIG. 1 is a diagram showing an example of the configuration of a liquid analysis system including a liquid analysis device (HPLC device) 10 according to this embodiment. The HPLC apparatus 10 shown in FIG. 1 is an HPLC apparatus using only one pump, similar to the HPLC apparatus 10A shown in FIG. 16 described above, and parts having the same configuration as the HPLC apparatus 10A shown in FIG. 16 are given the same reference numerals as in FIG. 16. Specifically, the configuration of the HPLC apparatus 10 shown in FIG. 1, except for the switching valve 14, the exhaust pressure section 34, and the pressure gauge 35, is the same as that of the HPLC apparatus 10A shown in FIG.

[0045] The HPLC device 10 includes, in this order, a pump 11, a switching valve 14 having ports on multiple sides, and an HPLC column 13 (corresponding to the column of the present invention). The HPLC device 10 also includes an eluent flow path 21, a sample flow path 22 (liquid sample flow path), a first flow path 23, a second flow path 24, a third flow path 26, a fourth flow path 27, a drainage flow path 25, and an air introduction path 28. The HPLC device 10 also includes a detector 31, a control unit 32, a data processing unit 33, an exhaust pressure unit 34, and a pressure gauge 35.

[0046] The eluent flow path 21 is a flow path for introducing an eluent 41a stored in an eluent tank 41 into the first flow path 23, and the sample flow path 22 is a flow path for introducing a sample (liquid sample) 42a stored in a sample tank (corresponding to a liquid sample tank of the present invention) 42 into the first flow path 23. One end of the eluent flow path 21 and one end of the sample flow path 22 are each connected to a switching valve 14.

[0047] The first flow path 23 is a flow path that connects the pump 11 and the switching valve 14, and the second flow path 24 is a flow path that connects the switching valve 14 and the HPLC column 13. The first flow path 23 and the second flow path 24 are flow paths through which the eluent 41a and the sample 42a flow simultaneously, and constitute the main flow path of the HPLC device 10. The third flow path 26 is a flow path that connects the HPLC column 13 and the detector 31, and the fourth flow path 27 is a flow path that connects the detector 31 and the exhaust pressure section . The drainage flow path 25 is a flow path for discharging the fluid in the flow path of the HPLC device 10 to a drainage tank 43 as a drain 43a. The air introduction channel 28 is a channel for introducing air outside the HPLC device 10 into the first channel 23. One end of the air introduction channel 28 is connected to the switching valve 14, and the other end of the air introduction channel 28 is an open air intake port.

[0048] The pump 11 is a liquid delivery section for delivering an eluent 41 a and / or a sample 42 a to the HPLC column 13 , and is connected to the HPLC column 13 via a switching valve 14 . The detector 31 is provided after the HPLC column 13 in the main flow path. The sample 42a introduced into the HPLC column 13 is separated into its constituent components by interaction with the stationary phase held inside the HPLC column 13. The detector 31 detects the components separated in the HPLC column 13, and sends the detected detection data to the data processing unit 33.

[0049] The data processing unit 33 analyzes the detection data detected by the detector 31, and identifies and quantifies the components of the sample (liquid specimen). The data processing unit 33 sends the analysis results to the control unit 32. The control unit 32 controls the operation of the pump 11 and the switching valve 14. By switching the switching valve 14, the control unit 32 can switch the flow path connected to the first flow path 23 to any one of the eluent flow path 21, the sample flow path 22, the second flow path 24, and the air introduction path 28. In addition, the control unit 32 can switch the flow direction of the fluid flowing in the flow path of the HPLC device 10 by controlling the suction operation and discharge operation of the pump 11. Furthermore, the control unit 32 can also control the operation of the pump 11 based on pressure data from a pressure gauge 35 provided in the first flow path 23, as will be described later. Furthermore, the control unit 32 can receive the analysis results analyzed by the data processing unit 33, and transmit them to the outside as necessary, or display the results on a monitor (not shown) or the like.

[0050] The operation process of the HPLC device 10 of this embodiment will be described below with reference to Figures 2 to 13. Note that the control unit 32 and the data processing unit 33 are omitted from Figures 2 to 8, 10, and 12. This operation process is broadly composed of three steps: preparation, sampling, and analysis.

[0051] (1) Preparation process The preparatory process includes a step of aspirating the eluent 41a (STEP 1), and a subsequent replacement step of replacing the inside of the HPLC column 13 with the eluent 41a (STEP 2). [STEP 1: Aspirating the eluent] FIG. 2 is a diagram illustrating the eluent aspiration step (STEP 1). In STEP 1, the control unit 32 (not shown) performs a switching operation of the switching valve 14 to connect the eluent flow path 21 and the first flow path 23, and then performs a suction operation of the pump 11 to suck up the eluent 41a from the eluent tank 41 and introduce the sucked up eluent 41a into the first flow path 23 via the eluent flow path 21 and the switching valve 14. The first flow path 23 between the switching valve 14 and the pump 11 functions as a storage flow path for the eluent 41a. In FIG. 2, the state in which the eluent flow path 21 and the first flow path 23 are filled with the eluent 41a is virtually shown by a thick line.

[0052] [STEP 2: Replacement with eluent] FIG. 3 is a diagram illustrating the replacement step (STEP 2) with an eluent. In STEP 2, the control unit 32 (not shown) performs a switching operation of the switching valve 14 to connect the first flow path 23 and the second flow path 24, and then performs a discharge operation of the pump 11 to introduce a part of the eluent 41a introduced into the first flow path 23 into the HPLC column 13 via the first flow path 23, the switching valve 14, and the second flow path 24. At least a part of the eluent 41a introduced into the HPLC column 13 is stored in the drain tank 43 via the third flow path 26, the detector 31, the fourth flow path 27, the exhaust pressure unit 34, and the drainage flow path 25. As a result, at least a portion of the first flow path 23, the second flow path 24, the HPLC column 13, the third flow path 26, the detector 31, the fourth flow path 27, the exhaust pressure section 34, and the drainage flow path 25 are filled with the eluent 41a. In FIG. 3, the first flow path 23, the second flow path 24, the third flow path 26, the fourth flow path 27 and the drainage flow path 25 are virtually shown by thick lines to be filled with the eluent 41a.

[0053] (2) Sampling process The sampling step following the preparatory step comprises a first air suction step (STEP 3), a liquid sample suction step (STEP 4), and a second air suction step (STEP 5) following the first air suction step. [STEP 3: First air suction step] FIG. 4 is a diagram illustrating the first air suction step (STEP 3). In STEP 3, the control unit 32 (not shown) performs a switching operation of the switching valve 14 to connect the first flow path 23 and the air introduction path 28, and then performs a suction operation of the pump 11 to introduce air A from the air intake port of the air introduction path 28 into the first flow path 23 via the switching valve 14. At this time, the air A is introduced into the first flow path 23 as air bubbles.

[0054] That is, the first flow path 23 is at least partially filled with the eluent 41a, and air (air bubbles) A ​​is introduced on the switching valve 14 side. The insides of the second flow path 24, the HPLC column 13, the third flow path 26, the detector 31, the fourth flow path 27, the exhaust pressure section 34, and the drainage flow path 25 are replaced with the eluent 41a. In addition, in FIG. 4, a state in which an air bubble A is introduced into the first flow path 23 on the switching valve 14 side is virtually shown.

[0055] Here, it is preferable to provide a period during which the operation of the pump 11 is stopped for a certain period between the replacement step with the eluent 41a (STEP 2) and the first air suction step (STEP 3). Generally, since the conductance of the HPLC column 13 is small, when the eluent 41a held in the first flow path 23 is introduced into the second flow path 24 and subsequent flow paths by the discharge operation of the pump 11 in STEP 2, the pressure in the first flow path 23 and the second flow path 24 increases. In this state, when the switching operation of the switching valve 14 is performed in STEP 3 to connect the first flow path 23 and the air introduction path 28, at least a part of the eluent 41a in the first flow path 23 may flow back into the air introduction path 28. In other words, the volume of air introduced into the first flow path 23 may not be the desired volume.

[0056] Therefore, after STEP 2 is completed, it is preferable to stop the operation of the pump 11 and perform STEP 3 after a certain waiting time has elapsed. As a result, the pressure in the first flow path 23, the switching valve 14, and the flow path from the second flow path 24 to the drainage flow path 25 gradually decreases as the drainage 43a flows into the drainage tank 43. By performing STEP 3 after the pressure in the flow path has sufficiently decreased (for example, to a pressure equivalent to atmospheric pressure), it becomes possible to introduce air A into the first flow path 23 without the eluent 41a in the first flow path 23 and the second flow path 24 flowing back into the air introduction path 28.

[0057] [STEP 4: Liquid sample aspiration step] FIG. 5 is a diagram illustrating the liquid sample suction step (STEP 4). In this STEP 4, the control unit 32, not shown in the figure, performs a switching operation of the switching valve 14 to connect the first flow path 23 and the sample flow path 22, and then performs a suction operation of the pump 11 to suck up the sample 42a from the sample tank 42, and introduces the sucked up sample 42a into the sample flow path 22 and the first flow path 23 via the switching valve 14.

[0058] In STEP 4, in the first flow path 23, the sample 42a is filled between the air bubbles A introduced into the first flow path 23 in STEP 3 and the switching valve 14. That is, in the first flow path 23, the eluent 41a filled on the pump 11 side and the sample 42a filled on the switching valve 14 side are separated by the air bubbles A, and the eluent 41a and the sample 42a are not mixed. In addition, the insides of the second flow path 24, the HPLC column 13, the third flow path 26, the detector 31, the fourth flow path 27, the exhaust pressure section 34, and the drainage flow path 25 are replaced with the eluent 41a. 5, a state in which the sample 42a is introduced into the first flow path 23 on the switching valve 14 side, and the eluent 41a and the sample 42a are separated by the air bubble A is virtually shown.

[0059] [STEP 5: Second air suction step] FIG. 6 is a diagram illustrating the second air suction step (STEP 5). In STEP 5, the control unit 32 (not shown) performs a switching operation of the switching valve 14 to connect the first flow path 23 and the air introduction path 28, and then performs a suction operation of the pump 11 to introduce air A from the air intake port of the air introduction path 28 into the first flow path 23 via the switching valve 14. At this time, the air A is introduced into the first flow path 23 as air bubbles.

[0060] In STEP 5, air bubbles A are introduced into the first flow path 23 between the sample 42a introduced into the first flow path 23 in STEP 4 and the switching valve 14. As a result, in the first flow path 23, the sample 42a is sandwiched between the air bubbles A, that is, becomes an air-segmented sample. In addition, the insides of the second flow path 24, the HPLC column 13, the third flow path 26, the detector 31, the fourth flow path 27, the exhaust pressure section 34, and the drainage flow path 25 are replaced with the eluent 41a. In addition, in FIG. 6, a state in which an air bubble A is introduced into the first flow path 23 on the switching valve 14 side, and an air-segmented sample is formed in the first flow path 23, is virtually shown.

[0061] (3) Analysis process The analysis step following the sampling step comprises an air segment sample ejection step (STEP 6), followed by a compression step (STEP 7), followed by a detection step (STEP 8). [STEP 6: Air segment sample discharge step] 7 and 8 are diagrams illustrating the discharge step (STEP 6) of discharging the air-fractionated sample from the first flow path 23 toward the HPLC column 13. FIG. In this STEP 6, the control unit 32 (not shown) performs a switching operation of the switching valve 14 to connect the first flow path 23 and the second flow path 24, and then performs a discharge operation of the pump 11 to discharge the bubbles A, sample 42a, bubbles A, and eluent 41a in the first flow path 23 to the second flow path 24 in that order. Note that Figure 7 shows the state immediately after the start of the discharge operation of pump 11, and the air segmentation samples (bubble A, sample 42a, bubble A) in first flow path 23 do not change significantly from the air segmentation samples shown in Figure 6 formed by STEP 6.

[0062] When the discharge operation of the pump 11 is continued from the state shown in FIG. 7, the air-fragmented sample present in the first flow path 23 is discharged and introduced into the second flow path 24, as shown in FIG. Fig. 9 is a diagram showing the state of the sample 42a introduced into the second flow path 24, and is an internal image diagram of the second flow path 24 indicated by reference numeral 51 in Fig. 8. As shown in Fig. 9, in the second flow path 24, the sample 42a is sandwiched at both ends by air bubbles A, and is separated from the eluent 41a by the air bubbles A. In other words, the eluent 41a and the sample 42a are not mixed.

[0063] [STEP 7: Compression step] 10 to 13 are diagrams for explaining the compression step (STEP 7). In STEP 7, the state of the switching valve 14 is the same as in STEP 6, in which the first flow path 23 and the second flow path 24 are connected to each other. As described above, since the conductance of the HPLC column 13 is generally small, the discharge operation of the pump 11 results in an increase in the pressure inside the first flow path 23 and the second flow path 24 . When the pump 11 performs a discharge operation to introduce the sample 42a into the HPLC column 13, the pressure in the first flow path 23 and the second flow path 24 increases, and the air-segmented sample introduced into the second flow path 24 is compressed. As a result, at least a part of the air bubbles A constituting the air-segmented sample dissolves in at least one of the eluent 41a and the sample 42a, and the volume of the air bubbles A decreases. Fig. 11 is an internal image diagram of the second flow path 24 indicated by the reference numeral 52 in Fig. 10. As shown in Fig. 11, the air-segmented sample is compressed, so that the volume of the air bubbles A is reduced below the volume of the air bubbles A introduced in the first flow path 23.

[0064] The pressure in the first flow path 23 and the second flow path 24 gradually increases from the time when the pump 11 starts discharging while the pump 11 is performing the discharging operation. This increase in pressure depends on the diameter and length of the flow path, the conductance of the flow path downstream of the HPLC column 13 including the HPLC column 13, the discharge amount of the pump 11 per unit time, etc. Therefore, in this embodiment, the volume of bubbles A introduced in STEP 3 and STEP 5 is set so that the pressure increase in the second flow path 24 causes bubbles A to completely dissolve in the eluent 41a and sample 42a (so that bubbles A disappear) just before the liquid inlet of the HPLC column 13 in the second flow path 24.

[0065] By introducing a preset volume of bubbles A as described above, when the pressure in the second flow path 24 increases due to the discharge operation of the pump 11, the bubbles A can be completely dissolved in the eluent 41a and sample 42a just before the HPLC column 13 in the second flow path 24, as shown in Figure 12. Fig. 13 is an internal image diagram of the second flow channel 24 indicated by reference numeral 53 in Fig. 12. As shown in Fig. 13, the air bubble A can be completely eliminated just before the HPLC column 13.

[0066] Here, the amount of the bubbles A introduced can be, for example, 1 μl (microliter) or more and 45 μl or less. For example, if the length of the bubbles A in the flow path is 1 mm, the eluent 41a and the sample 42a can be appropriately separated. Therefore, the minimum volume of the bubbles A is sufficient even if it is 1 microliter, although it depends on the diameter of the flow path. Also, the larger the amount of the bubbles A, the larger the pressure value for completely dissolving the bubbles A in the eluent 41a and the sample 42a, so although it depends on the discharge capacity of the pump 11, it is preferable that the maximum volume of the bubbles A is about 45 microliters. The amount of bubbles A to be introduced can be determined by prior experiments or simulations. For example, when determining the amount of bubbles A to be introduced by experiments, a transparent conduit (tube) may be used and an appropriate amount of bubbles A to be introduced may be determined by visually confirming that bubbles A disappear just before the HPLC column 13. Note that after the amount of bubbles A to be introduced is determined, the conduit (tube) does not need to be transparent.

[0067] In this embodiment, the amount of bubbles A introduced is set so that the bubbles A are completely dissolved in the eluent 41a and sample 42a just before the HPLC column 13 in the second flow path 24. However, the discharge amount per unit time of the pump 11 (hereinafter simply referred to as the "discharge amount") may be adjusted so that the bubbles A are completely dissolved in the eluent 41a and sample 42a just before the HPLC column 13 in the second flow path 24. The pressure in the first flow path 23 when a predetermined amount of bubbles A introduced into the first flow path 23 is completely dissolved in the eluent 41a or the sample 42a (hereinafter referred to as the "target pressure value") can be calculated in advance. Therefore, for example, the discharge rate of the pump 11 may be adjusted so that the pressure value measured by the pressure gauge 35 provided in the first flow path 23 matches (or approximately matches) the target pressure value when the bubbles A pass through the second flow path 24. Specifically, the pressure gauge 35 transmits the measured pressure values ​​(pressure values ​​of the first flow path 23 and the second flow path 24) to the control unit 32, and the control unit 32 compares the pressure value received from the pressure gauge 35 with a target pressure value and controls the operation of the pump 11 so that the pressure values ​​of the first flow path 23 and the second flow path 24 match (or approximately match) the target pressure value.

[0068] By adjusting the amount of the bubbles A introduced and the discharge rate of the pump 11 in this manner, only the sample 42a and the eluent 41a are introduced into the HPLC column 13, and the bubbles A are not introduced.

[0069] Through the above-mentioned steps, the sample 41a is transported as an air-separated sample through the first flow path 23 and the second flow path 24 until it reaches just before the liquid inlet of the HPLC column 13. In other words, the sample 42a is transported through the first flow path 23 and the second flow path 24 in a state separated from the eluent 41a by the air bubbles A arranged before and after it, and undesired diffusion within the first flow path 23 and the second flow path 24 is prevented.

[0070] On the other hand, by the above process, the air bubbles A disposed before and after the sample 42a constituting the air-segmented sample are dissolved in the eluent 41a and the sample 42a when the sample 42a reaches just before the liquid inlet of the HPLC column 13. Therefore, the air bubbles A are not introduced into the HPLC column 13 or the detector 31. Therefore, problems such as insufficient separation of the components to be detected that constitute the sample 42a in the HPLC column 13 and generation of noise in the detector 31 due to air (air bubbles) do not occur.

[0071] [STEP 8: Detection step] In STEP 8, the state of the switching valve 14 is the state in which the first flow path 23 and the second flow path 24 are connected, similarly to STEP 7. In STEP 8, the control unit 32 causes the pump 11 to continue the discharge operation, and introduces the eluent 41a and the sample 42a in the second flow path 24 into the HPLC column 13. When sample 42a flows through HPLC column 13 together with eluent 41a, each component constituting sample 42a moves while interacting with the stationary phase of HPLC column 13. The time at which each component is eluted from HPLC column 13 is determined by the difference in the strength of the interaction between each component and HPLC column 13. That is, each component contained in sample 42a is separated by utilizing the difference in the elution time of each component passing through HPLC column 13. The separated components are then detected by a detector 31 disposed downstream of the HPLC column 13. At this time, the detector 31 obtains a signal corresponding to each component in accordance with the elution time of each component eluted from the HPLC column 13. The fluid discharged from the detector 31 is discharged through the drainage flow path 25 into the drainage tank 43, and is held as drainage 43a.

[0072] In addition, a pressure exhaust section 34 having a small conductance may be provided downstream of the detector 31. The pressure exhaust section 34 may be configured, for example, by including a flow path having a cross-sectional area smaller than that of the third flow path 26 in at least a part of the fourth flow path 27. This pressure exhaust section 34 can maintain the pressure in the flow path downstream of the HPLC column 13, and can prevent the pressure of the fluid discharged from the HPLC column 13 from being released and causing bubbles A to be generated again. This can appropriately prevent air from being introduced into the detector 31.

[0073] Of the above steps, STEP 1 and STEP 2 correspond to the first step, STEP 3 to STEP 5 correspond to the second step, STEP 6 corresponds to the third step, STEP 7 corresponds to the fourth step, and STEP 8 corresponds to the fifth step.

[0074] As described above, the HPLC device 10 in this embodiment includes one pump 11, a switching valve 14, an HPLC column 13, a first flow path 23 that connects between the pump 11 and the switching valve 14 and through which the eluent 41a and the sample (liquid specimen) 42a flow simultaneously, a second flow path 24 that connects between the switching valve 14 and the HPLC column 13 and through which the eluent 41a and the sample 42a flow simultaneously, and an air introduction path 28 one end of which is connected to the switching valve 14. The HPLC device 10 also includes a detector 31 that detects components separated in the HPLC column 13, and a control unit 32 that controls the pump 11 and the switching valve 14.

[0075] The control unit 32 controls the pump 11 and the switching valve 14 to switch between the introduction of the eluent 41a and the sample 42a into the first flow path 23, and can introduce air bubbles from the air introduction path 28 between the eluent 41a and the sample 42a to generate an air-segmented sample (air-segmented liquid specimen) in which air bubbles A, the sample 42a, and air bubbles A are arranged in this order in the first fluid 23. The control unit 32 also controls the pump 11 and the switching valve 14 to flow the air-segmented sample from the first flow path 23 into the second flow path 24, and can dissolve the air bubbles A passing through the second flow path 23 in at least one of the eluent 41a and the sample 42a.

[0076] In this way, the introduction of the eluent 41a and the sample 42a into the first flow path 23 and the delivery of the mixture of the eluent 41a and the sample 42a to the HPLC column 13 can be realized by one pump 11. Therefore, it is not necessary to provide a plurality of pumps, and it is possible to realize a reduction in size and cost accordingly. Therefore, it is easy to spread to farmers who produce plants (crops such as vegetables). In addition, since the pump 11 is disposed outside the path that flows from the first flow path 23 through the switching valve 14 and the second flow path 24 into the HPLC column 13, it is possible to configure the fluid not to pass through the pump 11. Therefore, it is possible to prevent dispersion (diffusion) of the fluid caused by passing through the pump 11, and it is possible to prevent a decrease in the separation ability of the HPLC column 13.

[0077] Furthermore, in this embodiment, an air-segmented sample is generated in the first flow path 23 and the air-segmented sample is caused to flow toward the HPLC column 13 via the second flow path 24, so that the sample 42a can be delivered to the HPLC column 13 in a state in which the eluent 41a and the sample 42a are separated by the air bubbles A. Therefore, undesired diffusion of the sample 42a before the sample 42a is introduced into the HPLC column 13 can be appropriately suppressed, and a decrease in the separation ability in the HPLC column 13 can be prevented.

[0078] Furthermore, in this embodiment, the air bubbles A constituting the air segment sample can be dissolved in at least one of the eluent 41a and the sample 42a in the second flow path 24. That is, the air bubbles A can be eliminated before being introduced into the HPLC column 13 and the detector 31. Therefore, problems such as a decrease in the resolution in the HPLC column 13 and noise generation in the detector 31 due to the air bubbles A can be appropriately avoided. Here, if the air bubbles A are dissolved in the eluent 41a or the sample 42a immediately before the liquid inlet of the HPLC column 13, undesired diffusion of the sample 42a within the flow path can be more appropriately suppressed.

[0079] The control unit 32 controls at least one of the volume of air bubbles A introduced from the air inlet path 28 between the eluent 41a and the sample 42a, and the discharge volume of the pump 11 when the air-segmented sample flows from the first flow path 23 to the HPLC column 13 via the second flow path 24, thereby dissolving the air bubbles A passing through the second flow path 24 in the eluent 41a and the sample 42a.

[0080] Specifically, the control unit 32 introduces a volume of air bubbles A between the eluent 41a and the sample 42a such that the air bubbles A passing through the second flow path 23 are dissolved in the eluent 41a and the sample 42a due to the pressure inside the second flow path 24 generated when the air-segmented sample is flowing from the first flow path 23 to the second flow path 24 by the discharge operation of the pump 11. Here, the pressure inside the second flow path 24 generated when the air-fractionated sample is flowing from the first flow path 23 to the second flow path 24 is determined based on the discharge amount of the pump 11 when the air-fractionated sample is flowing from the first flow path 23 to the second flow path 24, the conductance of the HPLC column 13, the diameters of the first flow path 23 and the second flow path 24, and the lengths of the first flow path 23 and the second flow path 24. Therefore, by introducing a volume of air bubbles A set based on these conditions, it is possible to reliably dissolve the air bubbles A in the second flow path 24.

[0081] In addition, the control unit 32 may control the discharge volume of the pump 11 so that, when the air-segmented sample is flowing from the first flow path 23 to the HPLC column 13 via the second flow path 24, a pressure (target pressure value) is applied inside the second flow path 24 such that air bubbles A passing through the second flow path 24 are dissolved in the eluent 41a or the sample 42a. Here, the pressure at which the air bubbles A introduced from the air inlet path 28 are dissolved in the eluent 41a or the sample 42a is determined based on the volume of the air bubbles A introduced from the air inlet path 28, the conductance of the HPLC column 13, the diameters of the first flow path 23 and the second flow path 24, and the lengths of the first flow path 23 and the second flow path 24. Therefore, the air bubbles A can be reliably dissolved in the second flow path 24 by controlling the discharge rate of the pump 11 so as to apply a target pressure value set based on these conditions.

[0082] In this embodiment, the case where the air bubbles A are completely dissolved just before the liquid inlet of the HPLC column 13 has been described, but the air bubbles A do not have to be completely dissolved. It is sufficient that the air bubbles A are small enough to allow for the separation resolution of each component in the HPLC column 13 and the noise in the detector 31.

[0083] As described above, the HPLC device 100 of the present embodiment is a liquid sample analyzer that uses liquid chromatography, and can improve the separation resolution and measurement accuracy of each component while achieving miniaturization and cost reduction of the device.

[0084] Although specific embodiments have been described above, the embodiments are merely illustrative and are not intended to limit the scope of the present invention. The apparatus and method described in this specification can be embodied in forms other than those described above. Furthermore, the above-described embodiments can be appropriately omitted, substituted, and modified without departing from the scope of the present invention. Such omitted, substituted, and modified forms are included in the scope of the claims and their equivalents, and belong to the technical scope of the present invention. [Explanation of symbols]

[0085] 10...Liquid sample analyzer (HPLC device), 11...Pump, 12...Switching valve, 13...HPLC column, 14...Switching valve, 21...Eluant flow path, 22...Sample flow path (liquid sample flow path), 23...First flow path, 24...Second flow path, 25...Drainage flow path, 26...Third flow path, 27...Fourth flow path, 28...Air introduction path, 31...Detector, 32...Control unit, 33...Data processing unit, 41...Eluant tank, 41a...Eluant, 42...Sample tank (sample tank), 42a...Sample (liquid sample), 43...Drainage tank, 43a...Drainage, A...Air (air bubbles)

Claims

1. A pump for sucking and discharging a fluid; A switching valve having ports on multiple sides; a column into which a liquid containing an eluent and a liquid sample is supplied and which separates components of the liquid sample by interaction with a stationary phase held therein; a first flow path that connects the pump and the switching valve and through which the eluent and the liquid sample simultaneously flow; a second flow path that connects the switching valve and the column and through which the eluent and the liquid sample simultaneously flow; an air introduction passage having one end connected to the switching valve; a detector for detecting components separated in the column; a control unit for controlling the pump and the switching valve, The control unit is The pump and the switching valve are controlled to switch between introduction of the eluent and the liquid sample into the first flow path, and air bubbles are introduced from the air introduction path between the eluent and the liquid sample to generate an air-segmented liquid sample in which the air bubbles, the liquid sample, and the air bubbles are arranged in this order in the first flow path having the eluent therein; A liquid chromatograph characterized in that the pump and the switching valve are controlled to flow the eluent and the air-segmented liquid sample from the first flow path to the second flow path, and the volume of the air bubbles introduced from the air introduction path between the eluent and the liquid sample is controlled, thereby dissolving the air bubbles passing through the second flow path in at least one of the eluent and the liquid sample.

2. 2. The liquid chromatograph of claim 1, wherein the volume of the air bubbles introduced between the eluent and the liquid sample is set based on the discharge volume per unit time of the pump when the air-segmented liquid sample flows from the first flow path to the second flow path, the conductance of the column, the diameters of the first flow path and the second flow path, and the lengths of the first flow path and the second flow path.

3. 2. The liquid chromatograph according to claim 1, wherein a volume of the air bubbles introduced between the eluent and the liquid sample is set to be equal to or greater than 1 microliter and equal to or less than 45 microliters.

4. A pump for sucking and discharging a fluid; A switching valve having ports on multiple sides; a column into which a liquid containing an eluent and a liquid sample is supplied and which separates components of the liquid sample by interaction with a stationary phase held therein; a first flow path that connects the pump and the switching valve and through which the eluent and the liquid sample simultaneously flow; a second flow path that connects the switching valve and the column and through which the eluent and the liquid sample simultaneously flow; an air introduction passage having one end connected to the switching valve; a detector for detecting components separated in the column; A control unit that controls the pump and the switching valve; a back pressure section disposed downstream of the detector, The control unit is The pump and the switching valve are controlled to switch between introduction of the eluent and the liquid sample into the first flow path, and air bubbles are introduced from the air introduction path between the eluent and the liquid sample to generate an air-segmented liquid sample in which the air bubbles, the liquid sample, and the air bubbles are arranged in this order in the first flow path having the eluent therein; Controlling the pump and the switching valve to flow the eluent and the air-segmented liquid sample from the first flow path to the second flow path, and dissolving the air bubbles passing through the second flow path in at least one of the eluent and the liquid sample; a third flow path that is provided on a flow path downstream of the detector and that has a cross-sectional area smaller than a cross-sectional area of ​​the third flow path that connects the column and the detector.

5. A pump for sucking and discharging a fluid; A switching valve having ports on multiple sides; a column into which a liquid containing an eluent and a liquid sample is supplied and which separates components of the liquid sample by interaction with a stationary phase held therein; a first flow path that connects the pump and the switching valve and through which the eluent and the liquid sample simultaneously flow; a second flow path that connects the switching valve and the column and through which the eluent and the liquid sample simultaneously flow; an air introduction passage having one end connected to the switching valve; a detector for detecting components separated in the column; A control unit that controls the pump and the switching valve; an eluent flow path for collecting the eluent stored in the eluent tank; a liquid sample flow path for collecting the liquid sample stored in the liquid sample tank; The control unit is The pump and the switching valve are controlled to switch between introduction of the eluent and the liquid sample into the first flow path, and air bubbles are introduced from the air introduction path between the eluent and the liquid sample to generate an air-segmented liquid sample in which the air bubbles, the liquid sample, and the air bubbles are arranged in this order in the first flow path having the eluent therein; Controlling the pump and the switching valve to flow the eluent and the air-segmented liquid sample from the first flow path to the second flow path, and dissolving the air bubbles passing through the second flow path in at least one of the eluent and the liquid sample; a liquid chromatograph, wherein one end of the eluent flow path and one end of the liquid sample flow path are each connected to the switching valve;

6. The control unit is A liquid chromatograph as described in claim 4 or 5, characterized in that the discharge amount per unit time of the pump when the air-segmented liquid sample is flowing from the first flow path through the second flow path into the column is controlled, thereby dissolving the air bubbles passing through the second flow path into at least one of the eluent and the liquid sample.

7. 7. The liquid chromatograph of claim 6, wherein the discharge volume per unit time of the pump when the air-segmented liquid sample flows from the first flow path through the second flow path into the column is set based on the volume of the air bubbles introduced from the air inlet path, the conductance of the column, the diameters of the first flow path and the second flow path, and the lengths of the first flow path and the second flow path.

8. The control unit is 8. The liquid chromatograph according to claim 1, wherein the air bubbles are dissolved in at least one of the eluent and the liquid sample immediately before the liquid inlet of the column in the second flow path.

9. A pump for sucking and discharging a fluid; A switching valve having ports on multiple sides; a column into which a liquid containing an eluent and a liquid sample is supplied and which separates components of the liquid sample by interaction with a stationary phase held therein; a first flow path that connects the pump and the switching valve and through which the eluent and the liquid sample simultaneously flow; a second flow path that connects the switching valve and the column and through which the eluent and the liquid sample simultaneously flow; an air introduction passage having one end connected to the switching valve; A liquid sample analysis method in a liquid chromatograph comprising: a first step of controlling the pump and the switching valve to introduce a part of the eluent from the first flow path through the second flow path into the column, thereby filling the inside of the column with the eluent; a second step of controlling the pump and the switching valve to introduce the liquid sample and the air bubbles from the air introduction path into the first flow path having the eluent therein, thereby generating an air-segmented liquid sample in which the air bubbles, the liquid sample, and the air bubbles are arranged in this order in the first flow path having the eluent therein; a third step of controlling the pump and the switching valve to flow the eluent and the air-segmented liquid sample from the first flow path to the second flow path; a fourth step of dissolving the air bubbles passing through the second flow path in at least one of the eluent and the liquid sample; a fifth step of separating the eluent and the liquid sample in the column and detecting the liquid sample in the detector; In the second step, A liquid sample analysis method, comprising: introducing a volume of bubbles that is set based on the discharge amount per unit time of the pump in the third step, the conductance of the column, the diameters of the first flow path and the second flow path, and the lengths of the first flow path and the second flow path.

10. A pump for sucking and discharging a fluid; A switching valve having ports on multiple sides; a column into which a liquid containing an eluent and a liquid sample is supplied and which separates components of the liquid sample by interaction with a stationary phase held therein; a first flow path that connects the pump and the switching valve and through which the eluent and the liquid sample simultaneously flow; a second flow path that connects the switching valve and the column and through which the eluent and the liquid sample simultaneously flow; an air introduction passage having one end connected to the switching valve; A liquid sample analysis method in a liquid chromatograph comprising: a first step of controlling the pump and the switching valve to introduce a part of the eluent from the first flow path through the second flow path into the column, thereby filling the inside of the column with the eluent; a second step of controlling the pump and the switching valve to introduce the liquid sample and the air bubbles from the air introduction path into the first flow path having the eluent therein, thereby generating an air-segmented liquid sample in which the air bubbles, the liquid sample, and the air bubbles are arranged in this order in the first flow path having the eluent therein; a third step of controlling the pump and the switching valve to flow the eluent and the air-segmented liquid sample from the first flow path to the second flow path; a fourth step of dissolving the air bubbles passing through the second flow path in at least one of the eluent and the liquid sample; a fifth step of separating the eluent and the liquid sample in the column and detecting the liquid sample in the detector; In the first step, controlling the pump and the switching valve to introduce the eluent from an eluent flow path, one end of which is connected to the switching valve and which collects the eluent stored in an eluent tank, into the first flow path via the switching valve; Next, the pump and the switching valve are controlled to introduce a part of the eluent from the first flow path through the second flow path into the column, thereby filling the inside of the column with the eluent; In the second step, A liquid sample analyzing method, comprising: introducing the air bubbles in a volume of 1 microliter or more and 45 microliters or less between the eluent and the liquid sample.

11. In the second step, controlling the pump and the switching valve to introduce air bubbles from the air introduction path through the switching valve into the first flow path having the eluent therein; Next, the pump and the switching valve are controlled to introduce the liquid sample from a liquid sample flow path, one end of which is connected to the switching valve and which collects the liquid sample stored in a liquid sample tank, into the first flow path via the switching valve; 11. The liquid sample analyzing method according to claim 9, further comprising controlling the pump and the switching valve to introduce air bubbles from the air introduction path through the switching valve into the first flow path.

12. 12. The method for analyzing a liquid sample according to claim 9, wherein, after the first step, operation of the pump is stopped, and the second step is performed after a certain waiting time has elapsed.

13. In the fourth step, 12. A liquid sample analysis method according to claim 9, further comprising controlling the discharge amount per unit time of the pump based on the volume of the air bubbles introduced from the air inlet path in the second step, the conductance of the column, the diameters of the first flow path and the second flow path, and the lengths of the first flow path and the second flow path.

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