Liquid chromatograph and analytical method using liquid chromatograph

Abstract

The control unit of the liquid chromatograph is configured to: after the high-pressure valve is brought to a load state, a sample is sucked into the front end of a needle and held in a sampling flow path, the sampling flow path is connected to an injection port, and the high-pressure valve is brought to an injection state, a mobile phase is supplied from a liquid supply unit, and thus the sample held in the sampling flow path is injected into an analysis flow path; and after the sample injection ends, the high-pressure valve is brought to the load state, the mobile phase is supplied from the liquid supply unit, and thus the sample injected into the analysis flow path is separated by a separation column, and in a case where at least a prescribed condition is satisfied, after the analysis ends, the sampling flow path is connected to the injection port, the high-pressure valve is brought to the injection state, the mobile phase and / or a cleaning liquid is supplied from the liquid supply unit, and thus the inside of a liquid flow path from the sampling flow path to the analysis flow path is cleaned.

Description

TECHNICAL FIELD

[0001] The present application relates to a liquid chromatograph and an analysis method using the liquid chromatograph. BACKGROUND

[0002] In a liquid chromatograph, a sample flow path in which a needle for sampling is provided at a front end, a mobile phase supply unit that supplies a mobile phase, an analysis flow path in which a separation column is provided, and a high-pressure valve or the like for switching whether or not the sample flow path is interposed between the mobile phase supply unit and the analysis flow path are provided (see Patent Literature 1). A sample as an analysis target is sucked from the front end of the needle and held in the sample flow path, and is introduced into the analysis flow path together with the mobile phase supplied from the mobile phase supply unit by interposing the sample flow path between the mobile phase supply unit and the analysis flow path.

[0003] [Related Art Documents]

[0004] [Patent Literature]

[0005] [Patent Literature 1] International Publication No. 2019 / 211930 SUMMARY

[0006] [Problems to be Solved by the Invention]

[0007] In a case where a plurality of samples are analyzed using the same analysis flow path in a liquid chromatograph, in order to improve the reliability of the analysis result, it is important to reduce so-called carry over in which the previous sample remains in the system at the time of starting the analysis of the next sample. Although the carry over also depends on the kind of the sample and the like, on average, it is known that about 20% comes from the sample flow path (particularly, the needle), and the remaining about 80% comes from the analysis flow path. Therefore, the following countermeasure is taken: after the sample is introduced into the analysis flow path, the sample flow path and the analysis flow path are disconnected, the inside of the sample flow path is cleaned with a cleaning liquid or the like, and a liquid such as a mobile phase is sufficiently flowed in the analysis flow path after the analysis of the sample is finished.

[0008] However, it is known that in a case where the concentration of the sample introduced into the analysis flow path is a certain level or more, even if the sample flow path and the analysis flow path are separated as described and the respective cleanings are sufficiently performed, the carry over that affects the analysis result of the next sample can occur.

[0009] The present application was made in view of the above-described problem, and an object thereof is to provide a liquid chromatograph in which the carry over that affects the analysis of the next sample does not easily occur.

[0010] [Technical Means to Solve the Problem]

[0011] The present inventors have obtained the following insight from the results of experiments: in a case where a sample remains in a high-pressure valve that switches whether or not a sampling flow path and an analysis flow path are connected, particularly in a case where the concentration of the sample is above a certain level, the sample remaining in the high-pressure valve becomes a carryover and affects the analysis result of the next sample. The present invention is based on this insight and is directed to removing a sample remaining in a high-pressure valve after the end of analysis of a sample.

[0012] The liquid chromatograph of the present invention includes: a sampling flow path in which a needle for sampling is provided at a front end; a needle moving mechanism that moves the needle three-dimensionally; a liquid supply portion configured to be able to supply at least a mobile phase; an analysis flow path in which a separation column for separating components in a sample is provided; a high-pressure valve having an injection port that is fluidly connected to the sampling flow path by insertion of the needle, and a port to which the analysis flow path is connected, and configured to selectively switch to a load state for fluidly connecting the liquid supply portion and the analysis flow path without passing through the sampling flow path, and an injection state for fluidly connecting the liquid supply portion and the analysis flow path via the sampling flow path when the front end of the needle is inserted into the injection port; and a control portion that controls the needle moving mechanism, the liquid supply portion, and the high-pressure valve. The liquid supply portion, the analysis flow path, and the high-pressure valve constitute one analysis portion for performing analysis of a sample. The control portion is configured to perform, as an analysis operation of the analysis portion: a sample injection step of bringing the high-pressure valve to the load state, sucking a sample from the front end of the needle and retaining the sample in the sampling flow path, and then connecting the sampling flow path and the injection port and bringing the high-pressure valve to the injection state, and supplying a mobile phase from the liquid supply portion, thereby injecting the sample retained in the sampling flow path via the injection port into the analysis flow path; and an analysis step of bringing the high-pressure valve to the load state after the end of the sample injection step, and supplying the mobile phase from the liquid supply portion, thereby separating the sample injected into the analysis flow path by the separation column; and in a case where at least a prescribed condition is satisfied, a system cleaning step of connecting the sampling flow path and the injection port and bringing the high-pressure valve to the injection state after the end of the analysis step, and supplying the mobile phase and / or a cleaning liquid from the liquid supply portion, thereby cleaning the inside of a liquid flow path from the sampling flow path to the analysis flow path.

[0013] The analysis method of the present application is an analysis method using a liquid chromatograph. The liquid chromatograph includes a liquid supply portion configured to be able to supply at least a mobile phase; an analysis flow path provided with a separation column for separating components in a sample; and a high-pressure valve having an injection port fluidly connected with the sampling flow path by insertion of a needle and a port connected with the analysis flow path, and configured to selectively switch to a load state for fluidly connecting the liquid supply portion and the analysis flow path without passing through the sampling flow path, and an injection state for fluidly connecting the liquid supply portion and the analysis flow path via the sampling flow path when the tip of the needle is inserted into the injection port. The analysis method includes a sample injection step of connecting the sampling flow path and the injection port and switching the high-pressure valve to the injection state after sucking a sample from the tip of the needle and retaining the sample in the sampling flow path with the high-pressure valve in the load state, supplying a mobile phase from the liquid supply portion, and thereby injecting the sample retained in the sampling flow path via the injection port into the analysis flow path; and an analysis step of switching the high-pressure valve to the load state after the sample injection step ends, supplying the mobile phase from the liquid supply portion, and thereby separating the sample injected into the analysis flow path with the separation column. The analysis method includes a system purge step of connecting the sampling flow path and the injection port and switching the high-pressure valve to the injection state after the analysis step ends, supplying the mobile phase and / or a purge liquid from the liquid supply portion, and thereby purging the inside of a liquid flow path from the sampling flow path to the analysis flow path, in at least a case where a prescribed condition is satisfied.

[0014] [Effects of the Invention]

[0015] The liquid chromatograph according to the present application is configured to execute a system purge step, i.e., a system purge step of connecting the sampling flow path and the injection port and switching the high-pressure valve to the injection state after the analysis step ends, supplying the mobile phase and / or a purge liquid from the liquid supply portion, and thereby purging the inside of a liquid flow path from the sampling flow path to the analysis flow path, in at least a case where a prescribed condition is satisfied, such as a case where the concentration of a sample is equal to or higher than a prescribed level. Therefore, when the high-pressure valve is switched to the injection state, the flow path in the high-pressure valve (i.e., a groove provided on a rotor) that connects the sampling flow path and the analysis flow path is purged with the mobile phase and / or the purge liquid supplied from the liquid supply portion, and a sample remaining in the high-pressure valve is removed before the analysis of the next sample starts. Thus, a liquid chromatograph that is less likely to cause a sample remaining that affects the analysis of the next sample is provided.

[0016] According to the analysis method of the present application, in the case where the concentration of the sample is equal to or higher than a prescribed level or the like, at least in the case where a prescribed condition is satisfied, after the analysis step is completed, a system cleaning step of connecting the sampling flow path to the injection port and bringing the high-pressure valve to the injection state, and supplying the mobile phase and / or the cleaning liquid from the liquid supply portion, thereby cleaning the inside of the liquid flow path from the sampling flow path to the analysis flow path, is executed, and therefore, when the high-pressure valve is brought to the injection state, the flow path inside the high-pressure valve, which connects the sampling flow path and the analysis flow path, is cleaned by the mobile phase and / or the cleaning liquid supplied from the liquid supply portion, and the sample remaining in the high-pressure valve is removed before the analysis of the next sample is started. Thus, a liquid chromatograph in which the remaining liquid is less likely to affect the analysis of the next sample is provided. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is a structural diagram showing an embodiment of a liquid chromatograph.

[0018] Figure 2 is a diagram showing an example of the flow path structure at the time of sample injection of the embodiment.

[0019] Figure 3 is a diagram showing an example of the flow path structure at the time of needle cleaning of the embodiment.

[0020] Figure 4 is a diagram showing an example of the flow path structure at the time of system cleaning of the embodiment.

[0021] Figure 5 is a flowchart for explaining an example of the operation of the embodiment.

[0022] Figure 6 is a time chart showing the liquid supply state of each liquid at the time of cleaning of the embodiment.

[0023] Figure 7 is a graph showing the verification results of the relationship between the number of blank injections and the remaining amount.

[0024] Figure 8 is a graph showing the verification results of the remaining elimination effect due to system cleaning. DETAILED DESCRIPTION

[0025] Hereinafter, embodiments of a liquid chromatograph and an analysis method of the present application will be described with reference to the drawings.

[0026] USAGES Figure 1An embodiment of a liquid chromatograph will be described. Furthermore, a multiplex liquid chromatograph (LC) including a plurality of analysis sections 3 that perform separation of samples and configured to be able to perform separation analysis of samples in parallel at the same time will be described as an example here, but the present application is not limited thereto, and is also applicable to a liquid chromatograph provided with only one liquid flow.

[0027] The liquid chromatograph 1 of the embodiment includes a sampling flow path 2, a plurality of analysis sections 3, a needle moving mechanism 13, a selection valve 26, a selection valve 28, a low-pressure valve 34, a metering pump 36, and a control section 50.

[0028] The proximal end of the sampling flow path 2 is connected to a common port of the selection valve 26, and includes a needle 12 for sampling at the distal end. The needle 12 performs three-dimensional movement by the needle moving mechanism 13, and the proximal end of the sampling flow path 2 is fluidly connected to the metering pump 36, so that the needle 12 can suck and hold a sample contained in a sample container 24 disposed at a prescribed position from the distal end. Furthermore, the needle 12 can be moved to a position of the injection port 16 of the high-pressure valve 10 provided in each analysis section 3. The injection port 16 of each analysis section 3 is fluidly connected to the sampling flow path 2 by the distal end of the needle 12 inserted into the injection port 16 of each analysis section 3.

[0029] In the drawing, reference symbols are attached only to one of the plurality of analysis sections 3, but each analysis section 3 has the same structure as each other.

[0030] The analysis section 3 includes an analysis flow path 4, a liquid supply section 6, and a high-pressure valve 10. The high-pressure valve 10 is a two position valve that has six ports on the same circumference and switches the connection state between mutually adjacent ports. One port of the high-pressure valve 10 is an injection port 16. One of the ports adjacent to the port leading to the injection port leads to a drain, and the other is connected to the upstream end of the analysis flow path 4. The port of the ports connected to the upstream end of the analysis flow path 4 on the opposite side from the port communicating with the injection port 16 is connected to the liquid supply section 6. The remaining two ports of the high-pressure valve 10 are respectively connected to a flow path 25 and a flow path 30.

[0031] The analysis flow path 4 includes a separation column 14 for separating components in a sample. Although not shown in the drawing, the analysis flow path 4 leads to a mass spectrometer or the like detector for detecting components separated in the separation column 14. The liquid supply section 6 includes a pump 18 for delivering a mobile phase, a pump 20 for supplying a washing liquid, and a switching valve 22, and is configured to be able to switch the liquid to be supplied between the mobile phase and the washing liquid by the switching valve 22. Furthermore, in the drawing, the liquid supply section 6 is depicted as supplying one kind of mobile phase and one kind of washing liquid, but can also be configured to be able to supply a plurality of kinds of mobile phases and washing liquids at the same time or switched.

[0032] The selector valve 26 has multiple selector ports, each of which is connected via a flow path 25 to a selector port of a high-pressure valve 10 of each of the multiple analysis units 3. The selector valve 26 can selectively connect the common port to one of the multiple selector ports. That is, the selector valve 26 is used to select the connection destination of the base end of the sampling flow path 2 from the high-pressure valves 10 provided in each of the multiple analysis units 3.

[0033] The selector valve 28 includes a common port and multiple selector ports. Each selector port of the selector valve 28 is connected via a flow path 30 to a selector port of a high-pressure valve 10 of each of the multiple analysis units 3. The common port of the selector valve 28 is connected via a flow path 32 to a common port of a low-pressure valve 34. A metering pump 36 and a liquid delivery flow path 38 are connected to the selector port of the low-pressure valve 34. The liquid delivery flow path 38 is connected via a switching valve 40 to a mobile phase supply flow path 42 and a cleaning fluid supply flow path 44, through which the mobile phase and / or cleaning fluid are delivered. The mobile phase and cleaning fluid supplied via the mobile phase supply flow path 42 and the cleaning fluid supply flow path 44 can be the same as the mobile phase and cleaning fluid supplied from the liquid supply unit 6.

[0034] The high-pressure valve 10 of the analysis unit 3 can be switched to a loading state for fluidly connecting the liquid supply unit 6 and the analysis flow path 4 without passing through the sampling flow path 2. Figure 1 The state of the sampling flow path 2, and the injection state in which the sampling flow path 2 is located between the liquid supply section 6 and the analytical flow path 4. Figure 2 (State of operation). In the loading state, the high-pressure valve 10 fluidly connects the port connected to the liquid supply unit 6 and the port connected to the analysis flow path 4. In the injection state, the high-pressure valve 10 fluidly connects the injection port 16 with the port connected to the analysis flow path 4, and also fluidly connects the port connected to the liquid supply unit 6 with the port connected to the flow path 25.

[0035] The control unit 50 is implemented through electronic circuitry including a central processing unit (CPU) and an information storage device. The control unit 50 controls the operation of each analysis unit 3 (including the high-pressure valve 10, pump 18, pump 20, and switching valve 22 of each analysis unit 3), the needle movement mechanism 13, the selection valve 26, the selection valve 28, the low-pressure valve 34, the metering pump 36, the switching valve 40, the pump 46, and the pump 48. The control unit 50 is configured to perform the sample injection step, the analysis step, and the system cleaning step as the analytical actions of each analysis unit 3.

[0036] In the sample injection step, after the sample contained in the sample container 24 is collected into the sampling flow path 2, as follows: Figure 2As shown, an analytical section 3 is selected using selection valves 26 and 28. A needle 12 is inserted into the injection port 16 of the selected analytical section 3, and the high-pressure valve 10 of the analytical section 3 is set to injection mode, supplying mobile phase from the liquid supply section 6. The mobile phase supplied from the liquid supply section 6 flows towards the analytical flow path 4 within the sampling flow path 2, thereby introducing the sample held in the sampling flow path 2 along with the mobile phase into the analytical flow path 4.

[0037] In the analysis steps, such as Figure 3 As shown, the high-pressure valve 10 is in a loaded state, and the sample guide separation column 14, which is introduced into the analytical flow path 4 by the mobile phase from the liquid supply unit 6, is introduced into the detector connected to the analytical flow path 4 to detect the components that have separated from each other and dissolved from the separation column 14.

[0038] During the execution of the analytical step, the sampling flow path 2 is disconnected from the mobile phase delivery section 6 and the analytical flow path 4. Therefore, as Figure 3 As shown, a needle cleaning step can be performed simultaneously, supplying cleaning fluid from the cleaning fluid supply path 44 to the sampling path 2 while the tip of the needle 12 is inserted into the injection port 16. In this needle cleaning step, the composition of the liquid flowing in the sampling path 2 can be changed over time using a mobile phase and cleaning fluid. For example, as... Figure 6 As shown, a stepwise method can be used to switch the liquid supplied to the sampling flow path 2 at fixed time intervals (e.g., 0.1 minutes to 0.5 minutes), or a gradient method can be used to change the concentration ratio of each liquid over time. By performing the needle cleaning step, the interior of the sampling flow path 2, including the needle 12, and the interior of the injection port 16 can be cleaned.

[0039] Alternatively, the sample injection step can be performed in another analysis unit 3 while the analysis step is being performed in one analysis unit 3.

[0040] During the system cleaning step, the system cleaning unit 3 to be cleaned is selected using selector valves 26 and 28, such as... Figure 4 As shown, needle 12 is inserted into injection port 16 of the selected analysis unit 3, and the high-pressure valve 10 of the analysis unit 3 is set to injection mode, supplying cleaning fluid from the liquid supply unit 6. In the system cleaning step, similar to the needle cleaning step, the composition of the liquid supplied from the liquid supply unit 6 can be changed using a mobile phase and cleaning fluid.

[0041] The system cleaning step can be executed at any time in the analysis section 3 after the analysis step ends. In the system cleaning step, not only the sampling flow path 2 and the analysis flow path 4 but also the flow path (groove of a rotor for fluidly connecting a port connected to the analysis flow path 4 and the injection port 16) in the high-pressure valve 10 can be cleaned. The sample remaining in the high-pressure valve 10 can be removed. However, the system cleaning step is not necessarily executed in the analysis section 3 at the end of the analysis step, but can be executed only when a prescribed condition is satisfied. As the condition for executing the system cleaning, there can be cited that the concentration of the sample is equal to or higher than a prescribed level, the system cleaning is set by the user to be executed after the analysis of the sample, a solvent (blank) not containing the sample is injected to the analysis flow path 4 after the analysis step ends, the output signal of the detector obtained thereby exceeds a prescribed level, and the like.

[0042] The flowchart of FIG. 10 illustrates a series of actions related to the analysis implemented by the control section 50. Figures 1 to 4 Figure 5 The flowchart of FIG. 10 illustrates a series of actions related to the analysis implemented by the control section 50.

[0043] At the time of the analysis of the sample, the control section 50 executes a sample injection step of selecting the analysis section 3 to be used for the analysis of the sample by the selection valve 26 and the selection valve 28, collecting the sample from the sample container 24 by the needle 12, and inserting the needle 12 into the injection port 16 of the selected analysis section 3. Then, as shown in FIG. 11, the high-pressure valve 10 of the selected analysis section 3 is switched to the injection state, and the mobile phase is supplied from the liquid supply section 6, so that the sample injection step of injecting the sample into the analysis flow path 4 of the analysis section 3 is executed (step 101). Figure 2

[0044] The control section 50 judges that the sample held in the sampling flow path 2 is entirely injected into the analysis flow path 4 when a prescribed time elapses after the high-pressure valve 10 is switched to the injection state (the sample injection step ends), and switches the high-pressure valve 10 to the load state (the state of FIG. 12) to start the analysis step (step 102). In the analysis step, the sample injected into the analysis flow path 4 is guided to the separation column 14 by the mobile phase supplied from the liquid supply section 6, and the components in the sample are separated from each other and detected by the detector. Figure 1

[0045] ​​​In the analysis step, the control section 50 performs a needle cleaning step (step 103) of supplying the cleaning solution (and the mobile phase) from the cleaning solution supply flow path 44 to the sample flow path 2 in a state where the tip of the needle 12 is inserted into the injection port 16 of the analysis section 3. Further, the control section 50 selects the analysis section 3 (another analysis section) to be used in analysis of the next sample by the selection valves 26 and 28, collects the next sample from the tip of the needle 12, and injects it into the analysis flow path 4 of the other analysis section 3 (step 104). The control section 50 starts the analysis step in the other analysis section 3 after the sample injection step in the other analysis section 3 is completed by switching the high-pressure valve 10 of the other analysis section 3 to the loading state (step 105). Then, the control section 50 supplies the cleaning solution (and the mobile phase) from the cleaning solution supply flow path 44 to the sample flow path 2, for example, in a state where the tip of the needle 12 is inserted into the injection port 16 of the other analysis section 3, thereby performing the needle cleaning step (step 106).

[0046] Then, after the sample analysis in the analysis section 3 in which the sample injection step is performed in step 101 is completed (step 107), the control section 50 determines whether or not to perform the system cleaning (step 108), and performs the system cleaning (step 109) in a case where the system cleaning is to be performed.

[0047] Whether or not the system cleaning is to be performed can be determined, for example, on the basis of information on the sample (for example, information on whether or not the system cleaning is to be performed after the analysis of the sample is completed in addition to the concentration of the sample) that is input in advance by the user or on the basis of a determination result of whether or not the output signal of the detector exceeds a prescribed level obtained by the analysis of the sample. That is, whether or not the system cleaning is to be performed after the analysis of the sample is completed can be determined depending on whether or not the concentration of the sample injected in the sample injection step of step 101 is equal to or higher than a prescribed level. Alternatively, the system cleaning can be determined to be performed after the analysis of the sample is completed depending on whether or not the level of the residue in the system exceeds a prescribed level. For example, after the analysis of the sample in the analysis section 3 is completed, a "blank injection" of introducing a blank solution (for example, a mobile phase, a solvent, or the like) that does not contain the sample into the analysis flow path 4 is performed as with a normal sample, the output signal of the detector is obtained with respect to the blank solution, and if the obtained output signal exceeds a prescribed level, it can be determined that the system cleaning is to be performed. Further, the control section 50 can be configured to perform the system cleaning once after the analysis step in the analysis section 3 is completed.

[0048] Here, as the blank injection, the following two actions can be cited. One action of the blank injection is an action of connecting the needle 12 to the injection port 16 in a state where the blank liquid is held in the sample flow path 2, and introducing the blank liquid held in the sample flow path 2 to the analysis flow path 4 by making the high-pressure valve 10 into the loaded state, with the solvent not containing the sample as the blank liquid. Another action of the blank injection is an action of connecting the needle 12 to the injection port 16, and introducing the mobile phase as the blank liquid to the analysis flow path 4 by making the high-pressure valve 10 into the loaded state, with the mobile phase not containing the sample as the blank liquid. The "blank injection" below can be either of the two actions.

[0049] Then, the control section 50 performs the blank injection, and determines the presence or absence of the carryover based on the level of the output signal of the detector for the blank liquid obtained at that time (step 110, step 111). According to the result of the blank liquid measurement, in the case where it is determined that there is no carryover, the series of analysis actions of the sample in the analysis section 3 is ended. The analysis section 3 in which the series of analysis actions is ended becomes a state in which it can be used for the analysis of other samples. On the other hand, in the case where it is determined that there is the carryover in step 111 by the control section 50, the system purge is performed again (step 108). In addition, as shown by the dotted arrow in the figure, the control section 50 can also be configured to repeatedly perform the blank injection until it is determined that there is no carryover in step 111. By performing the blank injection, the carryover component remaining in the liquid flow path from the sample flow path 2 to the analysis flow path 4 through which the sample passes can be washed out to some extent, and thus by repeatedly performing the blank injection, the elimination of the carryover can be achieved to some extent. Figure 5

[0050] In addition, Figure 5 The order and the number of times of the steps shown are only examples, and can be freely replaced as needed. For example, in the example of Figure 5 , the sample injection step (step 104) is performed in the other analysis section 3 and the analysis step (step 105) is started after the analysis step in the analysis section 3 is started in step 102 until the analysis step is ended. Thereby, the following effects can be obtained: the efficiency of the analysis of a plurality of samples is achieved, and the time required until the analysis of all samples is ended is shortened. However, the present application is not limited to this, and it is not necessarily required to perform the analysis steps in the plurality of analysis sections 3 in parallel. In addition, the steps 110 and 111 are not necessarily steps that must be performed, and can be omitted.

[0051] Figure 7 indicates the result of performing the blank injection a plurality of times continuously after the analysis of the sample is performed in the two analysis sections 3 (FP1 and FP2). In Figure 7 , the vertical axis is the peak area value, and the horizontal axis is the number of times of the blank injection.

[0052] ​As can be seen from either FP1 or FP2, when blank injections are performed multiple times to reduce the residual amount to a certain level, the reduction rate of the residual amount becomes smaller, or even if blank injections are performed further, it becomes a stagnant state where the residual amount does not decrease. In the stagnant state, the high-pressure valve 10 is switched to the injection state, so that the sampling flow path 2 is interposed between the liquid supply section 6 and the analysis flow path 4. In the high-pressure valve 10, the mobile phase flows in the rotor groove that fluidly communicates between the port connected to the analysis flow path 4 and the injection port 16. After maintaining this state for a certain period of time, when blank injection is performed (after the 8th time), the residual amount decreases. According to the results, in the high-pressure valve 10, the residual components remain only in the flow path portion where the solution flows when the sample is injected into the analysis flow path 4. By not allowing the solution to flow through this flow path portion simultaneously with the sample injection, the residual amount can be reduced.

[0053] Figure 8 This is a graph representing the validation results of the residue removal effect brought about by system cleaning. The vertical axis of the graph represents the proportion of residue relative to the limit of quantitation (LOQ) (area % relative to the lowest calibrator value), and the horizontal axis represents the number of blank injections. In this validation, two solvents were used as the mobile phase (Mobile Phase A: formic acid). + Ammonium formate aqueous solution + (ammonium formate in water), Mobile phase B: methanol + Acetonitrile (Methanol) + After analyzing a sample containing a certain component at a high concentration using Acetonitrile, the detector (mass analyzer) signal during blank injection is used to confirm whether any residue remains. In the needle cleaning, the cleaning of the analytical flow path 4 (with the high-pressure valve 10 in a loaded state, supplying cleaning fluid and mobile phase from the liquid supply unit 6), and the system cleaning, in addition to the aforementioned mobile phases A and B, acetonitrile / acetone and formic acid are used as cleaning fluids. The composition of the liquid flowing in the sampling flow path 2 and / or the analytical flow path 4 is determined by the order of mobile phase B, mobile phase A, and cleaning fluid. Figure 6 That step-by-step method switches at 0.5-minute intervals.

[0054] exist Figure 8In the verification, in Method 1, needle washing after the end of sample injection to the analysis flow path 4 was performed, and system washing after the end of analysis was not performed, and blank injection was performed. In Method 2, needle washing after the end of sample injection to the analysis flow path 4 was not performed, and system washing after the end of analysis was performed, and then blank injection was performed. In Method 3, after needle washing after the end of sample injection to the analysis flow path 4 and washing of the analysis flow path 4 after the end of analysis were performed, blank injection was performed. In Method 4, needle washing after the end of sample injection to the analysis flow path 4 was not performed, and after washing of the analysis flow path 4 after the end of analysis and system washing were performed, blank injection was performed.

[0055] If the results of Method 1 and Method 2 are compared, it is understood that by performing system washing in which the cleaning liquid and the mobile phase flow in a state in which the sampling flow path 2 is interposed between the liquid supply portion 6 and the analysis flow path 4, not only the sampling flow path 2 and the analysis flow path 4 but also the rotor groove in the high-pressure valve 10 can be cleaned, and a high carryover elimination effect can be obtained as a whole. In addition, according to the result of Method 3, it is understood that by combining needle washing and washing of the analysis flow path 4, a certain carryover elimination effect can be obtained, but if the result of Method 4 is compared with Method 3, it is understood that by combining system washing and washing of the analysis flow path 4, a higher carryover elimination effect can be obtained. In Method 3 and Method 4, in the four blank injections, the proportion of carryover with respect to LOQ was reduced to 10% or less, and it can be evaluated that the elimination of carryover was sufficiently achieved.

[0056] In addition, the above-described embodiments are merely examples of the implementation of the liquid chromatograph and the analysis method of the present application. The implementation of the liquid chromatograph and the analysis method of the present application is as follows.

[0057] In one embodiment of the liquid chromatograph of the present application, there are included: a sample flow path 2 provided with a needle 12 for sampling at a front end; a needle moving mechanism 13 that moves the needle 12 three-dimensionally; a liquid supply section 6 configured to be able to supply at least a mobile phase; an analysis flow path 4 provided with a separation column 14 for separating components in a sample; a high-pressure valve 10 having an injection port 16 that is fluidly connected with the sample flow path 2 by insertion of the needle 12, and a port to which the analysis flow path 4 is connected, and configured to selectively switch to a load state for fluidly connecting the liquid supply section 6 with the analysis flow path 4 without passing through the sample flow path 2, and an injection state for fluidly connecting the liquid supply section 6 with the analysis flow path 4 via the sample flow path 2 when the front end of the needle 12 is inserted into the injection port 16; and a control section 50 that controls the needle moving mechanism 13, the liquid supply section 6, and the high-pressure valve 10. The liquid supply section 6, the analysis flow path 4, and the high-pressure valve 10 constitute one analysis section 3 for performing sample analysis. Also, the control section 50 is configured to perform, as an analysis operation of the analysis section 3: a sample injection step of causing the high-pressure valve 10 to be in the load state, sucking a sample from the front end of the needle 12 and holding the sample in the sample flow path 2, and then connecting the sample flow path 2 with the injection port 16 and causing the high-pressure valve 10 to be in the injection state, and supplying a mobile phase from the liquid supply section 6, thereby injecting the sample held in the sample flow path 2 into the analysis flow path 4; and an analysis step of causing the high-pressure valve 10 to be in the load state after the sample injection step ends, and supplying the mobile phase from the liquid supply section 6, thereby separating the sample injected into the analysis flow path 4 by the separation column 14, and in a case where at least a prescribed condition is satisfied, performing: a system purge step of connecting the sample flow path 2 with the injection port 16 and causing the high-pressure valve 10 to be in the injection state after the analysis step ends, and supplying the mobile phase and / or a purge liquid from the liquid supply section 6, thereby purging the inside of a liquid flow path from the sample flow path 2 to the analysis flow path 4.

[0058] In a first mode of the one embodiment of the liquid chromatograph, the liquid supply section 6 is configured to be able to switch the liquid to be supplied between the mobile phase and the purge liquid by switching a switching valve 22, and the control section 50 is configured to supply at least the purge liquid from the liquid supply section 6 in the system purge step.

[0059] In the first aspect, the control section 50 can be configured to supply the mobile phase and the cleaning liquid alternately or simultaneously from the liquid supply section 6 during the system cleaning step. If so, a high cleaning effect in the liquid flow path from the sample flow path 2 to the analysis flow path 4 can be obtained, and carryover can be efficiently eliminated.

[0060] In a second aspect of the embodiment of the liquid chromatograph, the prescribed condition includes that the concentration of the sample injected into the analysis flow path 4 is equal to or higher than a prescribed level. The second aspect can be combined with the first aspect.

[0061] In the second aspect, the control section 50 is configured to detect the concentration level of the sample injected into the analysis flow path 4 based on information input by a user or based on an output signal of a detector connected to the analysis flow path 4, to perform a determination as to whether the detected concentration level is equal to or higher than the prescribed level, and to determine whether the prescribed condition is satisfied based on the result of the determination.

[0062] In a third aspect of the embodiment of the liquid chromatograph, the control section 50 is configured to perform a blank measurement step in which, after the analysis step, the high-pressure valve 10 is brought to the loading state, a sample-free blank liquid is sucked from the tip of the needle 12 and held in the sample flow path 2, the sample flow path 2 is connected to the injection port 16 and the high-pressure valve 10 is brought to the injection state, a mobile phase is supplied from the liquid supply section 6, and thus the blank liquid held in the sample flow path 2 is injected into the analysis flow path 4, an output signal of a detector connected to the analysis flow path 4 with respect to the blank liquid is acquired, a determination is performed as to whether the output signal acquired in the blank measurement step is equal to or higher than a prescribed level, and whether the prescribed condition is satisfied is determined based on the result of the determination. The third aspect can be combined with the first aspect and / or the second aspect.

[0063] In the fourth modification of the above-described embodiment of the liquid chromatograph, the liquid chromatograph 1 includes at least one other analysis section 3 provided separately from the analysis section 3, the other analysis section 3 having the same structure as the analysis section 3, and the control section 50 is configured to execute the analysis operation including the sample injection step, the analysis step, and the system cleaning step in the other analysis section 3 as well as in the analysis section 3. Further, the control section 50 is configured to, in a case where the system cleaning step is executed as the analysis operation of the analysis section 3, execute the sample injection step of the other analysis section 3 after the end of the sample injection step of the analysis section and before the start of the system cleaning step, and execute the system cleaning step of the analysis section 3 during the execution of the analysis step of the other analysis section 3. According to this modification, it is possible to start the analysis step by executing the sample injection step of the other analysis section 3 during the execution of the analysis step in a certain analysis section 3, and it is possible to execute the system cleaning step in the analysis section 3 during the execution of the analysis step of the other analysis section 3, so that the analysis efficiency of a plurality of samples is improved.

[0064] In one embodiment of the analysis method using a liquid chromatograph according to the present application, the liquid chromatograph 1 includes a sampling flow path 2 provided with a needle 12 at a front end for sampling, a liquid supply portion 6 configured to be able to supply at least a mobile phase, an analysis flow path 4 provided with a separation column 14 for separating components in a sample, and a high-pressure valve 10 having an injection port 16 fluidly connected to the sampling flow path 2 by insertion of the needle 12 and a port to which the analysis flow path 4 is connected, and configured to selectively switch to a load state for fluidly connecting the liquid supply portion 6 to the analysis flow path 4 without passing through the sampling flow path 2 and an injection state for fluidly connecting the liquid supply portion 6 to the analysis flow path 4 via the sampling flow path 2 when the front end of the needle 12 is inserted into the injection port 16. The analysis method includes a sample injection step of bringing the high-pressure valve 10 to the load state, sucking a sample from the front end of the needle 12 and retaining the sample in the sampling flow path 2, connecting the sampling flow path 2 to the injection port 16 and bringing the high-pressure valve 10 to the injection state, and supplying a mobile phase from the liquid supply portion 6 to thereby inject the sample retained in the sampling flow path 2 into the analysis flow path 4, and an analysis step of bringing the high-pressure valve 10 to the load state after the sample injection step ends, supplying the mobile phase from the liquid supply portion 6 to thereby separate the sample injected into the analysis flow path 4 by the separation column 14, and in a case where at least a prescribed condition is satisfied, the analysis method includes a system cleaning step of connecting the sampling flow path 2 to the injection port 16 and bringing the high-pressure valve 10 to the injection state after the analysis step ends, and supplying the mobile phase and / or a cleaning liquid from the liquid supply portion 6 to thereby clean the inside of a liquid flow path from the sampling flow path 2 to the analysis flow path 4.

[0065] In a first aspect of the one embodiment of the analysis method, the cleaning liquid is supplied from the liquid supply portion 6 in the system cleaning step.

[0066] In the first aspect, the mobile phase and the cleaning liquid can be alternately or simultaneously supplied from the liquid supply portion 6 in the system cleaning step. This makes it possible to obtain a high cleaning effect in the inside of the liquid flow path from the sampling flow path 2 to the analysis flow path 4, and thereby efficiently eliminate carryover.

[0067] In a second aspect of the one embodiment of the analysis method, the prescribed condition includes a concentration of the sample injected into the analysis flow path 4 being equal to or higher than a prescribed level. The second aspect can be combined with the first aspect.

[0068] In the second aspect, the concentration level of the sample injected into the analysis flow path 4 can be detected based on the output signal of the detector connected to the analysis flow path 4, and it can be determined whether the detected concentration level is equal to or higher than the predetermined level. Then, it can be determined whether the predetermined condition is satisfied based on the result of the determination.

[0069] In the third aspect of the one embodiment of the analysis method, a blank measurement step is performed in which, after the analysis step is completed, the high-pressure valve 10 is brought to the loading state, a blank solution not containing a sample is sucked from the tip of the needle 12 and held in the sampling flow path 2, then the sampling flow path 2 is connected to the injection port 16 and the high-pressure valve 10 is brought to the injection state, a mobile phase is supplied from the liquid supply unit 6, and thus the blank solution held in the sampling flow path 2 is injected into the analysis flow path 4, and an output signal of the detector connected to the analysis flow path 4 with respect to the blank solution is acquired, and it is determined whether the output signal acquired in the blank measurement step is equal to or higher than a predetermined level, and it is determined whether the predetermined condition is satisfied based on the result of the determination. The third aspect can be combined with the first aspect and / or the second aspect.

[0070] In the fourth aspect of the one embodiment of the analysis method, in the liquid chromatograph 1, the liquid supply unit 6, the analysis flow path 4, and the high-pressure valve 10 constitute one analysis unit 3 for performing analysis of a sample, and at least one other analysis unit 3 having the same structure as the analysis unit 3 is provided separately from the analysis unit 3, and the analysis method is configured to include the sample injection step, the analysis step, and the system cleaning step as steps performed in the analysis unit 3 and the other analysis unit 3, respectively, and in a case where the system cleaning step is performed in the analysis unit 3, the sample injection step of the other analysis unit 3 is performed after the end of the sample injection step in the analysis unit 3 and before the start of the system cleaning step, and the system cleaning step of the analysis unit 3 is performed during the analysis step of the other analysis unit 3. According to this aspect, it is possible to perform the sample injection step of the other analysis unit 3 and start the analysis step during the analysis step of the analysis unit 3, and it is possible to perform the system cleaning step in the analysis unit 3 during the analysis step of the other analysis unit 3, and thus the analysis efficiency of a plurality of samples is improved.

[0071] [Explanation of Symbols]

[0072] 1: Liquid chromatograph

[0073] 2: Sampling flow path

[0074] 3: Analysis unit

[0075] 4: analysis flow path

[0076] 6: liquid supply section

[0077] 10: high-pressure valve

[0078] 12: needle

[0079] 14: separation column

[0080] 16: injection port

[0081] 18, 46: mobile phase pump

[0082] 20, 48: cleaning liquid pump

[0083] 22, 40: switching valve

[0084] 24: sample container

[0085] 26, 28: selection valve

[0086] 34: low-pressure valve

[0087] 36: metering pump

[0088] 42: mobile phase supply flow path

[0089] 44: cleaning liquid supply flow path

[0090] 50: control section

Claims

1. A liquid chromatograph, comprising: The sampling flow path is equipped with a sampling needle at the front end; A needle-moving mechanism enables the needle to move in three dimensions; The liquid supply unit is configured to supply at least the mobile phase; The analytical flow path is equipped with a separation column for separating the components in the sample; A high-pressure valve has an injection port that is fluidly connected to the sampling flow path via insertion of a needle, and a port connected to the analytical flow path, and is configured to selectively switch between a loading state and an injection state. The loading state allows the liquid supply section to be fluidly connected to the analytical flow path without passing through the sampling flow path, while the injection state allows the liquid supply section to be fluidly connected to the analytical flow path via the sampling flow path when the tip of the needle is inserted into the injection port. The control unit controls the needle movement mechanism, the liquid supply unit, and the high-pressure valve, wherein... The liquid supply unit, the analytical flow path, and the high-pressure valve constitute an analytical unit for performing sample analysis. The control unit is configured to execute the analysis action of the analysis unit: In the sample injection step, the high-pressure valve is in the loaded state, the sample is drawn from the tip of the needle and held in the sampling flow path, the sampling flow path is connected to the injection port and the high-pressure valve is in the injection state, the mobile phase is supplied from the liquid supply section, thereby injecting the sample held in the sampling flow path into the analytical flow path. as well as In the analytical step, after the sample injection step is completed, the high-pressure valve is brought to the loaded state, and the mobile phase is supplied from the liquid supply unit, thereby using the separation column to separate the sample injected into the analytical flow path. Perform the following steps under at least the specified conditions: After the analysis step is completed, connect the sampling flow path to the injection port and put the high-pressure valve into the injection state, supply the mobile phase and / or cleaning fluid from the liquid supply unit, thereby cleaning the liquid flow path from the sampling flow path to the analysis flow path, and cleaning the rotor groove and port of the high-pressure valve used in the injection state.

2. The liquid chromatograph according to claim 1, wherein, The liquid supply unit is configured to switch the supplied liquid between the mobile phase and the cleaning fluid by switching a switching valve. The control unit is configured to supply at least the cleaning fluid from the liquid supply unit during the system cleaning step.

3. The liquid chromatograph according to claim 2, wherein, The control unit is configured to alternately or simultaneously supply the mobile phase and the cleaning liquid from the liquid supply unit during the system cleaning step.

4. The liquid chromatograph according to claim 1, wherein, The specified conditions include that the concentration of the sample injected into the analytical flow path is above a specified level.

5. The liquid chromatograph according to claim 4, wherein, The control unit is configured to detect the concentration level of the sample injected into the analysis flow path based on information input by the user or based on the output signal of the detector connected to the analysis flow path, determine whether the detected concentration level is above the specified level, and determine whether the specified conditions are met based on the result of the determination.

6. The liquid chromatograph according to claim 1, wherein, The control unit is configured to, after the analysis step is completed, execute a blank liquid measurement step in which the high-pressure valve is in the loading state, blank liquid without sample is drawn from the tip of the needle and held in the sampling flow path, the sampling flow path is connected to the injection port and the high-pressure valve is in the injection state, a mobile phase is supplied from the liquid supply unit, thereby injecting the blank liquid held in the sampling flow path into the analysis flow path, and a blank liquid measurement step is performed to obtain the output signal of the detector connected to the analysis flow path related to the blank liquid, and to determine whether the output signal obtained in the blank liquid measurement step is above a specified level, and to determine whether the specified condition is met based on the result of the determination.

7. The liquid chromatograph according to claim 1, wherein, The liquid chromatograph includes at least one other analytical unit disposed separately from the analytical unit, the other analytical unit having the same structure as the analytical unit. The control unit is configured to perform the same analytical actions as the analytical actions of the other analytical units, including the sample injection step, the analytical steps, and the system cleaning step. The control unit is configured to perform the system cleaning step of the other analytical unit after the sample injection step of the analytical unit has ended and before the system cleaning step begins, and to perform the system cleaning step of the analytical unit while the other analytical unit is performing the analytical step.

8. An analytical method, which is an analytical method using a liquid chromatograph, wherein... The liquid chromatograph includes: The sampling flow path is equipped with a sampling needle at the front end; The liquid supply unit is configured to supply at least the mobile phase; The analytical flow path is equipped with a separation column for separating components in the sample; and A high-pressure valve has an injection port that is fluidly connected to the sampling flow path via insertion of a needle, and a port connected to the analytical flow path. It is configured to selectively switch between a loading state and an injection state. In the loading state, the liquid supply section is fluidly connected to the analytical flow path without passing through the sampling flow path. In the injection state, the liquid supply section is fluidly connected to the analytical flow path via the sampling flow path when the tip of the needle is inserted into the injection port. The analytical method includes: The sample injection step involves aligning the high-pressure valve to the loaded state, drawing in the sample from the tip of the needle and holding it within the sampling flow path, then connecting the sampling flow path to the injection port and aligning the high-pressure valve to the injection state, supplying mobile phase from the liquid supply unit, thereby injecting the sample held within the sampling flow path into the analytical flow path; and In the analytical step, after the sample injection step is completed, the high-pressure valve is brought to the loaded state, and the mobile phase is supplied from the liquid supply unit, thereby using the separation column to separate the sample injected into the analytical flow path. Under conditions that at least the specified conditions are met, the analytical method includes: a system cleaning step, wherein after the analytical step is completed, the sampling flow path is connected to the injection port and the high-pressure valve is put into the injection state, and the mobile phase and / or cleaning fluid is supplied from the liquid supply unit to clean the liquid flow path from the sampling flow path to the analytical flow path, and the rotor groove and port of the high-pressure valve used in the injection state are cleaned.

9. The analytical method according to claim 8, wherein, In the system cleaning step, at least the cleaning fluid is supplied from the liquid supply unit.

10. The analytical method according to claim 9, wherein, In the system cleaning step, the mobile phase and the cleaning liquid are supplied alternately or simultaneously from the liquid supply unit.

11. The analytical method according to claim 8, wherein, The specified conditions include that the concentration of the sample injected into the analytical flow path is above a specified level.

12. The analytical method according to claim 11, wherein, The concentration level of the sample injected into the analytical flow path is detected based on the output signal of the detector connected to the analytical flow path, and a determination is made as to whether the detected concentration level is above the specified level, and whether the specified conditions are met based on the result of the determination.

13. The analytical method according to claim 8, wherein, After the analysis step is completed, the high-pressure valve is put into the loading state, blank liquid without sample is drawn from the tip of the needle and held in the sampling flow path, the sampling flow path is connected to the injection port and the high-pressure valve is put into the injection state, the mobile phase is supplied from the liquid supply unit, thereby injecting the blank liquid held in the sampling flow path into the analysis flow path, and the output signal of the detector connected to the analysis flow path related to the blank liquid is obtained. The output signal obtained in the blank liquid determination step is determined to be above a specified level, and the specified condition is determined based on the result of the determination.

14. The analytical method according to claim 8, wherein, In the liquid chromatograph, the liquid supply unit, the analytical flow path, and the high-pressure valve constitute an analytical unit for analyzing the sample. At least one other analytical unit with the same structure as the analytical unit is disposed separately from the analytical unit. The analytical method is configured as follows: The steps performed respectively in the analytical unit and the other analytical units include the sample injection step, the analytical step, and the system cleaning step, and When the system cleaning step is performed in the analytical unit, the sample injection step of the other analytical unit is performed after the sample injection step of the analytical unit is completed and before the system cleaning step is started, and the system cleaning step of the analytical unit is performed during the execution of the analytical step in the other analytical unit.

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