Preparative isolation chromatograph system

Abstract

To prevent the reproducibility of chromatogram from reducing in stack injection preparative isolation.SOLUTION: A preparative isolation chromatograph system (1) includes a preparative isolation condition correction part (18) constituted so as to detect the change of chromatogram acquired by each of two or more injections of a sample during stack injection preparative isolation to correct preparative isolation conditions during the stack injection preparative isolation so as to approach the chromatogram to a state before the change. The preparative isolation condition correction part (18) is constituted so as to continue the stack injection preparative isolation on the basis of th preparative isolation conditions corrected after correcting the preparative isolation conditions.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a preparative chromatography system.

Background Art

[0002] A preparative chromatograph including a preparative liquid chromatograph (hereinafter, preparative LC) and a preparative supercritical fluid chromatograph (hereinafter, preparative SFC) is a device that separates a plurality of components in a sample from each other by chromatography using a liquid or a supercritical fluid, and collects the separated individual components into individual containers by a fraction collector.

[0003] In purification using a preparative chromatograph, stack injection is widely used to improve throughput (see Patent Document 1). In stack injection, the same sample is injected into the mobile phase multiple times at regular time intervals.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] In the preparative separation of components using stack injection in which the same sample is injected at regular time intervals (hereinafter referred to as stack injection preparative separation), high reproducibility is required for the chromatograms obtained in each injection. However, due to fluctuations in room temperature, an increase in pump pressure, etc., the retention time of the peaks changes, and the reproducibility of the chromatograms may decrease. When the reproducibility of the chromatograms decreases, problems such as insufficient separation of the sample and a decrease in the recovery rate of the components may occur. In addition, if the delay in the retention time of the peaks in the chromatogram becomes large, there is a risk that the peaks of the sample injected this time and the peaks of the sample injected next will overlap.

[0006] The present invention has been made in view of the above problems, and an object thereof is to suppress a decrease in the reproducibility of chromatograms in stack injection fractionation.

Means for Solving the Problems

[0007] The fractionation chromatography system according to the present invention includes a liquid feed pump for feeding a mobile phase, an autosampler provided downstream of the liquid feed pump for injecting a sample into the mobile phase, a separation column provided downstream of the autosampler for separating a plurality of components in the sample from each other, a detector provided downstream of the separation column for acquiring a chromatogram of the eluate from the separation column, a fraction collector provided with a plurality of collection containers and configured to collect fractions of the eluate from the separation column into the collection containers, a controller configured to perform stack injection fractionation in which the same sample is injected a plurality of times at preset time intervals and the components in the injected sample are fractionated by controlling the operations of the liquid feed pump, the autosampler, and the fraction collector according to preset fractionation conditions, a fractionation condition correction unit configured to detect a change in the chromatogram obtained in each of a plurality of injections of the sample during the stack injection fractionation and correct the fractionation conditions during the stack injection fractionation so that the chromatogram approaches the state before the change, The controller is configured to continue the stack injection fractionation based on the corrected fractionation conditions after the fractionation condition correction unit corrects the fractionation conditions.

Advantages of the Invention

[0008] According to the preparative chromatography system of the present invention, during preparative stack injection, changes in chromatograms obtained in each of a plurality of sample injections are detected, and preparative conditions are corrected during the preparative stack injection so that the chromatogram approaches the state before the change. After correcting the preparative conditions, the preparative stack injection is continued based on the corrected preparative conditions. Therefore, a decrease in the reproducibility of chromatograms obtained in each injection is suppressed.

Brief Description of Drawings

[0009]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Embodiments for Carrying Out the Invention

[0010] Hereinafter, an embodiment of a preparative chromatography system will be described with reference to the drawings.

[0011] As shown in FIG. 1, the preparative chromatography system 1 includes a liquid feed pump 2, an autosampler 4, a separation column 6, a detector 8, a fraction collector 10, a column oven 12, a controller 14, and an information processing device 16.

[0012] The liquid delivery pump 2 delivers the mobile phase toward the separation column 6. The autosampler 4 is provided downstream of the liquid delivery pump 2 and injects a sample into the mobile phase delivered by the liquid delivery pump 2. The separation column 6 separates a plurality of components in the sample injected into the mobile phase by the autosampler 4 from each other in time. The detector 8 is provided downstream of the separation column 6 and is for obtaining a chromatogram by outputting a signal with an intensity corresponding to the component concentration in the eluate from the separation column 6. The separation column 6 is housed in the column oven 12. The column oven 12 is a device for adjusting the temperature of the separation column 6 to a set temperature. The fraction collector 10 is a device provided with a plurality of collection containers 24 and collecting the fraction of the eluate from the separation column 6 passing through the detector 8 by dropping it from the nozzle 20 into the collection container 24. A three-way solenoid valve 22 is provided upstream of the nozzle 20, and it is possible to switch whether to guide the eluate to the nozzle 20 or to the drain by switching the three-way solenoid valve 22.

[0013] The controller 14 is configured to control the operations of the liquid delivery pump 2, the autosampler 4, the detector 8, the fraction collector 10, and the column oven 12 according to the fractionation conditions given from the information processing device 16 described later, and to perform fractionation of the components in the sample. The controller 14 is realized by an electronic circuit device including a CPU (Central Processing Unit) or the like.

[0014] The information processing device 16 is a computer device communicably connected to the controller 14. The user sets the fractionation conditions on the information processing device 16. The fractionation conditions can include the flow rate of the mobile phase, the sample injection volume, the injection interval of the stack injection, the column temperature, the purge time of the nozzle 20, the peak detection conditions for fractionation by waveform processing of the chromatogram, and the time program for fractionation by the time program. The information processing device 16 transmits the fractionation conditions set by the user to the controller 14.

[0015] The information processing device 16 reads the signal output from the detector 8 through the controller 14 to create a chromatogram, and performs detection of peaks appearing in the chromatogram, calculation of the retention time of each peak, and the like.

[0016] Furthermore, the information processing device 16 includes a fractionation condition correction unit 18. The fractionation condition correction unit 18 is a function obtained when the CPU executes a program. During the execution of stack injection fractionation, the fractionation condition correction unit 18 determines whether there is a change in the chromatogram obtained for each injection of the sample, that is, whether the chromatogram obtained by the current injection of the sample has changed from a reference chromatogram (for example, the chromatogram obtained by the first injection or the chromatogram obtained by the previous injection). When a change in the chromatogram is detected, the fractionation condition correction unit 18 corrects the fractionation conditions so that the chromatogram approaches the reference chromatogram.

[0017] For the determination of whether there is a change in the chromatogram, determination parameters such as the retention time of the peak, the height of the peak, the peak width, and the interval between peaks are used, and it can be determined that there is a change in the chromatogram when those determination parameters differ from the reference chromatogram by a predetermined ratio or more.

[0018] For example, as shown in FIG. 2, when the retention time of the peak in the chromatogram obtained by the second injection is slower than the retention time of the same peak in the chromatogram obtained by the first injection by a predetermined ratio or more, the fractionation condition correction unit 18 increases the flow rate of the mobile phase set as the fractionation condition so that the retention time of the peak approaches the chromatogram obtained by the first injection. The information processing device 16 transmits the fractionation conditions corrected by the analysis condition correction unit 18 to the controller 14. The controller 14 continues the stack injection fractionation using the corrected fractionation conditions. As a result, the chromatogram obtained by the third injection has a shape closer to the chromatogram obtained by the first injection than the chromatogram obtained by the second injection.

[0019] When a change is detected in any of the determination parameters, the fractionation condition correction unit 18 corrects the fractionation conditions immediately for the fractionation conditions associated with the changed determination parameter and can reflect it in the control by the controller 14. For example, in the example of FIG. 2, when it is detected that the retention time of the first peak A of the chromatogram obtained by the second injection is delayed from the retention time of the first peak of the chromatogram obtained by the first injection, the fractionation condition correction unit 18 can immediately increase the flow rate of the mobile phase. By doing so, the retention time of the second peak B of the chromatogram obtained by the second injection can be advanced, and the chromatogram can be brought closer to the chromatogram obtained by the first injection during the fractionation of the components in the sample injected the second time.

[0020] Also, as another correction method, the fractionation condition correction unit 18 corrects to increase the injection interval so that the component peaks of the samples injected at different timings do not overlap each other. The correction of the injection interval can be performed when the delay in the retention time of the peak cannot be eliminated even by correcting the flow rate of the mobile phase or the like.

[0021] Next, an example of the operation of the stack injection fractionation by the fractionation chromatograph system 1 will be described with reference to the flowchart of FIG. 3 together with FIG. 1.

[0022] When the user sets the fractionation conditions for the stack injection fractionation and inputs an instruction to start to the information processing device 16, the fractionation conditions are transmitted from the information processing device 16 to the controller 14, and the controller 14 starts the stack injection fractionation according to the transmitted fractionation conditions.

[0023] In the stack injection fractionation, the same sample is injected multiple times at the injection interval set as the fractionation condition (step 101). In the first injection of the sample, fractionation is performed according to the set fractionation conditions (step 105).

[0024] On the other hand, in the second and subsequent injections, the fractionation condition correction unit 18 determines whether the obtained chromatogram has changed from the reference chromatogram (step 103). When a change in the chromatogram is detected (step 103: Yes), the fractionation conditions are corrected so as to bring the chromatogram closer to the reference chromatogram (step 104). The corrected fractionation conditions are transmitted to the controller 14, and the controller 14 performs fractionation according to the corrected fractionation conditions (step 105). Conversely, when the fractionation condition correction unit 18 does not detect a change in the chromatogram (step 103: No), fractionation continues without correcting the fractionation conditions (step 105). The above steps 101 to 105 are repeatedly performed until the number of sample injections reaches the set number, and the stack injection fractionation ends when the number of sample injections reaches the set number (step 106).

[0025] Here, in the stack injection fractionation, the controller 14 stores the location of the collection container 24 used for collecting each component, the capacity of each collection container 24, and the amount of the eluate collected in each collection container 24, so that the same component can be continuously collected in the same collection container 24 until the collection container 24 is full of the eluate. Then, when the used collection container 24 is filled with the eluate, the collection of the corresponding component can be continued using a new empty collection container 24. Specifically, it is provided with a collection container storage unit that stores the number or position information of the collection container collected for each peak of the chromatogram. When the collection container collecting the peak becomes full, the number or position information of the collection container stored in the collection container storage unit is updated to the number or position information of the next used container. Thereby, the number of collection containers can be saved.

[0026] For example, when the fraction collector 10 is configured such that the collection containers 24 are arranged in a 6-row × 4-column matrix as shown in FIG. 4, and when eluates containing two components A and B included in the sample are collected in the collection containers 24 respectively, for the collection of component A eluting first from the separation column 6, the collection container 24 arranged at the position 1-1 (the first column from the left in the first row from the bottom) is continuously used until it is full. For the collection of component B eluting second from the separation column 6, the collection container 24 arranged at the position 2-1 (the first column from the left in the second row from the bottom) can be continuously used until it is full. At this time, the collection container storage unit stores that component A is collected in the 1-1 (first) collection container and component B is collected in the 2-1 (second) collection container.

[0027] And, for example, when the collection container 24 arranged at the position 1-1 becomes full, as shown in FIG. 5, a new empty collection container 24 (the collection container 24 arranged at the position 3-1 (the first column in the third row) in the figure) can be used for the collection of component A. Further, when the collection container 24 arranged at the position 2-1 becomes full, a new empty collection container 24 (for example, the collection container 24 arranged at the position 4-1 (the first column in the fourth row)) can be used for the collection of component B. At this time, the information stored in the collection container storage unit is updated such that component A is collected in the 3-1 (third) collection container and component B is collected in the 4-1 (fourth) collection container.

[0028] By the way, when the purge of the nozzle 20 is executed each time the fraction of the eluate is collected in the collection container 24, a specific position can be set as the purge location of the nozzle 20. In the examples of FIGS. 4 and 5, the drain port 26 is set as the purge location.

[0029] The purge operation of the nozzle 20 will be described with reference to FIG. 4. After the controller 14 drips the eluate containing component A from the nozzle 20 into the collection container 24 arranged at the position 1-1, the nozzle 20 is moved to the position of the drain port 26, and the eluate is dripped from the nozzle 20 to the drain port 26 for the purge time set as the fractionation condition to purge the nozzle 20. Then, after moving the nozzle 20 to the position above the collection container 24 arranged at the position 2-1 and dripping the eluate containing component B from the nozzle 20, the nozzle 20 is moved again to the position of the drain port 26, and the eluate is dripped from the nozzle 20 to the drain port 26 for the set purge time to purge the nozzle 20.

[0030] In addition to the drain port 26, it is also possible to set the collection container 24 arranged at a specific position (for example, the position of the 4th column in the 6th row) among the plurality of collection containers 24 arranged in a matrix as the purge location of the nozzle 20.

[0031] As described above, when the purge location is set at a specific position such as the drain port 26, depending on the length of the set purge time, the movement of the nozzle 20 to the position where the next eluted component should be collected may not be in time, and there is a possibility of failing to collect some or all of that component. As shown in the upper diagram of FIG. 6, when the purge time of T seconds is set, after collecting component A in the collection container 24 and moving the nozzle 20 to a predetermined purge location and performing the purge of the nozzle 20 for T seconds, a part of component B cannot be collected.

[0032] Therefore, the fractionation condition correction unit 18 is configured to correct the purge time of T seconds set for the second and subsequent injections to a purge time of T' seconds so that the collection of each component of the sample injected later is not failed, based on the movement time of the nozzle 20 and the interval between the peak of component A and the peak of component B in the chromatogram obtained by the first injection, as shown in the lower diagram of FIG. 6.

[0033] Note that the embodiments described above are merely examples of embodiments of the preparative chromatography system according to the present invention. Embodiments of the preparative chromatography system according to the present invention are as follows.

[0034] In one embodiment of the preparative chromatography system (1) according to the present invention, a liquid delivery pump (2) for delivering a mobile phase, an autosampler (4) provided downstream of the liquid delivery pump (2) for injecting a sample into the mobile phase, a separation column (6) provided downstream of the autosampler (4) for separating a plurality of components in the sample from each other, a detector (8) provided downstream of the separation column (6) for acquiring a chromatogram of the eluate from the separation column (6), a fraction collector (10) provided with a plurality of collection containers (24) and configured to collect fractions of the eluate from the separation column (6) into the collection containers (24), a controller (14) configured to perform stack injection fractionation in which the same sample is injected multiple times at preset time intervals and the components in the injected sample are fractionated by controlling the operations of the liquid delivery pump (2), the autosampler (4), and the fraction collector (10) according to preset fractionation conditions, a fractionation condition correction unit (18) configured to detect changes in the chromatogram obtained in each of the multiple injections of the sample during the stack injection fractionation and correct the fractionation conditions during the stack injection fractionation so that the chromatogram approaches the state before the change, and the controller (14) is configured to continue the stack injection fractionation based on the corrected fractionation conditions after the fractionation condition correction unit (18) corrects the fractionation conditions.

[0035] In aspect [1] of the above-described embodiment of the preparative chromatography system (1), the fractionation conditions include the flow rate of the mobile phase, The fractionation condition correction unit (18) is configured to correct the flow rate of the mobile phase so that the retention time of the peak appearing in the chromatogram of the sample injected after the second time approaches the retention time of the corresponding peak in the chromatogram of the sample injected before the previous time.

[0036] In aspect [2] of the above-described embodiment of the preparative chromatography system (1), the fractionation conditions include the injection interval of the sample, the fractionation condition correction unit is configured to increase the injection interval when the retention time of the peak appearing in the chromatogram of the sample injected after the second time is later than the retention time of the same peak appearing in the chromatogram of the sample injected previously. This aspect [2] can be combined with the above aspect [1].

[0037] In aspect [3] of the above-described embodiment of the preparative chromatography system (1), the controller (14) recognizes the amount of the eluate collected in each collection container (24) of the fraction collector in the stack injection fractionation, collects the same component in the same collection container (24) until the used collection container (24) is full, and uses a new empty collection container (24) when the used collection container (24) is filled. This aspect [3] can be combined with the above aspects [1] and / or [2].

[0038] In aspect [4] of the above-described embodiment of the preparative chromatography system (1), the fraction collector (10) includes a nozzle (20) for dropping the fraction of the eluate into the collection container (24), the controller (14) is configured to drop the eluate to a purge location set at a specific position when purging the nozzle (20). This aspect [4] can be combined with the above aspects [1], [2], and / or [3].

[0039] In the above aspect [4], the controller (14) is configured to perform the purge of the nozzle (20) each time a fraction of the eluate corresponding to the peak portion of the chromatogram is collected in the stack injection fractionation into the collection container (24). The fractionation conditions include a purge time for the purge of the nozzle (20). The fractionation condition correction unit (18) is configured to correct the purge time using the peak interval of the chromatogram of the sample injected for the first time in the stack injection fractionation.

Explanation of Reference Numerals

[0040] 1 Fractionation chromatograph system 2 Liquid delivery pump 4 Autosampler 6 Separation column 8 Detector 10 Fraction collector 12 Column oven 14 Controller 16 Information processing device 18 Fractionation condition setting unit 20 Nozzle 22 Three-way solenoid valve 24 Fraction collector

Claims

1. A liquid delivery pump for delivering a mobile phase, An autosampler provided downstream of the liquid delivery pump for injecting a sample into the mobile phase, A separation column provided downstream of the autosampler for separating a plurality of components in the sample from each other, A detector provided downstream of the separation column for acquiring a chromatogram of the eluate from the separation column, A fraction collector configured to collect fractions of the eluate from the separation column into the collection vessels, with a plurality of collection vessels provided, A controller configured to perform stack injection fractionation by controlling the operations of the liquid delivery pump, the autosampler, and the fraction collector according to preset fractionation conditions, injecting the same sample multiple times at preset time intervals, and fractionating the components in the injected sample, A fractionation condition correction unit configured to detect changes in the chromatogram obtained in each of the multiple injections of the sample during the stack injection fractionation, and correct the fractionation conditions during the stack injection fractionation so that the chromatogram approaches the state before the change, The fractionation chromatograph system, wherein the controller is configured to continue the stack injection fractionation based on the corrected fractionation conditions after the fractionation condition correction unit corrects the fractionation conditions.

2. The fractionation conditions include the flow rate of the mobile phase, The fractionation condition correction unit is configured to correct the flow rate of the mobile phase so that the retention time of the peak appearing in the chromatogram of the sample injected in the second and subsequent injections approaches the retention time of the corresponding peak in the chromatogram of the sample injected before the previous injection. The fractionation chromatograph system according to Claim 1.

3. The fractionation conditions include the injection interval of the sample, The fractionation condition correction unit is configured to increase the injection interval when the retention time of the peak appearing in the chromatogram of the sample injected in the second and subsequent injections is later than the retention time of the same peak appearing in the chromatogram of the sample injected previously. The fractionation chromatograph system according to Claim 1.

4. The controller recognizes the amount of the eluate collected in each collection container of the fraction collector in the stack injection fractionation, collects the same components in the same collection container until the in-use collection container is full, and is configured to use a new empty collection container when the in-use collection container is filled. The fractionation chromatography system according to claim 1.

5. The fraction collector includes a nozzle for dropping the fraction of the eluate into the collection container. The controller is configured to drop the eluate to a purge location set at a specific position when purging the nozzle. The fractionation chromatography system according to claim 1.

6. The controller is configured to perform the purge of the nozzle each time the fraction of the eluate corresponding to the peak portion of the chromatogram is collected in the collection container in the stack injection fractionation. The fractionation conditions include a purge time for the purge of the nozzle. The fractionation condition correction unit is configured to correct the purge time using the peak interval of the chromatogram of the sample injected for the first time and the movement time of the nozzle in the stack injection fractionation. The fractionation chromatography system according to claim 5.

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