JP2010014559A - Preparative liquid chromatograph - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To prevent other constituents from being mixed into the preparative isolation of an aimed constituent, and to avoid the loss of a precious sample by performing preparative isolation without causing even unexpected constituents to leak. <P>SOLUTION: A chromatogram preparation section 21 prepares a chromatogram in real time from a detection signal by an MS detector 14, and a peak detection section 22 detects peak start/end points. Although a demarcation control section 24 controls a fraction collector 9 so that time demarcation of an equal time interval is performed until a peak is detected, the demarcation control section 24 finishes demarcation at that point immediately when a peak start point detection signal is obtained, and transfers to the next demarcation. Then, until the peak end point is detected, time demarcation for prescribed amount of time or one demarcation is executed, and the demarcation at that point is completed immediately when the peak end point detection signal is obtained before returning to the time demarcation of the original equal time interval, thus preventing the aimed constituent from being mixed with other constituents even if they approach for flowing out and collecting all constituents without any omission. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a preparative liquid chromatograph for fractionating an eluate containing components separated by a column and collecting the fraction in a plurality of containers.

  2. Description of the Related Art A so-called preparative liquid chromatograph apparatus that fractionates and collects a plurality of components contained in a liquid sample using a high performance liquid chromatograph (HPLC) has been known (Patent Document 1, Non-Patent Document). Reference 1 etc.). In such a preparative liquid chromatograph apparatus, it is necessary to manually or automatically determine the timing for fractionating the eluate with the fraction collector.

  When a relatively large amount of sample can be prepared, a preliminary LC analysis is performed on the sample to create a chromatogram, and the operator determines the timing of fractionation by viewing the chromatogram. Then, a program for fraction control can be created, and fractionation and fractionation can be performed while performing LC analysis on the same sample again according to the program. However, for example, when there are only a small amount of sample, or when it is expensive and valuable, preliminary LC analysis cannot be performed, and it is necessary to fractionate and fractionate the eluate separated in one LC analysis. There is.

  In such a case, the fraction control method is roughly divided into time fractions for fractionation at predetermined time intervals or random time intervals irrespective of the presence or absence of peaks due to sample components, and peak start and end points. A peak fraction that automatically detects and extracts and fractionates the peak portion is known. In the peak fractionation, the target component can be fractionated without mixing other components if the peak detection conditions are appropriately set. However, if the peak detection conditions are not set appropriately, a small amount of components cannot be recovered, and valuable samples are lost. On the other hand, time fractionation is often used especially when it is desired to collect all components including the target component without omission, and even when there is a small amount of component that the operator does not expect at all. There is an advantage that it can be collected. However, in the time fractionation, when another impurity component is eluted in time proximity to the target component, there is a possibility that the target component and the impurity component are mixed.

JP-A-3-29850 "Shimadzu Application News No.C56 Fractionation by Preparative LC-MS and Fraction by Ultra High Speed LC-MS", [online], Shimadzu Corporation, [Search June 27, 2008], Internet < URL: http://www.an.shimadzu.co.jp/support/lib/an/200710/c56.pdf>

  The present invention has been made in view of the above problems, and the main purpose of the present invention is to collect the target component without leaking other unexpected components, and to separate the target component. An object of the present invention is to provide a preparative liquid chromatograph apparatus capable of preventing mixing of other components.

In order to solve the above-mentioned problems, the present invention includes a column for separating components in a sample, a detector for detecting components in the eluate from the column, and different containers by fractionating the eluate. In a preparative liquid chromatograph apparatus comprising a fraction collector collected in
a) Peak detection means for detecting at least one of a peak start point and an end point on a chromatogram created based on detection signals sequentially obtained by the detector as the analysis of the target sample proceeds,
b) Time fractionation is executed at one or more preset time intervals during a period when there is no peak, and at least one of the peak start point and end point is detected by the peak detection means Control means for controlling the timing of fractionation by the fraction collector so as to end the fractionation executed in step 1 and shift to the next fractionation,
It is characterized by having.

  As one aspect of the preparative liquid chromatograph apparatus according to the present invention, the control means performs the fractionation by the fraction collector so that the period from the start point to the end point of a certain peak is one fraction. It can be configured to control timing.

  Moreover, as another aspect of the preparative liquid chromatograph apparatus according to the present invention, the control means is configured such that, during a period in which there is a peak from the start point to the end point of each peak, at a time interval different from a period in which there is no peak. It is also possible to control the fractionation timing by the fraction collector so as to execute the time fractionation.

  That is, in the preparative liquid chromatograph apparatus according to the present invention, in a period in which there is no peak on the chromatogram, that is, in a period excluding a period from when the peak detecting means detects the peak start point to when the peak end point is detected. The control means controls the fraction collector to perform time fractionation. However, in a period where there is no peak, it can be clearly determined that there are no significant components, for example, a period from when a sample is injected into the mobile phase until the first component is eluted, or all In the period after the elution of the components, the eluate may be discarded without performing fractionation.

  As described above, in the period when there is no peak (strictly speaking, there is no peak of intensity that can be detected by the peak detection means), time fractionation is basically executed, so a small amount of components that are not detected as peaks. If present, the components are always dispensed into either container. For example, when the peak detection means detects the peak start point, the fraction collector under the control of the control means ends the previous fraction and shifts to the next fraction. It is definitely fractionated from the previously eluted components. When the peak detecting means detects the peak end point, the fraction collector completes the immediately preceding fraction and shifts to the next fraction under the control of the controlling means. The components eluted later are also reliably fractionated. In other words, the component having the peak is surely fractionated from the components existing before and after in time and is not mixed.

  As described above, according to the preparative liquid chromatograph apparatus according to the present invention, components corresponding to small peaks that cannot be detected by the peak detection means can be collected in the container without omission, while detected by the peak detection means. The fraction of the target component corresponding to a relatively large peak can be prevented from being mixed with other components. Thereby, the target component can be efficiently separated. In addition, since it is possible to sort out components that were not originally scheduled to be collected or unknown components that are not anticipated by the operator, it is possible to conduct detailed surveys on these items later. Loss can be prevented.

  Hereinafter, a preparative liquid chromatograph apparatus according to an embodiment of the present invention will be described with reference to the accompanying drawings. FIG. 1 is a configuration diagram of a main part of a preparative chromatograph apparatus according to the present embodiment.

  In this preparative liquid chromatograph apparatus, the liquid feed pump 2 sucks the mobile phase stored in the mobile phase container 1 and feeds it to the separation column 4 through the injector 3 at a constant flow rate. When the sample liquid is injected into the mobile phase in the injector 3, the sample liquid rides on the mobile phase, is introduced into the separation column 4, and is separated and eluted in the time direction while passing through the separation column 4. This eluate is introduced into the fraction collector 9 through the elution channel 5, through the splitter 6 and the ultraviolet-visible spectroscopic (UV) detector 8. A loop pipe 7 for delaying the flow of the eluate is provided in the elution flow path 5 on the downstream side of the splitter 6. The UV detector 8 detects a sample component in the eluate that passes therethrough and sends a detection signal to a data processing unit 20 described later.

  The fraction collector 9 moves the dispensing valve 91 and the drain valve 95 for turning on and off the eluate, the container rack 93 in which a large number of containers 92 are accommodated, and the dispensing nozzle for dropping the eluate in two axial directions. And a drive unit 94 for switching the container for collecting the eluate and a waste liquid container 96 for collecting the eluate when the drain valve 9 is turned on. In the fraction collector 9, the drive unit 94 appropriately moves the dispensing nozzle in accordance with an instruction from the fractionation control unit 23, whereby the sequentially flowing eluate can be sequentially sorted into a plurality of containers. In this specification, “to shift to the next fraction” means to move the dispensing nozzle onto the next container in this example. Of course, instead of moving the dispensing nozzle, the container may be moved, or “transition to the next fraction” can be performed by switching the flow path using a valve.

  The makeup pump 11 sucks the mobile phase stored in another mobile phase container 10 and flows it to the detection flow path 12. The detection flow path 12 is connected to a photodiode array (PDA) detector 13 and a mass spectrometry (MS) detector 14 via the splitter 6. The splitter 6 is a so-called active splitter, and a part of a high flow rate of the eluate flowing in the elution channel 5 is divided by a predetermined split ratio and guided to the detection channel 12. Part of the divided eluate rides on the flow of the mobile phase fed by the makeup pump 11 and is smoothly introduced into the PDA detector 13 and the MS detector 14. Both the PDA detector 13 and the MS detector 14 detect sample components in the divided eluate supplied via the detection flow path 12.

  Detection signals from the UV detector 8, the PDA detector 13, and the MS detector 14 are all sent to the data processing unit 20. In the apparatus of this embodiment, the detection signal from the PDA detector 13 or the MS detector 14 is used for fraction control of the eluate in the fraction collector 9, and the detection signal from the UV detector 8 is eluted into the fraction collector 9. This is used for confirming the arrival time of the liquid and for confirming the change of the peak shape by the loop tube 7.

  In the data processing unit 20, a chromatogram creation unit 21 creates a chromatogram in real time based on detection signals sequentially obtained by the PDA detector 13 or the MS detector 14. When the detection signal from the MS detector 14 is used, a mass chromatogram based on the detection result of ions in a predetermined m / z or m / z range or a total ion chromatogram based on the detection result of all ions is created. The peak detector 22 detects the peak start and end points in real time for the chromatogram. Here, a threshold value LV of the signal intensity is set in advance, a time point when the signal intensity of the chromatogram exceeds the threshold value LV is determined as a peak start point, and then a time point when the signal intensity falls below the threshold value LV Judge. Of course, in addition to this, the slope of the peak curve may be judged to detect the peak start point and end point, respectively, and the peak start point and end point may be combined with the signal strength and peak curve slope. A point may be detected.

  When the peak detection unit 22 detects the start point or the end point of the peak, it immediately informs the fractionation control unit 23 accordingly. The fraction control unit 23 determines the fraction switching timing based on the fractionation conditions set by the fractionation condition setting unit 24 and the detection signal of the peak start point and the end point, and the dispensing valve 91 and the The operation of the drive unit 94 is controlled.

  Before explaining the fractionation operation in the preparative liquid chromatograph apparatus of the present embodiment, general time fractionation at equal time intervals will be briefly explained with reference to FIG. FIG. 3 is a diagram showing an example of a chromatogram waveform (a) and fractionation timing (b) in time fractionation at equal time intervals. Here, as shown in FIG. 3A, it is assumed that a peak Pb of another component is present in time proximity to the peak Pa of the target component. As shown in the figure, when time fractionation is performed on an eluate containing such components at a constant time ta interval, the fraction collector supports chromatograms for each fractionation period of [# 1] to [# 8]. The eluates containing the components are dispensed into different containers. Therefore, not only the target component but also other components are mixed in the eluate collected corresponding to the fraction period of [# 5].

  The preparative liquid chromatograph apparatus of the present example was able to reliably separate all the components including the components that the operator did not expect while preventing the mixing of the target component and other components as described above. Is. The control operation of the fraction will be described with reference to FIG. (A) is the same chromatogram waveform as FIG. 3 (a), (b)-(d) is a figure which shows the example of a different fractionation timing, respectively. First, the example of FIG. 2B will be described.

  Prior to the sorting operation, the operator performs the fractionation conditions including the time interval ta for the time fraction in the period without the peak, the time interval tb for the time fraction in the period with the peak, the threshold LV as the peak detection condition, and the like. Is input from the fractionation condition setting unit 24. The input threshold value LV is stored inside the peak detector 22, and the time intervals ta and tb of the time fraction are stored inside the fraction controller 23. When the sorting operation is started, the sample liquid is injected from the injector 3 into the mobile phase that is fed at a constant flow rate by the liquid feed pump 2 as described above. The fraction collector 9 may start fractionation of the eluate simultaneously with this sample injection. However, since it takes some time until the fastest sample component actually arrives, the start timing of the fractionation is shifted. Also good. Until the separation is started, the drainage valve 95 is opened to collect the eluate in the waste liquid container 96.

  Here, it is assumed that sorting is started from the time indicated by t0 in FIG. That is, at time t0, the fraction control unit 23 moves the dispensing nozzle onto a predetermined container by the driving unit 94, closes the drain valve 95, and opens the dispensing valve 91. As a result, the eluate that has reached the fraction collector 9 via the elution channel 5 is separated into the first container. When the time interval ta elapses, the fractionation control unit 23 moves the dispensing nozzle onto the next predetermined container by the driving unit 94, and dispenses the eluate into the container. Thus, during a period when there is no peak, the eluate is collected in different containers in a predetermined order each time the time interval ta elapses.

  Various components in the sample liquid injected into the mobile phase are separated while passing through the separation column 4 and are eluted from the separation column 4 with a time difference. A part of the eluate is divided by the splitter 6, and the components in the divided eluate are detected by the MS detector 14. The chromatogram creation unit 21 creates a mass chromatogram in real time based on the detection signal from the MS detector 14, and the peak detection unit 22 is the peak start point when the signal intensity of the chromatogram exceeds the threshold LV (time t1). The peak start point detection signal is sent to the fraction control unit 23.

  As soon as the fraction control unit 23 receives the peak start point detection signal, the fraction control unit 23 ends the fractionation performed at that time and proceeds to the next fractionation. That is, the dispensing nozzle is moved onto the next container by the drive unit 94. Therefore, as shown in FIG. 2B, the fractionation period [# 3] immediately before that is shorter than the time interval ta for time fractionation, and the amount of eluate collected in the container at this time is also small. Become. After receiving the peak start point detection signal, the fraction control unit 23 starts time fractionation in which the time interval is changed from ta to tb. That is, every time the time interval tb elapses, the drive unit 94 moves the dispensing nozzle onto the next predetermined container, and dispenses the eluate into the container.

  When time elapses from the peak start detection time and the signal intensity of the chromatogram falls below the threshold LV (time t2), the peak detector 22 determines that it is the peak end point, and the peak end point detection signal is fractionally controlled. Send to part 23. As soon as the peak end point detection signal is received, the fraction control unit 23 ends the fractionation performed at that time and shifts to the next fraction, as in the case of detecting the peak start point. That is, the dispensing nozzle is moved onto the next container by the drive unit 94. Therefore, as shown in FIG. 2 (b), the fraction period [# 6] immediately before that is shorter than the time interval tb of the time fraction during the peak period, and the eluate collected in the container at this time The amount is also reduced.

  After receiving the peak end point detection signal, the fraction control unit 23 executes time fractionation in which the time interval is returned to ta again. However, in the example of FIG. 2A, the peak start point detection signal of the next peak Pb is obtained by the peak detection unit 22 before ta elapses, and the fractionation control unit 23 responds accordingly by the fraction control unit 23. Ends the image and moves to the next fraction. Therefore, the eluate corresponding to a short fractionation period [# 7] from time t2 which is the end point of peak Pa to time t3 which is the start point of peak Pb is fractionated into one container, and the start point of peak Pb The eluate after is detected is dispensed into another container. Since the end point of the peak Pb is detected before tb elapses from the start point of the peak Pb, there is only one eluate corresponding to the fractionation period [# 8] from the start point to the end point of the peak Pb. Collected only in containers. Then, after the end point of the peak Pb is detected, time fractionation for each time interval ta is executed again.

  According to the above fraction control operation, the eluate containing the target component having the peak Pa is fractionated into three containers corresponding to the fraction periods of [# 4], [# 5], and [# 6], respectively. The eluate is free from other components. In addition, the eluate containing the component causing the peak Pb is collected in only one container corresponding to the fractionation period of [# 8], and no other components are mixed in the eluate. Thereby, fractionation of a highly pure component is attained. In addition, the component that caused a small peak so that the signal intensity does not exceed LV is always sorted into one of the containers, so even if a small amount of an unexpected component is contained by the operator, it is not discarded. Can be used for investigation later.

  By executing time fractionation even during a period when there is a peak as described above, the maximum amount of eluate collected in the container can be determined in advance. That is, the maximum amount is determined by the flow rate (flow velocity) of the mobile phase and the time intervals ta and tb. Therefore, it can be avoided that the capacity of the container is insufficient and the eluate overflows. On the other hand, the eluate containing one component may be divided into a plurality of containers, and the number of necessary containers increases.

  Therefore, during the period when the peak is present, that is, during the period from the start point to the end point of one peak, it is possible to control to dispense into one container without performing time fractionation as described above. Good. This example is shown in FIG. That is, from the peak start point to the end point for the peak Pa is one fractionation period [# 4], and all the eluate corresponding to this fractionation period is dispensed into one container. Therefore, if the capacity of the container is insufficient or the peak spread is large and the time from the start point to the end point becomes unexpectedly long, the eluate may overflow without entering the container. It is necessary to pay attention to this point.

  In addition, the peak detector 22 detects a peak top other than the peak start point and end point, and if a peak top is detected as shown in FIG. 2 (d) (time t5) You may make it complete | finish the fraction to a time point and may transfer to the following fraction. Thereby, since the eluate containing the component of peak Pa is collected in two containers, at least the problem of overflow of the eluate as described above is reduced.

  In the above description, the fraction control is performed using the peak appearing in the mass chromatogram created based on the detection signal from the MS detector 14, but it is created based on the detection signal from the PDA detector 13. It is also possible to control fractionation using the peak appearing in the chromatogram.

  Further, the component in the eluate divided by the splitter 6 is not detected, but the component in the eluate before reaching the fraction collector 9 is directly detected and appears in the chromatogram created based on the detection signal. The fraction can be controlled using the peak. That is, the detection signal of the UV detector 8 in FIG. 1 can also be used. However, in that case, the loop tube 7 for adjusting the time is provided between the UV detector 8 and the fraction collector 9.

  In addition, it is obvious that other changes, modifications, and additions within the scope of the present invention are included in the scope of the claims of the present application.

The block diagram of the principal part of the preparative liquid chromatograph apparatus by one Example of this invention. The chromatogram waveform diagram (a) for explaining the fractionation operation | movement in the preparative liquid chromatograph apparatus by a present Example, and a fraction timing figure (b), (c), (d). A chromatogram waveform diagram (a) and a fractionation timing diagram (b) in a general time fractionation operation.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1, 10 ... Mobile phase container 2 ... Liquid feed pump 11 ... Makeup pump 12 ... Detection flow path 13 ... PDA detector 14 ... MS detector 20 ... Data processing part 21 ... Chromatogram preparation part 22 ... Peak detection part 23 ... Fraction control unit 24 ... Fraction condition setting unit 3 ... Injector 4 ... Separation column 5 ... Elution flow path 6 ... Splitter 7 ... Loop tube 8 ... UV detector 9 ... Fraction collector 91 ... Dispensing valve 92 ... Container 93 ... Container Rack 94 ... Drive unit 95 ... Drain valve 96 ... Drain container

Claims (3)

A preparative liquid chromatograph comprising: a column for separating components in a sample; a detector for detecting components in the eluate from the column; and a fraction collector for fractionating the eluate and collecting it in different containers. In the graph device,
a) Peak detection means for detecting at least one of a peak start point and an end point on a chromatogram created based on detection signals sequentially obtained by the detector as the analysis of the target sample proceeds,
b) Time fractionation is executed at one or more preset time intervals during a period when there is no peak, and at least one of the peak start point and end point is detected by the peak detection means Control means for controlling the timing of fractionation by the fraction collector so as to end the fractionation executed in step 1 and shift to the next fractionation,
A preparative liquid chromatograph apparatus comprising: The preparative liquid chromatograph device according to claim 1,
The preparative liquid chromatograph apparatus, wherein the control means controls a fractionation timing by the fraction collector so that a period from a start point to an end point of a certain peak is one fraction. . The preparative liquid chromatograph device according to claim 1,
The control means performs fractionation by the fraction collector so as to execute time fractionation at a time interval different from a period without a peak during a period from the start point to the end point of each peak. A preparative liquid chromatograph characterized by controlling timing.

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