JP2008185558A - Two-dimensional liquid chromatography analysis method and preparative channel switching unit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a two-dimensional liquid chromatography analysis method capable of performing two-dimensional liquid chromatography analysis in a short time, and a preparative channel switching unit usable preferably for the analysis method. <P>SOLUTION: This two-dimensional liquid chromatography analysis method is constituted of the first process for separating and fractionating a sample component, and the second process for performing liquid chromatography analysis of the fractionated component. In the second process, one or a plurality of components fractionated respectively are isolated separately into some of a plurality of holding parts based on information at a rising start time from a base line and a reset finish time to the base line of a peak of each component in a chromatogram in the first process acquired beforehand, and a component in each holding part whose fractionation is completed is subjected successively to the liquid chromatography analysis. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention is used in the case where it is difficult to accurately separate and analyze components in a sample with a single column, and the components separated and fractionated by a liquid chromatography apparatus are separated by liquid chromatography. The present invention relates to a two-dimensional chromatographic analysis method for analysis.

  In liquid chromatography, the liquid sent into the analysis system is used as the mobile phase and the column filler is used as the stationary phase, and a sample containing multiple components is introduced into the analysis system. This is a method for separating and analyzing a plurality of components by utilizing the difference in distribution between phases.

However, for example, samples containing amino acids containing optical isomers and samples such as rat urine cannot accurately separate multiple components contained in a sample with a single column. It takes a long time and it is difficult to perform efficient and accurate analysis.
For this reason, an analysis method is widely adopted in which components separated and fractionated by liquid chromatography are further analyzed using a liquid chromatography apparatus. Hereinafter, this is referred to as a two-dimensional liquid chromatography analysis method.

As a two-dimensional liquid chromatography analysis method, generally, after each component in the sample separated and fractionated by the first-stage liquid chromatography is collected in a lump, the first-stage liquid chromatography is completed. The column can be exchanged to perform the second-stage liquid chromatography analysis, or alternatively, the liquid chromatography analysis can be performed using a second-stage liquid chromatography apparatus different from that used for the first-stage liquid chromatography. Has been done.
However, in any of the above methods, it takes at least an analysis and processing time for combining the first stage liquid chromatography and the second stage liquid chromatography until a series of analysis is completed. The analysis of the sample as described above, which originally requires a long time for liquid chromatography, requires a great deal of time.
In addition, when performing a second-stage liquid chromatography analysis using a single liquid chromatography device with a mobile phase or column being replaced, additional time is required for setting up the replacement of the column in addition to the net analysis time. In addition, there is a problem that the setup operation becomes complicated.

  In view of the above problems, the present applicant has developed a liquid chromatography system capable of automatically performing the above-described two-dimensional liquid chromatography analysis by combining two liquid chromatography apparatuses (for example, Patent Documents). 1).

This liquid chromatography system includes a flow path for transferring a component (sample) to be analyzed (target) flowing out from a first column used in the first liquid chromatography apparatus to a holding unit, The operation of fractionating the components in the sample by switching the flow path for discharging the mobile phase that does not contain the component to the outside of the system, and the liquid chromatography using the second column used in the second liquid chromatography device In order to analyze, an operation of switching a flow path for feeding a mobile phase different from the mobile phase used in the first liquid chromatography apparatus to a holding unit that has been separated and a flow path in an analysis standby state are 1 It is configured to be realized by automatically switching and controlling the six six-way switching valves. The six-way switching valve and its control mechanism are collectively referred to as a column selection unit. For example, a high-pressure switching six-way valve (model number 3011) manufactured by Shiseido can be used.
In addition, this liquid chromatography system employs, for example, a column selection system (C5-2000EMTD) manufactured by Valcoins Instruments, so that each component in the sample separated and fractionated by the first-stage liquid chromatography can be fractionated. can do. This column selection system has a structure in which six loops having a volume of 150 μL are connected and control valves that can be automatically controlled are provided at both ends of each loop.
With this liquid chromatography system, two-dimensional liquid chromatography analysis can be performed substantially automatically and efficiently.
JP 2005-3558 A

  However, since the liquid chromatography system performs liquid chromatography analysis on each separated component after completion of the separation of each component in the sample, two-dimensional liquid chromatography analysis of one sample is performed. From the viewpoint of performing in as short a time as possible, it is not always sufficient.

  The present invention has been made in view of the above problems, and a two-dimensional liquid chromatography analysis method capable of performing two-dimensional liquid chromatography analysis in a short time, and a fractionation that can be suitably used for this analysis method. An object is to provide a flow path switching unit.

The two-dimensional liquid chromatography analysis method according to the present invention includes a first step of separating and fractionating a sample containing a plurality of components by liquid chromatography using a first column, and the fractionated components. A two-dimensional chromatography analysis method comprising a second step of liquid chromatography analysis using a second column,
In the second step, each of the fractionated components based on the information on the start of rising from the baseline of the peak of each component of the chromatogram in the first step obtained in advance and the end of return to the baseline. The components are separately separated into any of a plurality of holding units, and the components of the holding units that have been separated are sequentially subjected to liquid chromatography analysis.

  In the two-dimensional liquid chromatography analysis method according to the present invention, preferably, the baseline of the peak of the previous component of the two components adjacent to each other in the chromatogram obtained in advance in the second step is the baseline. At the end of the return when the time from the end of the return to the start of the rise of the peak of the subsequent component from the baseline exceeds the time required for the liquid chromatography analysis of the previous component obtained in advance, Switching from the flow path for transferring and separating the previous component flowing out from the first column to the flow path for discharging the mobile phase of the first step out of the system, and in the analysis standby state The mobile phase of the second step bypasses the holding unit and is directly fed to the second column, and then the second phase is transferred to the holding unit that has been separated for liquid chromatography analysis on the second column. Second phase mobile phase It forced in, further characterized in that for switching the mobile phase of the second step of flowing out from the holding portion to the flow path fed to said second column at a single hexagonal switching valve.

  In the two-dimensional liquid chromatography analysis method according to the present invention, preferably, the time obtained by subtracting the loss time associated with the switching from the time from the end of the return to the start of the rise exceeds the time required for the analysis. The switching is performed at the end of the return at the time.

The sorting flow path switching unit according to the present invention separates the fractionated components from the outlet side of the first column of the first liquid chromatography device for separating and fractionating a sample containing a plurality of components. A holding part to be taken, and a liquid chromatographic analysis of components separated in the holding part, provided to be connected to three of a second liquid chromatography apparatus having a second column,
For switching the flow path for transferring the components flowing out from the first column to the holding section and for switching the flow path for discharging the mobile phase out of the system, and for liquid chromatography analysis in the second column The mobile phase of the second liquid chromatography device is fed into the holding unit that has been separated, and the flow path for feeding the component flowing out from the holding unit into the second column and the analysis standby A single six-way switching valve for switching the flow path of the mobile phase of the second liquid chromatography device when in a state, bypassing the holding section and directly feeding into the second column;
Control for controlling the switching operation of the six-way switching valve based on the information on the start of rising from the baseline of each component peak of the chromatogram of the first liquid chromatography device obtained in advance and the end of return to the baseline. And
It is characterized by having.

  In the sorting flow path switching unit according to the present invention, preferably, the control unit supplies the peak of the previous component to the baseline of the two components adjacent to each other in the chromatogram obtained in advance. At the end of the return when the time from the end of the return to the start of the rise of the peak of the subsequent component from the baseline exceeds the time required for the liquid chromatography analysis of the previous component obtained in advance, Switching from the flow path for transferring and separating the previous component flowing out from the column to the holding section to the flow path for discharging the mobile phase not containing the component out of the system, and in the analysis standby state, the second The mobile phase of the liquid chromatography device is transferred from the flow path that directly feeds into the second column, bypassing the holding unit, to the holding unit that has been separated for chromatographic analysis on the second column. Second liquid The mobile phase of the chromatography apparatus is fed, and the mobile phase of the second liquid chromatography apparatus flowing out from the holding unit is further switched to the flow path for feeding the second column. The switching operation of the six-way switching valve is controlled.

  In the sorting flow path switching unit according to the present invention, preferably, the control unit subtracts a loss time associated with the switching from a time from the end of the return to the start of the rise. The above switching is performed at the end of the return when the analysis required time is exceeded. The time obtained by subtracting the loss time associated with the switching from the time from the start of the rise to the end of the return is liquid in the second column. The switching operation of the six-way switching valve is controlled so that the switching is performed at the end of the return when the time required for the chromatographic analysis is exceeded.

The two-dimensional liquid chromatography analysis method according to the present invention is a second step of chromatographic analysis, in which the rise of the peak of each component of the chromatogram in the first step obtained in advance from the baseline and the baseline is obtained. Based on the return completion information, each of the fractionated components is separately separated into one of a plurality of holding units, and the components of the holding units that have been separated are sequentially subjected to liquid chromatography analysis. It is possible to shorten the total time for the first-stage preparative treatment and the second-stage analysis in the two-dimensional liquid chromatography analysis as compared with the conventional technique.
The sorting flow path switching unit according to the present invention separates the fractionated components from the outlet side of the first column of the first liquid chromatography device for separating and fractionating a sample containing a plurality of components. A holding part to be removed, and a component separated in the holding part to be subjected to liquid chromatography analysis. Separation was completed for switching the flow path for separating the component flowing out from the column to the holding section and the flow path for discharging the mobile phase out of the system, and for liquid chromatography analysis in the second column The mobile phase of the second liquid chromatography device is sent to the holding unit, and the second liquid chromatography is in the analysis standby state and the flow path for sending the component flowing out from the holding unit to the second column. Equipment A single six-way switching valve that switches the flow phase for directly feeding the mobile phase to the second column, bypassing the holding section, and each component of the chromatogram of the first liquid chromatography device obtained in advance. And a control unit that controls the switching operation of the six-way switching valve based on the information about the start of the peak from the baseline and the end of the return to the baseline, so that the two-dimensional liquid chromatography analysis method according to the present invention is provided. Can be suitably used.

  Embodiments of the present invention will be described below.

First, the two-dimensional liquid chromatography analysis method (two-dimensional liquid chromatography method, two-dimensional liquid chromatography) according to the present embodiment will be described.
The two-dimensional liquid chromatography analysis method according to the present embodiment includes a first step of separating and fractionating a sample containing a plurality of components for each component by liquid chromatography using a first column. And a second step of performing liquid chromatography analysis on each component (sample) using a second column.
In the two-dimensional liquid chromatography analysis method according to the present embodiment, in the second step, the rise of the peak of each component of the chromatogram in the first step obtained in advance from the baseline and the baseline to the baseline are obtained. Based on the return completion information, each of the fractionated components is separately sorted into one of a plurality of holding units, and the components of the holding units that have been sorted are sequentially subjected to liquid chromatography analysis.

  Here, as for the peak information of each component of the chromatogram obtained in advance, the sample obtained in the preliminary test may be used when the separability of each component is good. Similarly to the above, the information of the peak of the standard chromatogram obtained by processing using the apparatus used for processing the sample for the compound containing the standard substance whose content is known is used.

  According to the two-dimensional liquid chromatography analysis method according to the present embodiment, based on the information on the peak of each component in the chromatogram obtained in advance, the first sample for fractionating the information on the peak of each component is collected. Two-dimensional analysis can be performed in parallel for analysis of the components that have been separated in some of the fractionation / sorting time required until the process is completed for all components. It is possible to shorten the total time of the first-stage preparative treatment and the second-stage analysis of the liquid chromatography analysis as compared with the conventional case.

The two-dimensional liquid chromatography analysis method according to the present embodiment will be described in more detail.
The two-dimensional liquid chromatography analysis method according to the present embodiment is performed using, for example, the chromatography analysis system shown in FIG.
The chromatographic analysis system shown in FIG. 1 includes a first mobile phase supply unit 10, a first pump 12 that sucks and delivers the first mobile phase of the first mobile phase supply unit 10, and injects a sample. A sample injection section 14, a first column 16, a column oven 17 in which the first column 16 is disposed, and a first detector 18 for detecting components separated and fractionated by the first column 16. And a first recorder 20 for recording detection data, and a first liquid chromatography apparatus for separating and fractionating a sample. However, in the present embodiment, the first detector 18 and the first recorder 20 are omitted in performing the operation of separating and fractionating the sample except when collecting standard chromatogram data. be able to.
The chromatographic analysis system also includes a second mobile phase supply unit 22, a second pump 24 that sucks and delivers the second mobile phase of the second mobile phase supply unit 22, and a second column 26. A column oven 27 in which the second column 26 is arranged, a second detector 28 for detecting the components separated and fractionated in the second column 26, and a second recorder 30 for recording the detection data. And a second liquid chromatography apparatus for liquid chromatography of fractionated components in the sample.

  The chromatography analysis system further includes a column selection unit (sorting channel switching unit) 32 and a multi-loop unit (plural holding units) 34 according to the present embodiment between the two liquid chromatography devices. Have.

The multi-loop unit 34 will be described first. As shown in a detailed configuration in FIG. 2, the multi-loop unit 34 includes, for example, six loops (holding units) 36, and the selected loop 36 is connected to the column selection unit 32. It has switching means 37a and 37b which connect and form a flow path.
Thereby, one fractionated component or a plurality of continuous components can be individually held in the loop 36. The loop 36 serves as the sample injection unit 14 in the first liquid chromatography apparatus.

Next, the column selection unit 32 controls the operation of the single six-way switching valve 35 and the six-way switching valve 35 that switch the fluid flow path between the three liquid chromatography devices and the multi-loop unit 34. The control unit 38 is configured. The control unit 38 also controls the operations of the switching units 37a and 37b. However, the present invention is not limited to this. For example, the operation of the switching units 37a and 37b may be controlled by a main control unit described later.
In FIG. 1, the hexagonal switching valve 35 forms a flow path (solid line portion of the hexagonal switching valve 36) for transferring the components flowing out from the first column to the loop and sorting them, and also in the analysis standby state. The mobile phase of the second liquid chromatography apparatus is in a preparative mode in which a flow path (solid line portion of the six-way switching valve 35) is formed that bypasses the loop and is directly fed to the second column.
On the other hand, in FIG. 3, the six-way switching valve 35 has a flow path (solid line part of the six-way switching valve 35) that discharges the mobile phase that flows out of the first column and does not contain the components in the sample. A flow path (hexagonal) that feeds the mobile phase of the second liquid chromatography device into the loop (holding section) that has been formed and that has been separated, and further feeds the components flowing out of the loop into the second column A solid line portion of the switching valve 35) is in an analysis mode.

The control unit 38 determines whether the six-way switching valve 35 is based on information obtained when the peak of each component of the chromatogram of the first liquid chromatography device obtained in advance is started from the baseline and when the peak is returned to the baseline. The switching operation is controlled. That is, for example, ideally, the time between the start of rising of the peak of each component from the baseline and the end of return to the baseline is the above-described sorting mode, while the two adjacent components are The time between the end of the return to the baseline of the peak and the start of the rise from the baseline is the analysis mode. However, in this case, depending on the sorting and analysis conditions, the time from the end of the return of the peak of two adjacent components to the baseline to the start of rising from the baseline may not be balanced with the analysis time. And the processing of either one of the analyzes can be disrupted.
Therefore, the control unit 38 preferably restores the peak of the previous component to the baseline of the two component peaks adjacent to each other in the chromatogram obtained in advance by the first liquid chromatography device obtained in advance. The time from the end of the peak of the subsequent component to the start of the rise from the baseline is the time required for liquid chromatography analysis of the previous component obtained in advance on the second column (required analysis time) At the end of the peak recovery of the previous component when exceeding, the component flowing out from the first column is transferred to the holding unit and separated from the flow channel to the flow channel for discharging the sample not containing the above component In the analysis standby state, a liquid phase that is different from the mobile phase of the first liquid chromatography device bypasses the holding unit and is directly fed to the second column from the flow path. The other mobile phase is sent to the holding section where the fractionation has been completed for tomographic analysis, and further, the mobile phase flowing out from the holding section is switched to the flow path for sending it to the second column. In addition, the switching operation of the six-way switching valve 35 is controlled.
More preferably, the control unit 38 is configured so that the time obtained by subtracting the loss time associated with the switching from the time from the start of rising to the baseline to the end of return to the baseline exceeds the time required for analysis. The switching operation of the six-way switching valve 35 is controlled so as to perform the switching at the end of the return. Here, the loss time associated with switching is mainly the residence time of the sample in the pipe and the six-way switching valve 35 until the sample sent from the first column reaches the holding unit, but is necessary. Depending on the situation, all loss times associated with switching operations such as analysis preparation time may be taken into consideration.

  In the two-dimensional liquid chromatography analysis method according to the present embodiment, for example, the main control unit (not shown) of the chromatography analysis system performs the switching operation of the six-way switching valve, the first liquid chromatography device, and the second liquid. By linking the specified operation of the chromatography device, at least a part of the sample can be collected within the time to collect all the samples without interfering with the separation process and the analysis process. The total time of the preparative processing and analysis can be shortened compared with the conventional one.

  Examples in which two-dimensional liquid chromatography analysis was performed using the above-described chromatography analysis system will be described below.

A first liquid chromatography apparatus having the following specifications was used, and the following operating conditions were used.
As the mobile phase, THF (tetrahydrofuran) / TFA (trifluoroacetic acid) /water=25/0.05/75 (v / v) was used. The pump (mobile phase feed pump) was an inert pump (NANOSPACE SI-2 3001), and the flow rate was 75 &micro; L / min. As the sample injector, an HTS autosampler (NANOSPACE SI-2 3033) was used. The sample used was NBD of valine (Val), alloisoleucine (allo-Ile), isoleucine (Ile), leucine (Leu) and phenylalanine (Phe). Here, NBD conversion refers to one of fluorescent derivatization methods in which NBD-F (4-fluoro-7-nitrobenzofurazane) is reacted with an amino acid to increase fluorescence sensitivity. A column oven (column thermostat) (not shown) was held at 40 ° C. using NANOSPACE SI-2 3004. As the first column, a reversed-phase microcolumn (Capcell pak C18 MG II manufactured by Shiseido Co., Ltd., 1 mm id × 150 mm) was used. As the detector, a fluorescence detector (NANOSPACE SI-2 3013 ex. 470 nm, em 530 nm) was used.
The second liquid chromatograph was used with the following specifications, and with the following operating conditions.
As the mobile phase, acetonitrile / methanol = 50/50 (v / v) containing 10 mM citric acid was used. The pump (mobile phase feed pump) used an inert pump (NANOSPACE SI-2 3001), and the flow rate was 75 μL / min. As the sample injector, an HTS autosampler (NANOSPACE SI-2 3033) was used. A column oven (column thermostat) (not shown) was held at 40 ° C. using NANOSPACE SI-2 3004. The second column was a chiral column (QN-1-AX 4 mm id X 150 mm, manufactured by Chiral Technologies Europe). As the detector, a fluorescence detector (NANOSPACE SI-2 3013 ex. 470 nm, em 530 nm) was used.

FIG. 4 shows a time table of the operation of the column selection unit (sorting flow path switching unit) of the embodiment that advances the sorting and analysis.
In FIG. 4, HPV in the source section indicates the above-described hexagonal switching valve, and VICI indicates the switching means of the multi-loop unit. Also, START indicates the start of analysis when sequentially analyzing samples containing each component that has been sequentially collected. Further, PosA in the section of Value indicates that HPV has a flow path configuration in the analysis mode, and PosB indicates that HPV has a flow path configuration in the sorting mode. In addition, Pos02, Pos03,... Indicate that the switching unit sequentially switches the holding unit holding the sample to the second loop and the third loop and is in a selected state. In FIG. 1, the initial condition for selecting the first loop holding valine (Val) described below is omitted. The time required for analysis of valine is confirmed to be 10 minutes or less in a chromatogram obtained in advance, for example, in FIG.
The initial condition of the system, that is, the valine is entered after 25 minutes and 40 seconds from the start of the fractionation operation by entering the fractionation mode in which the valine is fractionated from the beginning of the rise of the valin (Val) peak from the baseline The preparative mode is continued until the return of the peak to the baseline is completed. As described above, the time required for analysis of valine is sufficiently shorter than 25 minutes and 40 seconds. Next, the valine analysis mode is entered at 28 minutes and 40 seconds after the switching operation loss time has elapsed. Next, after the end of the valine analysis, 43 minutes and 40 seconds have elapsed since the start of the sorting operation, that is, after 18 minutes of sorting time has elapsed, the sorting mode is returned again. As described above, this fractionation time is sufficiently longer than the time required for analysis of valine. In this way, basically, these switching operations are repeated according to the components that are sequentially collected. However, in the examples, each component other than valine (Val) does not shift to analysis immediately after sorting because the time from the start of the rise from the baseline to the end of the return to the baseline is very short. In addition, the sorting is continued, and at this time, a plurality of components are held in different loops. Then, the plurality of components are collectively analyzed. In addition, allo-leucine (allo-Ile) is retained in a loop that is empty after the analysis of valine (Val) is completed in order to use the loop effectively.

  FIG. 5 shows a standard chromatogram obtained in advance in the first column of the chromatographic analysis system. Moreover, the chromatogram of each component obtained by a 2nd column is shown in FIG. In FIG. 6, D-form and L-form of optical isomers of valine (Val), alloisoleucine (allo-Ile), isoleucine (Ile), leucine (Leu) and phenylalanine (Phe) were separated in order from the left. It is a chromatogram.

According to the embodiment, after all the components have been separated in 59 minutes from the start of the preparative operation, the analysis of all the components has been completed by 99 minutes. Compared to the method in which each component is sequentially analyzed after the completion of the fractionation, the total time for fractionation and analysis is about 10 minutes, which corresponds to the time required for analysis of valine.
At this time, as is apparent from FIG. 6, the analysis accuracy of each component is the same as that of the conventional method.

It is a figure which shows schematic structure of the chromatography analysis system used for the two-dimensional liquid chromatography analysis method which concerns on this Embodiment. It is for demonstrating the detailed structure of the multi-loop unit of FIG. 1, (a) shows a schematic diagram, (b) shows a front view, respectively. It is a figure which shows schematic structure of the chromatography analysis system for demonstrating the mode different from FIG. 1 of the six-way switching valve of FIG. It is a figure which shows the time table of operation | movement of the column selection unit of an Example. It is a graph which shows the example of the chromatogram obtained by the 1st column in an Example. It is a graph which shows the chromatogram of each component obtained by the 2nd column in an Example.

Explanation of symbols

10 First mobile phase supply unit 12 First pump 12
DESCRIPTION OF SYMBOLS 14 Sample injection part 16 1st column 17, 27 Column oven 18 1st detector 20 1st recorder 22 2nd mobile phase supply part 24 2nd pump 26 2nd column 28 2nd detector 30 Second recorder 32 Column selection unit 34 Multi-loop unit 35 Six-way switching valve 36 Loop 37a, 37b Switching means 38 Control unit

Claims (6)

A first step of separating and fractionating a sample containing a plurality of components by liquid chromatography using a first column, and a liquid chromatography analysis of each fractionated component using a second column A two-dimensional chromatographic analysis method comprising two steps,
In the second step, each of the fractionated components based on the information on the start of rising from the baseline of the peak of each component of the chromatogram in the first step obtained in advance and the end of return to the baseline. A two-dimensional liquid chromatography analysis method, wherein components are separately separated into any of a plurality of holding units, and components of the holding units that have been separated are sequentially subjected to liquid chromatography analysis.   In the second step, the rising of the peak of the subsequent component from the baseline begins after the return to the baseline of the peak of the previous component of the two components adjacent to each other in the chromatogram obtained in advance in the second step. When the time until the time exceeds the time required for liquid chromatography analysis of the previous component obtained in advance, the previous component flowing out from the first column is transferred to the holding unit at the end of the return. Switching from the flow path for sorting to the flow path for discharging the mobile phase of the first step to the outside of the system, and bypassing the holding portion for the mobile phase of the second step in the analysis standby state The mobile phase of the second step is sent from the flow path directly sent to the column to the holding section where the fractionation has been completed for liquid chromatography analysis in the second column, and further, the outflow from the holding section The mobile phase of the second step 2 dimensional liquid chromatographic analysis method according to claim 1, characterized in that the switching to the flow path fed to the arm by a single hexagonal switching valve.   The switching is performed at the end of the return when the time obtained by subtracting the loss time associated with the switch from the time from the end of the return to the start of the rise exceeds the time required for the analysis. Item 2. The two-dimensional liquid chromatography analysis method according to Item 2. The outlet side of the first column of the first liquid chromatography device for separating and fractionating a sample containing a plurality of components, the holding part for fractionating the fractionated components, and the components fractionated in the holding part Is connected to three of the second liquid chromatography apparatus having a second column for liquid chromatography analysis,
For switching the flow path for transferring the components flowing out from the first column to the holding section and for switching the flow path for discharging the mobile phase out of the system, and for liquid chromatography analysis in the second column The mobile phase of the second liquid chromatography device is fed into the holding unit that has been separated, and the flow path for feeding the component flowing out from the holding unit into the second column and the analysis standby A single six-way switching valve for switching the flow path of the mobile phase of the second liquid chromatography device when in a state, bypassing the holding section and directly feeding into the second column;
Control for controlling the switching operation of the six-way switching valve based on the information on the start of rising from the baseline of each component peak of the chromatogram of the first liquid chromatography device obtained in advance and the end of return to the baseline. And
A flow path switching unit for sorting, comprising:   From the end of returning to the baseline of the peak of the previous component of the two adjacent peaks in the chromatogram obtained in advance from the start of rising of the peak of the subsequent component from the baseline At the end of the return when the time required for liquid chromatography analysis of the previous component obtained in advance exceeds the time required for liquid chromatography analysis, the previous component flowing out from the first column is transferred to the holding unit and fractionated. Switching to a flow path for discharging the mobile phase not containing the component from the flow path to the outside of the system, and the mobile phase of the second liquid chromatography device in the analysis standby state, bypassing the holding unit, the second The mobile phase of the second liquid chromatography apparatus is sent from the flow path directly sent to the column to the holding unit where the fractionation has been completed for chromatographic analysis on the second column, and further the holding Part 5. The switching operation of the six-way switching valve is controlled so as to switch to a flow path for feeding the mobile phase of the second liquid chromatography device flowing out to the second column. The flow path switching unit for sorting as described.   The control unit performs the switching at the end of the return when the time obtained by subtracting the loss time associated with the switching from the time from the end of the return to the start of the rise exceeds the time required for the analysis. At the end of the return when the time obtained by subtracting the loss time associated with the switching from the time from the start of the rise to the end of the return exceeds the time required for the liquid chromatography analysis in the second column. 6. The sorting flow path switching unit according to claim 5, wherein a switching operation of the hexagonal switching valve is controlled so as to perform the switching.

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