JP2011052973A - High-speed liquid chromatograph and method of recycle analysis using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide high-speed liquid chromatograph allowing recycling with only a part of components of a specimen taken as an object without performing a separate recovery operation. <P>SOLUTION: A column switch mechanism is incorporated into this high-speed liquid chromatograph used for performing recycle analysis to switch recycling object components to a second column. Thereafter, the object components switched to the second column are passed through the second column a plurality of times by using a second moving phase. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a high performance liquid chromatograph (hereinafter also referred to as HPLC) having a column switch mechanism and a recycling mechanism, and a recycling analysis method using the apparatus.

  Recycle analysis of HPLC is used in the fields of organic purification and analytical chemistry because it can accurately isolate sample components that are difficult to isolate. The HPLC recycle analysis is performed as follows.

  FIG. 13 is an explanatory diagram showing an example of a conventional HPLC 500 having a recycling mechanism. In FIG. 13, reference numeral 501 denotes a mobile phase storage tank, and the internal mobile phase 503 is sucked by a liquid feed pump 511 via a pipe 505, a flow path switching valve 507, and a pipe 509. The ports 507a and 507b of the flow path switching valve 507 are always circulated. The mobile phase 503 is sent to the sample introduction device 513 by the liquid feed pump 511. The sample introduced from the sample introduction device 513 is sent to the column 515 together with the mobile phase 503. Each component in the sample is eluted from the column 515 at different rates due to the difference in partition coefficient between the mobile phase 503 and the stationary phase of the packing material packed in the column 515. After leaving the column 515, the mobile phase 503 containing each component eluted according to the distribution coefficient is sent to the detector 517, where each component is detected. The mobile phase 503 containing each component detected by the detector 517 is collected or recycled via the pipe 519.

  When recovering the mobile phase containing each component, by switching the flow path of the flow path switching valve 507 to the flow path 508 indicated by the dotted line, the flow between the pipe 519 and the pipe 521 is enabled, The flow of the flow path 510 indicated by the solid line between the pipe 519 and the pipe 509 is blocked. Thereby, the mobile phase containing each component is sent from the pipe 519 to the pipe 521, and the mobile phase containing each component is recovered in the recovery tank 523 as the recovery liquid 525. The recovered liquid 525 is usually a waste liquid, or the eluted components may be collected for each holding time by using a fraction collector (not shown) or the like.

  When the mobile phase 503 containing each component is recycled, the pipe 519 and the pipe 509 are connected by a flow path 510 indicated by a solid line by switching the flow path switching valve 507, so that the pipe 519 and Distribution with the pipe 509 is possible. On the other hand, the flow between the pipe 519 and the pipe 521 is blocked. As a result, the mobile phase containing each component is sent to the pipe 509 by the valve 507 and again introduced into the column 515, and the separation between the components is repeated.

  Recycling analysis is effective in increasing the degree of separation of each component in a sample. FIG. 7 is a chromatogram showing an example of the result of the conventional recycling analysis. One peak (containing two components) appearing at a retention time of about 3 minutes is split into two when recycling is repeated, and each component can be isolated.

  In the recycling analysis, the peak of each component appears repeatedly for each holding time. For example, when a component analysis having a retention time of about 3 minutes is subjected to recycling analysis, the second round peak appears at about 6 minutes and the third round peak appears at about 9 minutes.

  FIG. 8 is a chromatogram showing an example of the HPLC analysis result of a certain sample. In this chromatogram, a peak group having a retention time of about 3 minutes and a peak group having a retention time of about 6 minutes appear.

  FIG. 9 is a chromatogram showing an analysis result obtained by performing the recycling analysis of the sample of FIG. 8 for about 6 minutes. In this chromatogram, the second peak group of the peak group having a retention time of about 3 minutes overlaps the first peak group of the peak group having a retention time of about 6 minutes. In the case of such a sample, since components having different retention times are mixed by recycling, a desired peak cannot be separated. In order to solve this problem, only the target component for recycling is separated and recovered, and then the recovered target component is separately recycled. In the case of this method, the operation of collecting the components to be recycled becomes complicated. Moreover, the collection | recovery omission of the component for recycling may be produced, and the amount of component collection is reduced.

JP 05-223803 A

  The present invention has been made in view of the above circumstances, and an object of the present invention is to enable recycle analysis for only some components in a sample without performing a separate recovery operation.

  As a result of intensive studies to solve the above problems, the present inventor has found that the above problems can be solved by incorporating a column switch mechanism in HPLC for performing recycle analysis, and the present invention has been completed. The present invention for solving the above problems is described below.

[1]
From one end side to the other end side of the flow path of the first mobile phase, sequentially the first liquid feed pump, the sample introduction device, the first column, the first flow path switching valve group, the first detector,
While interposing
By switching the flow path switching valve of the first flow path switching valve group, the first mobile phase flow path is switched to a flow path having a second column interposed between the first column and the first detector. A flow path for one mobile phase;
From one end side to the other end side of the flow path of the second mobile phase, sequentially a second flow path switching valve group, a second liquid feeding pump, a first flow path switching valve group, a second detector, A two-channel switching valve group;
While interposing
By switching the flow path switching valve of the first flow path switching valve group, in the flow path of the second mobile phase, the flow path that interposes the second column between the second liquid feeding pump and the second detector. A flow path for the second mobile phase to be switched;
Have
The first flow path switching valve group includes a flow path switching valve that switches the mobile phase passing through the second column to either the first mobile phase or the second mobile phase, and the first flow path switching valve group includes: At the same time, each valve switches,
and,
By switching the flow path switching valve of the second flow path switching valve group, a second flow path switching valve group, a second liquid feed pump, a second column, a second detector, and a second flow path switching valve A high-performance liquid chromatograph comprising a mechanism for switching to a closed flow path circulating through a group.

[2]
From one end side to the other end side of the flow path of the first mobile phase, sequentially the first liquid feed pump, the sample introduction device, the first flow path switching valve group, the first detector,
While interposing
By switching the flow path switching valve of the first flow path switching valve group, the first mobile phase flow path is switched to a flow path having a first column interposed between the sample introduction device and the first detector. A flow path for one mobile phase;
From one end side to the other end side of the flow path of the second mobile phase, the second flow path switching valve group, the second liquid feeding pump, the first flow path switching valve group, the second column, A detector, a second flow path switching valve group,
While interposing
By switching the flow path switching valve of the first flow path switching valve group, the flow path of the second mobile phase is switched to the flow path that interposes the first column between the second liquid feeding pump and the second column. A flow path for the second mobile phase;
Have
The first flow path switching valve group includes a flow path switching valve that switches the mobile phase passing through the first column to either the first mobile phase or the second mobile phase, and the first flow path switching valve group is At the same time, each valve switches,
and,
By switching the flow path switching valve of the second flow path switching valve group, a second flow path switching valve group, a second liquid feeding pump, a first flow path switching valve group, a second column, and a second detector And a mechanism for switching to a closed flow path that circulates through the second flow path switching valve group.

  The invention described in [1] or [2] includes the following inventions.

[3]
The high-performance liquid chromatograph according to [1] or [2], wherein a sample introduction device is interposed between the flow path between the second liquid feeding pump and the second column.

[4]
The high-performance liquid chromatograph according to [1] or [2], wherein the first flow path switching valve group includes one valve having five or more openings.

[5]
The high-performance liquid chromatograph according to [1] or [2], wherein the second flow path switching valve group includes one valve having four or more openings.

[6]
Using the high-performance liquid chromatograph according to [1] or [2], after introducing a target component for recycling into the second column, a plurality of target components for recycling introduced into the second column using the second mobile phase. Recycle analysis method leading to the second column.

  If the HPLC of the present invention is used, it is possible to isolate even a sample that has been mixed with retention times during recycling. Therefore, it is suitable for separation and purification of various substances.

It is explanatory drawing which shows an example of HPLC of this invention. (A) And (b) is explanatory drawing which shows the flow path of the 1st mobile phase in HPLC of FIG. (A)-(c) is explanatory drawing which shows the flow path of the 2nd mobile phase in HPLC of FIG. It is a chromatogram which shows an example of the HPLC analysis result of a certain sample. FIG. 5 is a chromatogram showing a result of HPLC analysis performed on the sample of FIG. 4 using a column switch and detection by a first detector. FIG. It is a chromatogram which shows the result detected by the 2nd detector which performed the column switch for the recycle analysis of the sample of FIG. It is a chromatogram which shows an example of the recycle analysis result of a certain sample. It is a chromatogram which shows an example of the HPLC analysis result of a certain sample. It is a chromatogram which shows the HPLC analysis result which recycled the sample of FIG. It is explanatory drawing which shows the other example of HPLC of this invention. (A) And (b) is explanatory drawing which shows the flow path of the 1st mobile phase in HPLC of FIG. (A)-(c) is explanatory drawing which shows the flow path of the 2nd mobile phase in HPLC of FIG. It is explanatory drawing which shows an example of the conventional HPLC provided with the recycling mechanism.

  Hereinafter, the HPLC of the present invention and the analysis method using the apparatus will be described with reference to two embodiments.

<First embodiment>
First, a high performance liquid chromatograph (hereinafter, also simply referred to as HPLC of the present invention) having a column switch mechanism and a recycling mechanism of the present invention will be described.

  FIG. 1 is an explanatory diagram showing an example of the HPLC of the present invention. This HPLC flow path has a flow path through which the first mobile phase 13 flows and a flow path through which the second mobile phase 35 flows. The flow paths are related. The flow path through which the first mobile phase 13 flows is from the first mobile phase storage tank 11 toward the recovery tank 57 in order, the pipe 15, the first liquid feeding pump 17, the sample introduction device 19, the first column 21, The first flow path switching valve 23, a pipe 25, a first detector 27, and a pipe 28 are configured. By switching the first flow path switching valve 23, in the flow path in which the first mobile phase 13 flows, between the first column 21 and the pipe 25, the pipe 29 and the second flow path are connected via the first flow path switching valve 23. The two columns 31 and the pipe 55 are configured to be switched to a flow path interposed.

  In addition, the flow path through which the second mobile phase 35 flows is from the second mobile phase storage tank 33 toward the recovery tank 57, in order, a pipe 37, a second flow path switching valve 39, a pipe 41, and a second liquid feed pump. 43, a pipe 45, a first flow path switching valve 23, a pipe 47, a second detector 49, a pipe 51, a second flow path switching valve 39, and a pipe 53. By switching the first flow path switching valve 23, in the flow path through which the second mobile phase 35 flows, the piping 29 and the second column 31 are interposed between the piping 45 and the piping 47 via the first flow path switching valve 23. And it is comprised so that it can switch to the flow path in which the piping 55 is interposed. Further, by switching the second flow path switching valve 39, the pipe 51 and the pipe 41 can be circulated, and the circulation between the pipe 51 and the pipe 53 can be interrupted to be recycled. Yes.

  2 (a) and 2 (b) are explanatory diagrams showing the flow path of the first mobile phase in the HPLC of FIG. Of the flow paths in FIGS. 2 (a) and 2 (b), the flow path shown by a solid line indicates the flow path of the first mobile phase, and this flow path is used in the case of analysis without (1) column switch described later. To do.

  In FIG. 2A, reference numeral 11 denotes a first mobile phase storage tank, and an internal first mobile phase 13 is sucked by a first liquid feed pump 17 via a pipe 15. The first mobile phase 13 is sent to the sample introduction device 19 by the first liquid feeding pump 17. The first mobile phase 13 is sent to the inlet of the first column 21 together with the sample introduced from the sample introduction device 19. The first mobile phase 13 exiting from the outlet of the first column 21 is sent to the first detector 27 through the ports 23 a and 23 f of the first flow path switching valve 23. The mobile phase 13 that has exited the first detector 27 is sent to the recovery tank 57 via the pipe 28 and becomes the recovery liquid 59.

  FIG. 2B shows the flow path of the first mobile phase when the first flow path switching valve 23 of FIG. The first mobile phase 13 exiting from the outlet of the first column 21 passes through the ports 23 a and 23 b of the first flow path switching valve 23, and is sent to the inlet of the second column 31 via the pipe 29. The first mobile phase 13 exiting from the outlet of the second column 31 passes through the pipe 55, passes through the ports 23 e and 23 f of the first flow path switching valve 23, passes through the pipe 25, and passes through the first detector 27. Sent. The mobile phase 13 that has exited the first detector 27 is sent to the recovery tank 57 via the pipe 28 and becomes the recovery liquid 59.

  FIGS. 3A to 3C are explanatory views showing the flow path of the second mobile phase in the HPLC of FIG. Among the channels in FIGS. 3A to 3C, the channel indicated by the solid line indicates the channel of the second mobile phase.

  In FIG. 3A, the second mobile phase 35 sucked by the second liquid feeding pump 43 is sent to the second flow path switching valve 39 via the pipe 37. The ports 39a and 39d of the second flow path switching valve 39 can always flow. The second mobile phase 35 is sent to the first flow path switching valve 23 via the pipe 41, the second liquid feed pump 43, and the pipe 45. The first flow path switching valve 23 can circulate through ports 23c and 23b. Thereafter, the second mobile phase 35 is sent to the inlet of the second column 31 via the pipe 29. The second mobile phase 35 exiting from the outlet of the second column is sent to the first flow path switching valve 23 via the pipe 55. The first flow path switching valve 23 can circulate through ports 23e and 23d. The second mobile phase 35 exiting the first flow path switching valve 23 is sent to the second detector 49 via the pipe 47. After that, it is sent to the second flow path switching valve 39 through the pipe 51. Ports 39c and 39b can flow through the second flow path switching valve 39. The second mobile phase 35 exiting the second flow path switching valve 39 is sent to the recovery tank 57 via the pipe 53 and becomes the recovery liquid 59.

  FIG. 3B shows the flow path of the second mobile phase when the first flow path switching valve 23 of FIG. The first flow path switching valve 23 can circulate through ports 23c and 23d. The second mobile phase 35 exiting the first flow path switching valve 23 is sent to the second detector 49 via the pipe 47. The second mobile phase 35 exiting the second detector 49 is sent to the second flow path switching valve 39 via the pipe 51. When the ports 39c and 39d of the second flow path switching valve 39 are allowed to flow (the flow path in FIG. 3b), a recycle flow path is configured. That is, from the pipe 41, the second liquid feeding pump 43, the pipe 45, the first flow path switching valve 23, the pipe 47, the second detector 49, the pipe 51, and the second flow path switching valve 39. A closed flow path is formed. On the other hand, when the ports 39c and 39b of the second flow path switching valve 39 can be circulated (not shown), the second mobile phase 35 is sent to the recovery tank 57 via the pipe 53, and the recovered liquid 59 and Become.

  FIG. 3C shows a flow path of the second mobile phase when the second flow path switching valve 39 of FIG. Ports 39c and 39d can flow through the second flow path switching valve 39. Thereby, the piping 41, the 2nd liquid feeding pump 43, the piping 45, the 1st flow path switching valve 23, the piping 29, the 2nd column 31, the piping 55, the 1st flow path switching valve 23, and piping 47, a closed recycle flow path including the second detector 49, the pipe 51, and the second flow path switching valve 39 is formed.

  When the first flow path switching valve 23 can flow between the ports 23a and 23f, between the ports 23e and 23d, and between the ports 23c and 23b, between the ports 23f and 23e, Distribution between the ports 23d and 23c and between the ports 23b and 23a is blocked. Similarly, when the ports 23f and 23e, the ports 23d and 23c, and the ports 23b and 23a can be circulated, the ports 23a and 23f, the ports 23e and 23d, During this time, the flow between the ports 23c and 23b is blocked. That is, when the flow path of the first mobile phase is the flow path of FIG. 2 (a), the flow path of the second mobile phase is the flow path of FIG. 3 (a) or 3 (c). When the flow path of the first mobile phase is the flow path of FIG. 2B, the flow path of the second mobile phase is the flow path of FIG.

  Next, an analysis method using the HPLC of the present invention will be described.

  The HPLC of the present invention can perform (1) analysis without column switch, (2) analysis to perform column switch for specific components, and (3) analysis to recycle specific components of (2) above. The above (1)-(3) performed using the HPLC of the present invention will be described below.

(1) Analysis without the column switch The analysis without the column switch is performed using the first mobile phase. At this time, the flow path of the first mobile phase is a flow path indicated by a solid line in FIG. The sample introduced from the sample introduction device 19 is sent to the first column 21 together with the first mobile phase 13. In the first column 21, the sample is held in a stationary phase. Thereafter, the first mobile phase 13 continues to be supplied. In the first column 21, each component in the sample is eluted from the stationary phase in the first column 21 at a different rate due to the difference in the distribution coefficient between the mobile phase and the stationary phase. The first mobile phase 13 to which each component is distributed exits the column 21 and is then sent to the first detector 27 via the first flow path switching valve 23. The first mobile phase 13 containing each component detected by the first detector 27 is sent to the recovery tank 57 via the pipe 28, and the recovery liquid 59 is recovered. The recovered liquid 59 is used as a waste liquid or is collected every holding time by a fraction collector (not shown). At this time, the flow path of the second mobile phase 35 is the flow path of FIG. 3A or the flow path of FIG. In this analysis, since the second mobile phase 35 is not used, it is not necessary to distribute it.

  The analysis (1) in which the column switch is not performed is used when a normal analysis is performed or when the elution time of each component is measured in advance before performing the analysis for recycling.

(2) Analysis for performing column switch on specific component First, the analysis of (1) above is performed in advance to confirm the retention time of the specific component (target component of the column switch). Next, the sample is introduced from the sample introduction device 19 through the flow path indicated by the solid line in FIG. The switching operation of the first flow path switching valve 23 is performed with reference to the retention time of the specific component obtained in the analysis (1) in which the column switch is not performed. Components other than the specific component (components having a shorter retention time than the specific component) are sent to the collection tank 57 through the flow path indicated by the solid line in FIG. At this time, the second mobile phase 35 flows through the flow path indicated by the solid line in FIG. 3 (a) or FIG. 3 (c).

  Immediately before the specific component elutes from the first column 21, the first flow path switching valve 23 is switched so that the flow path of the first mobile phase is the flow path indicated by the solid line in FIG. As a result, the specific component is sent to the second column 31 and held in the second column 31. At this time, the second mobile phase 35 flows through the flow path shown by the solid line in FIG.

  When elution of the specific component from the first column 21 is completed, the first flow path switching valve 23 is switched to obtain a flow path indicated by a solid line in FIG. As a result, components other than the specific component (components having a longer retention time than the specific component) are sent to the collection tank 57. At this time, the second mobile phase 35 flows through the flow path shown by the solid line in FIG. 3A, and the specific component is eluted from the second column 31 by the second mobile phase 35. The specific component eluted from the second column 31 is detected by the second detector 49. The second mobile phase that has exited the second detector 49 passes through the second flow path switching valve 39 and is sent to the recovery tank 57 to be a recovery liquid 59.

  In this case, the specific component (consisting of a plurality of components) is separated into each component in the second column 31, and further recycling is not necessary.

(3) Analysis for Recycling Specific Component in (2) When eluting the specific component stocked in the second column 31 in (2), the second flow path switching valve 39 is switched to connect the piping 51 and the piping. 41 is allowed to flow, and the flow path is indicated by the solid line in FIG. Thereby, the piping 41, the 2nd liquid feeding pump 43, the piping 45, the 1st flow path switching valve 23, the piping 29, the 2nd column 31, the piping 55, the 1st flow path switching valve 23, and piping 47, a closed recycle flow path including the second detector 49, the pipe 51, and the second flow path switching valve 39 is formed.

  With reference to the output result of the second detector 49, when each component constituting the specific component is sufficiently separated and no recycling is required, the second flow path switching valve 39 is switched to switch the FIG. The flow path. As a result, the second mobile phase 35 from which each component has been eluted is sent to the recovery tank 57 via the pipe 53 and becomes the recovery liquid 59.

<Second embodiment>
First, another embodiment of the high performance liquid chromatograph (hereinafter also simply referred to as the HPLC of the present invention) having a column switch mechanism and a recycling mechanism of the present invention will be described.

  FIG. 10 is an explanatory view showing another example of the HPLC of the present invention. In the flow path of the first mobile phase 113, the pipe 115, the first liquid feed pump 117, the pipe 119, the sample introduction device 121, and the pipe 123 are sequentially directed from the first mobile phase storage tank 111 to the recovery tank 149. The first flow path switching valve 125, the pipe 133, the first detector 135, and the pipe 137 are interposed. By switching the first flow path switching valve 125, in the flow path of the first mobile phase 113, the first flow path switching valve 125, the pipe 127, the first column 129, between the pipe 123 and the pipe 133, The flow path is configured to be switched to a flow path that interposes the pipe 131 and the first flow path switching valve 125.

  Further, in the flow path of the second mobile phase 155, the pipe 157, the second flow path switching valve 159, the pipe 161, and the second liquid feed pump 163 are sequentially moved from the second mobile phase storage tank 153 toward the recovery tank 149. , Piping 165, first flow path switching valve 125, piping 167, second column 169, piping 171, second detector 173, piping 175, second flow path switching valve 159, piping 177 And is intervening. By switching the first flow path switching valve 125, in the flow path of the second mobile phase 155, the first flow path switching valve 125, the pipe 127, the first column 129, and the pipe are arranged between the pipe 165 and the pipe 167. 131 and the first flow path switching valve 125 are configured to be switched to a flow path. Further, by switching the second flow path switching valve 159, the pipe 175 and the pipe 161 can be circulated, and the circulation between the pipe 175 and the pipe 177 can be interrupted to be recycled. Yes.

  FIGS. 11A and 11B are explanatory views showing the flow path of the first mobile phase in the HPLC of FIG. Among the channels in FIGS. 11A and 11B, the channel indicated by the solid line indicates the channel of the first mobile phase.

  In FIG. 11A, reference numeral 111 denotes a first mobile phase storage tank, and the internal first mobile phase 113 is sucked by the first liquid feed pump 117 via the pipe 115. The first mobile phase 113 is sent to the sample introduction device 121 via the pipe 119 by the first liquid feeding pump 117. The first mobile phase 113 is sent to the first flow path switching valve 125 through the pipe 123 together with the sample introduced from the sample introduction device 121. The first mobile phase 113 is sent to the inlet of the first column 129 through the ports 125a and 125b of the first flow path switching valve. The first mobile phase 113 exiting from the outlet of the first column 129 is sent to the first detector 135 through the ports 125e and 125f of the first flow path switching valve 125. The mobile phase 113 exiting the first detector 135 is sent to the recovery tank 149 through the pipe 137 and becomes the recovery liquid 151.

  FIG.11 (b) has shown the flow path of the 1st mobile phase at the time of switching the 1st flow path switching valve 125 of Fig.11 (a). The first mobile phase 113 that exits the sample introduction device 121 passes through the pipe 123, passes through the ports 125 a and 125 f of the first flow path switching valve 125, and is sent to the first detector 135 via the pipe 133. The mobile phase 113 that has exited the first detector 135 is sent to the recovery tank 149 via the pipe 137 and becomes the recovered liquid 151.

  FIGS. 12A to 12C are explanatory views showing the flow path of the second mobile phase in the HPLC of FIG. Among the channels in FIGS. 12A to 12C, the channel indicated by the solid line indicates the channel of the second mobile phase.

  In FIG. 12A, the second mobile phase 155 sucked by the second liquid feeding pump 163 is sent to the second flow path switching valve 159 via the pipe 157. The ports 159a and 159d of the second flow path switching valve 159 can always flow. The second mobile phase 155 is sent to the first flow path switching valve 125 via the pipe 161, the second liquid feed pump 163, and the pipe 165. In the first flow path switching valve 125, ports 125c and 125d can flow. Thereafter, the second mobile phase 155 is sent to the inlet of the second column 169 via the pipe 167. The second mobile phase 155 exiting the second column 169 is sent to the second detector 173 via the pipe 171. Then, it is sent to the second flow path switching valve 159 via the pipe 175. In the second flow path switching valve 159, ports 159c and 159b can flow. The second mobile phase 155 exiting the second flow path switching valve 159 is sent to the recovery tank 149 via the pipe 177 and becomes the recovery liquid 151.

  FIG. 12B shows the flow path of the second mobile phase when the first flow path switching valve 125 of FIG. 12A is switched. In the first flow path switching valve 125, ports 125c and 125b can be circulated. The second mobile phase 155 exiting the first flow path switching valve 125 is sent to the first column 129 via the pipe 127. The second mobile phase 155 exiting the first column 129 is sent to the first flow path switching valve 125 via the pipe 131. Ports 125e and 125d can flow through the first flow path switching valve 125. Thereafter, the second mobile phase 155 is sent to the second column 169 via the pipe 167. The second mobile phase 155 exiting the second column 169 is sent to the second flow path switching valve 159 via the pipe 171, the second detector 173, and the pipe 175. In the second flow path switching valve 159, ports 159c and 159b can flow. The second mobile phase 155 is sent to the recovery tank 149 via the pipe 177 and becomes the recovery liquid 151.

  FIG. 12C shows the flow path of the second mobile phase when the second flow path switching valve 159 of FIG. 12A is switched. In the second flow path switching valve 159, ports 159c and 159d can flow. Thereby, the pipe 161, the second liquid feeding pump 163, the pipe 165, the first flow path switching valve 125, the pipe 167, the second column 169, the pipe 171, the second detector 173, and the pipe 175. A closed flow path consisting of the second flow path switching valve 159.

  When the first flow path switching valve 125 can flow between the ports 125a and 125f, between the ports 125e and 125d, and between the ports 125c and 125b, between the ports 125f and 125e, Distribution between the ports 125d and 125c and between the ports 125b and 125a is blocked. Similarly, when the ports 125f and 125e, the ports 125d and 125c, and the ports 125b and 125a can be circulated, the ports 125a and 125f, the ports 125e and 125d, In the meantime, the flow between the ports 125c and 125b is blocked. That is, when the flow path of the first mobile phase is the flow path of FIG. 11 (a), the flow path of the second mobile phase is the flow path of FIG. 12 (a) or FIG. 12 (c). When the flow path of the first mobile phase is the flow path of FIG. 11B, the flow path of the second mobile phase is the flow path of FIG.

  Next, an analysis method using this HPLC will be described.

  This HPLC can perform (4) analysis without column switch, (5) analysis with column switch for specific components, and (6) analysis with recycle for specific components in (5) above. The above (4) to (6) performed using this HPLC will be described below.

(4) Analysis without the column switch The analysis without the column switch is performed using the first mobile phase. At this time, the flow path of the first mobile phase is a flow path indicated by a solid line in FIG. The sample introduced from the sample introduction device 121 is sent to the first column 129 together with the first mobile phase 113. Within the first column 129, the sample is held in a stationary phase. Thereafter, the first mobile phase 113 continues to be supplied. The first mobile phase 113 containing each component eluted from the first column 129 exits the first column 129 and is then sent to the first detector 135. The first mobile phase 113 containing each component detected by the first detector 135 is sent to the recovery tank 149 via the pipe 137, and the recovery liquid 151 is recovered. The recovered liquid 151 is a waste liquid or is collected every holding time by a fraction collector (not shown). At this time, the flow path of the second mobile phase 155 is a flow path of FIG. 12A or a flow path of FIG. In this analysis, since the second mobile phase 155 is not used, it is not necessary to distribute it.

(5) Analysis for performing column switch on specific component First, the analysis of (4) above is performed in advance, and the retention time of the specific component (a mixture of plural components as the target component of the column switch) is confirmed. Next, the sample is introduced from the sample introduction device 121 through the flow path indicated by the solid line in FIG. The switching operation of the first flow path switching valve 125 is performed with reference to the holding time of the specific component. Components other than the specific component (components having a shorter retention time than the specific component) are sent to the recovery tank 149 through the flow path indicated by the solid line in FIG. At this time, the second mobile phase 155 flows through the flow path indicated by the solid line in FIG.

  Immediately before the specific component elutes from the first column 129, the first flow path switching valve 125 is switched to switch the flow path of the first mobile phase to the flow path indicated by the solid line in FIG. Thereby, the 2nd mobile phase 155 switches to the flow path shown with the continuous line of Drawing 12 (b). The specific component is eluted from the first column 129 by the second mobile phase 155. The second mobile phase 155 containing the specific component is sent to the second column 169 via the pipe 131 and the first flow path switching valve 125 and is held in the column.

  When the elution of the specific component is finished, the first flow path switching valve 125 is switched to obtain a flow path indicated by a solid line in FIG. As a result, components other than the specific component (components having a longer retention time than the specific component) are sent to the recovery tank 149. At this time, the second mobile phase 155 flows through the flow path indicated by the solid line in FIG. 12A, and the specific component is eluted from the second column 169 by the second mobile phase 155. The specific component eluted from the second column 169 is detected by the second detector 173. The second mobile phase 155 that has exited the second detector 173 is sent to the recovery tank 149 via the second flow path switching valve 159, and becomes the recovery liquid 151. In this case, each component in the specific component is sufficiently separated in the second column 169.

(6) Analysis for Recycling Specific Component in (5) When eluting the specific component stocked in the second column 169 in (5), the second flow path switching valve 159 is switched to connect the piping 175 and the piping. 161 is allowed to circulate, and a flow path indicated by a solid line in FIG. Thereby, the pipe 161, the second liquid feeding pump 163, the pipe 165, the first flow path switching valve 125, the pipe 167, the second column 169, the pipe 171, the second detector 173, and the pipe 175. A closed flow path consisting of the second flow path switching valve 159.

  With reference to the output result of the second detector 173, when each component is sufficiently separated and recycling becomes unnecessary, the second flow path switching valve 159 is switched to the flow path of FIG. . As a result, the second mobile phase 155 from which each component has been eluted is sent to the recovery tank 149 via the pipe 177 and becomes the recovery liquid 151.

  In FIG. 1 and FIG. 10, the sample introduction device is not interposed in the second flow path, but may be interposed. When the sample introduction device is interposed in the second channel, the first channel and the second channel can be used as two independent HPLCs. In FIG. 1, the first flow path is not provided with a recycling mechanism, but may be provided with a recycling mechanism. Thereby, two types of recycling target components can be recycled simultaneously.

  In the figure, the first flow path switching valve uses a 6-way valve, but a plurality of valves may be combined (a plurality of valves in this case are collectively referred to as a first flow path switching valve group). . Similarly, the second flow path switching valve may be configured by combining a plurality of valves (in this case, the plurality of valves are collectively referred to as a second flow path switching valve group). In these cases, it is necessary for the valve group to simultaneously switch the valves constituting each valve group. If they are not switched at the same time, the flow path is temporarily blocked and the inside becomes a high pressure, so that the apparatus may be damaged. Accordingly, the term “simultaneous” refers to a time within a range in which such blocking of the flow path is allowed.

  The first mobile phase and the second mobile phase may have the same composition or different compositions. When the first mobile phase and the second mobile phase have the same composition, the first mobile phase storage tank and the second mobile phase storage tank may be the same storage tank. Further, the first liquid feeding pump and the second liquid feeding pump may have the same liquid feeding speed or different liquid feeding speeds. The first detector and the second detector may be the same detection method or different detection methods. The first column and the second column may be the same column or different columns. As the column, a column generally used for HPLC such as a reverse phase column or normal phase column, or a GPC column or ion exchange column can be used. The mobile phase is appropriately selected from known ones according to the type of column used.

  Hereinafter, specific sample separation results by HPLC of the present invention will be described.

  First liquid feed pump: SSC-3461 (manufactured by Senshu Kagaku), second liquid pump: L-2130 (manufactured by Hitachi High-Tech), first detector: SSC-5410 (manufactured by Senshu Kagaku), second detector: L -450 (manufactured by Hitachi High-Tech), sample introduction apparatus: RHEODYNE7125 (manufactured by RHEODYNE), and first and second columns: ODS10φ × 150 mm (manufactured by Senshu Kagaku) were used to configure the apparatus of FIG.

  Using this apparatus, a sample containing multiple components (p-hydroxybenzoic acid (methyl, ethyl, butyl, n-butyl, isopropyl) using a water / acetonitrile (1/1) mixture as the first and second mobile phases , Caffeine, resorcinol, t-butylhydroquinone, 2 mg / mL each). The column temperature was 40 ° C. The mobile phase flow rate was 3 mL / min. As a result, the pressures in the first column and the second column were 3.3 MPa and 3.4 MPa, respectively. The detection wavelength of the first and second detectors was 254 nm.

  First, in the flow path shown by the solid line in FIG. 2A, HPLC analysis without recycling was performed, and the chromatogram shown in FIG. 4 was obtained. Of each component, separation of two peaks appearing at a retention time of about 8 minutes was insufficient. Therefore, recycling was performed for these two components. When this sample is recycled by the conventional HPLC shown in FIG. 13, it is impossible to separate a component having a retention time of about 8 minutes because it overlaps with a peak of a component having a retention time of about 2 minutes or a component having a retention time of about 4 minutes.

  Components having a retention time of 0 to 7 minutes were eluted in the flow path indicated by the solid line in FIG. In the holding time of 7 to 9 minutes, the column was switched by switching the channel to the channel indicated by the solid line in FIG. Thereafter, after the holding time of 9 minutes, the flow path was switched to the flow path indicated by the solid line in FIG. FIG. 5 is a chromatogram detected by the first detector after HPLC analysis of the same sample as FIG. The component having the retention time of 7 to 9 minutes is not detected by the first detector because the column switch is performed. The components stocked in the second column were subjected to a recycling analysis using a flow path indicated by a solid line in FIG. The chromatogram is shown in FIG.

  According to this chromatogram, a peak appears every 8 minutes from a retention time of 16 minutes. The peak with a retention time of 16 minutes is the peak of the component that has passed through the first column and further passed once through the second column (this is the first round). According to this chromatogram, separation of the two components was achieved with a retention time of about 56 minutes (sixth round).

100: HPLC of the present invention
DESCRIPTION OF SYMBOLS 11 ... 1st mobile phase storage tank 13 ... 1st mobile phase 17 ... 1st liquid feeding pump 19 ... Sample introduction apparatus 21 ... 1st column 23 ... 1st flow-path switching valve 23a ˜f: Valve port 27: First detector 31: Second column 33: Second mobile phase storage tank 35: Second mobile phase 39: Second flow path switching valve 39a to d ... Valve port 43 ... Second liquid feed pump 49 ... Second detector 57 ... Recovery tank 59 ... Recovery liquid 15, 25, 28, 29, 37, 41, 45, 47, 51, 53, 55 ... Pipe 200 ... HPLC of the present invention
DESCRIPTION OF SYMBOLS 111 ... 1st mobile phase storage tank 113 ... 1st mobile phase 117 ... 1st liquid feeding pump 121 ... Sample introduction apparatus 125 ... 1st flow-path switching valve 125a-f ... Valve Port 129 ... First column 135 ... First detector 149 ... Recovery tank 151 ... Recovery liquid 153 ... Second mobile phase storage tank 155 ... Second mobile phase 159 ... First Two-channel switching valve 159a-d ... Valve port 163 ... Second liquid feed pump 169 ... Second column 173 ... Second detector 115, 119, 123, 127, 131, 133, 137, 157, 161, 165, 167, 171, 175, 177 ... Piping 500 ... HPLC with conventional recycling mechanism
501 ... Mobile phase storage tank 503 ... Mobile phase 507 ... Flow path switching valve 507a, 507b, 507c, 507d ... Valve port 511 ... Liquid feed pump 513 ... Sample introduction device 515 ..Column 517 ... Detector 523 ... Recovery tank 525 ... Recovered liquid 505, 509, 519, 521 ... Piping

Claims (6)

From one end side to the other end side of the flow path of the first mobile phase, sequentially the first liquid feed pump, the sample introduction device, the first column, the first flow path switching valve group, the first detector,
While interposing
By switching the flow path switching valve of the first flow path switching valve group, the first mobile phase flow path is switched to a flow path having a second column interposed between the first column and the first detector. A flow path for one mobile phase;
From one end side to the other end side of the flow path of the second mobile phase, sequentially a second flow path switching valve group, a second liquid feeding pump, a first flow path switching valve group, a second detector, A two-channel switching valve group;
While interposing
By switching the flow path switching valve of the first flow path switching valve group, in the flow path of the second mobile phase, the flow path that interposes the second column between the second liquid feeding pump and the second detector. A flow path for the second mobile phase to be switched;
Have
The first flow path switching valve group includes a flow path switching valve that switches the mobile phase passing through the second column to either the first mobile phase or the second mobile phase, and the first flow path switching valve group includes: At the same time, each valve switches,
and,
By switching the flow path switching valve of the second flow path switching valve group, a second flow path switching valve group, a second liquid feed pump, a second column, a second detector, and a second flow path switching valve A high-performance liquid chromatograph comprising a mechanism for switching to a closed flow path circulating through a group. From one end side to the other end side of the flow path of the first mobile phase, sequentially the first liquid feed pump, the sample introduction device, the first flow path switching valve group, the first detector,
While interposing
By switching the flow path switching valve of the first flow path switching valve group, the first mobile phase flow path is switched to a flow path having a first column interposed between the sample introduction device and the first detector. A flow path for one mobile phase;
From one end side to the other end side of the flow path of the second mobile phase, the second flow path switching valve group, the second liquid feeding pump, the first flow path switching valve group, the second column, A detector, a second flow path switching valve group,
While interposing
By switching the flow path switching valve of the first flow path switching valve group, the flow path of the second mobile phase is switched to the flow path that interposes the first column between the second liquid feeding pump and the second column. A flow path for the second mobile phase;
Have
The first flow path switching valve group includes a flow path switching valve that switches the mobile phase passing through the first column to either the first mobile phase or the second mobile phase, and the first flow path switching valve group is At the same time, each valve switches,
and,
By switching the flow path switching valve of the second flow path switching valve group, a second flow path switching valve group, a second liquid feeding pump, a first flow path switching valve group, a second column, and a second detector And a mechanism for switching to a closed flow path that circulates through the second flow path switching valve group.   The high-performance liquid chromatograph according to claim 1 or 2, wherein a sample introduction device is interposed between flow paths between the second liquid feeding pump and the second column.   The high-performance liquid chromatograph according to claim 1 or 2, wherein the first flow path switching valve group includes one valve having five or more openings.   The high-performance liquid chromatograph according to claim 1 or 2, wherein the second flow path switching valve group is composed of one valve having four or more openings.   Using the high-performance liquid chromatograph according to claim 1 or 2, after introducing the target component for recycling into the second column, the target component for recycling introduced into the second column is repeated a plurality of times using the second mobile phase. Recycling analysis method leading to two columns.

Images