JPH0375069B2 - - Google Patents

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a liquid chromatography device,
In particular, the present invention relates to a liquid chromatography apparatus equipped with an in-line analysis means, in which the recovery rate of a target substance is improved by controlling the elution rate during analysis.

[Prior Art] Liquid chromatography is mainly composed of four parts: a liquid feeding section, a sample injection section, a separation section, and a detection section. Then, when the moving phase liquid is caused to flow by the liquid sending pump installed in the liquid sending section and the sample is injected from the injection section, each component in the sample is eluted in order of decreasing affinity with the fixed layer in the column. Therefore, a chromatogram can be obtained by detecting this eluted component with a detection section. One type of chromatography is preparative liquid chromatography, which separates the fluid flowing out from a chromatography column according to the components contained in the fluid.

In conventional preparative liquid chromatography, in order to efficiently analyze the eluate from the preparative column, the eluate is collected in fractions using a fraction collector, and automatically analyzed using an autosampler. I try to do this.

[Problems to be solved by the invention] In such conventional preparative liquid chromatography, the preparative operation and the analytical operation are independent, and it is difficult to grasp the situation of each fraction during preparative collection. I couldn't do it. In addition, the number of samples analyzed,
When the number of preparative collections increases, a great deal of time and effort is required to prepare preparative containers, sample bottles for autosamplers, etc., and to transfer samples. Furthermore, since analysis using an autosampler is performed after the preparative separation process is completed, there are various problems such as the inability to feed back the data to the same test to perform accurate preparative separation.

In order to solve these problems, the applicant has developed a preparative column filled with a packing material, a fluid supply means for supplying a sample, a developing agent, an eluent, etc. to the preparative column, and a preparative column. In a chromatography apparatus comprising a system for taking out an outflow fluid from a system, and an outflow direction changing means for changing the outflow direction of the system for taking out an outflow fluid into a plurality of directions, the system for taking out an outflow fluid is provided with a micro-volume injector; An in-line analysis comprising an analysis means for analyzing a trace sample taken out by the trace injector using a chromatographic analysis column, etc., and a controller for controlling the outflow direction changing means based on a signal from the analysis means. discovered a chromatography device of the same type and filed a patent application (Japanese Patent Application No. 153757-1988, hereinafter referred to as the "prior application").

According to the device of the prior application, a small amount of sample is taken out from the fluid flowing out from the preparative column, this small amount of sample is analyzed by the analytical means, and the outflow direction changing means is controlled based on this analysis data, thereby eliminating the need for complicated separation. It is possible to save labor by automatically performing the collection and analysis operations.

By the way, in order to further improve the recovery rate of the target substance in such an in-line analysis type liquid chromatography apparatus, it is necessary to control the operation of the outflow direction changing means of the outflow fluid with respect to the elution of the target substance depending on the time required for analysis. It is necessary to make the feed as small as possible.

That is, when a multi-component system is separated using a preparative column with a relatively low number of plates, the purity of the target substance in the effluent gradually increases and the preparative separation is started when the desired purity is reached. The rise in purity at this time can be monitored using an in-line fluid chromatography device as in the previous application, and based on this, fractionation of the target substance can be started. In order to more accurately understand the rise in purity,
Although it is necessary to increase the sampling frequency sufficiently, the sampling interval is limited by the time required for analysis.

On the other hand, in liquid chromatography, the solvent strength of the preparative solvent is usually selected based on the maximum level that provides the required separation. For this reason, the rise in purity of the target substance becomes extremely rapid, and elution of the target substance at a high concentration begins before the rise in purity can be clearly monitored through sufficient sampling and analysis. For this reason, in the device of the previous application, the operation of the outflow direction changing means for the outflow fluid always tends to be delayed with respect to the elution of the target substance, and the results are not sufficiently satisfactory in terms of the recovery rate of the target substance. It was hard.

[Means for Solving the Problems] The present invention has been made to solve these problems, and includes a preparative column packed with a packing material, a sample, a strong solvent, or a preparative column packed with a packing material. A fluid supply means with a flow path switching mechanism that supplies one of the weak solvents, a means for taking out the outflow fluid from the preparative column, a means for changing the outflow direction of a system for taking out the outflow fluid into a plurality of directions, and one of the outflow fluids. A fluid chromatography apparatus comprising: an analysis means for intermittently extracting and analyzing a sample; and a controller for changing the flow direction of the flow direction changing means based on a signal from the analysis means, the fluid supply means The flow path switching mechanism provided in the is configured to supply the weak solvent to the preparative column when the target component is first detected by the analysis means.
A liquid chromatography apparatus characterized in that it is controlled by the controller.

In the present invention, a strong solvent is a solvent with high solvent strength, that is, a solvent with a short retention time of the target component,
A weak solvent refers to a solvent with a strong elution effect, and a weak solvent is a solvent with low solvent strength, that is, a solvent that retains the target component for a long time.
Refers to a solvent with a weak elution effect.

[Function] Compared to the case where a strong solvent is used, when a weak solvent is used, the retention time of the target component is longer, and the rise in purity of the target component in the effluent is gradual and the peak is small.

In the apparatus of the present invention, the solvent supplied to the preparative column is controlled to be switched from a strong solvent to a weak solvent when the target component is detected for the first time, so the elution of the target component is delayed and the purity is reduced. The rise will be fully monitored. Therefore, when the purity of the target component in the outflow fluid reaches the most appropriate value, it is possible to appropriately change the flow direction of the outflow fluid, and the target component of the desired purity can be fractionated with a high recovery rate. It becomes possible to do so.

[Example] Examples will be described below with reference to the drawings.

FIG. 1 is a system diagram showing the configuration of a liquid chromatography apparatus according to an embodiment of the present invention.

In FIG. 1, containers 1, 2, and 3 are used to store a sample solution, a strong solvent (80% methanol in this example), and a weak solvent (70% in this example), respectively.
Contains methanol). The liquids in these containers 1, 2, and 3 can be introduced into the preparative column 5 via the pipes 1a, 2a, 3a, the switching valve 4, and a liquid pump P provided in the middle of the pipe 14. . The switching valve 4 has three liquid inlets and one liquid outlet, and allows any one of the pipes 1a, 2a, and 3a to communicate with the pipe 14.

The preparative column 5 is filled with a packing material.
The liquid outflow side of the preparative column 5 is connected to a small amount injector or the like through a pipe 15, which injects a small amount of liquid into the pipe 1 at regular intervals.
6 is connected to a valve mechanism 6 which feeds the detector 7 .

This valve mechanism 6 is connected to a plurality of (three in this embodiment) pipes 11a, 1 via a pipe 17.
2a and 13a are connected, and each pipe 11a,
12a and 13a are connected to the container 1 via valves _V1 to _V3 .
1, 12, and 13 so as to send out liquid.

The detector 7 is equipped with a chromatographic analysis column, and the analytical column is filled with a packing material, and the effluent thereof is sent to the detector 7. The detection signal of the detector 7 is incorporated into the controller 8. The signal is input to a data processor, and the signal from the data processor is input to a controller 8. This controller 8
outputs signals for operating the switching valve 4, the valve mechanism 6, and the separation valves _V1 to _V3 .

The switching valve 4 selects a flow path so that a predetermined amount of the sample solution flows out from the container 1 to the preparative column 5, and then a strong solvent and a weak solvent are sent from the containers 2 and 3 to the preparative column 22.

The valves V ₁ to _{V 3} open and close according to changes in the components in the effluent of the preparative column 5, and control the flow such that the same component is sent to one container (one of the containers 11 to 13). Make route selection.

The valve mechanism 6 causes a minute amount of the preparative column effluent flowing from the piping 15 to flow into the piping 16 and introduce it into the analytical column of the detector 7 at predetermined time intervals.

In the above configuration, the sample solution in the container 1 is
First, a predetermined amount is introduced into the preparative column 5, and then the strong solvent in the container 2 is introduced into the preparative column 5, and the components in the sample are developed and eluted. Preparative column 5
The effluent is analyzed by a detector 7 at predetermined intervals,
Every time there is a change in the composition, the valves V ₁ to _{V 3} open and close, and the effluent containing only one component is transferred to the containers 11 to 11.
13.

In the apparatus of the present invention, when the target component is first detected in the detector 7, the controller 8 controls the switching valve 4 based on the detection signal of the detector 7, and instead of the strong solvent in the container 2, The weak solvent in the container 3 is supplied to the preparative column 5. This slows down the elution rate of the target component and increases the number of times the effluent is sampled in response to changes in purity, making it possible to sufficiently monitor the rise in purity of the target component in the effluent with a detector. Become.

In this case, it is more effective to change the solvent by one bed volume earlier than the point at which the elution is desired to be delayed.

Furthermore, it is more effective to control the pump P by the controller 8 to adjust the solvent supply rate, and to slow down the solvent supply rate when the purity of the target component increases.

In the present invention, after the detector 7 detects that the concentration of the target component in the effluent is equal to or higher than a predetermined value, the switching valve 4 is switched again to supply the strong solvent and perform fast elution. After that, when the detector 7 detects that the purity of the target component in the effluent is below a predetermined value, the switching valve 4 is switched again to feed the weak solvent and adjust the elution rate. You may try to slow it down. (Furthermore, the target component is 2
If more than 100% of the solvent is contained, after the end of outflow of the first target component is detected, the strong solvent may be supplied to the preparative column again and the above procedure may be repeated. ) By doing this, the rising and falling parts of the purity of the target component in the effluent, where the purity changes rapidly, are used as a weak solvent to slow down the elution rate and monitor the target component sufficiently. In the range where the purity peak appears, it is possible to increase the elution rate as a strong solvent and perform efficient elution.

According to the device of this embodiment, a sample solution, a strong solvent, and a weak solvent are accurately introduced into the preparative column 5 at a predetermined time, and the effluent from the preparative column 5 is fractionated according to the components contained. It is accommodated in containers 11-13. Since this fractionation is performed in parallel with the separation operation of passing the liquid through the preparative column 5, it is extremely accurate and reliable.

As is clear from the above description, in the apparatus of this embodiment, fluid supply means to the preparative column 5 is provided by the containers 1 to 3, the pipes 1a to 3a, the switching valve 4, the pipe 14, and the liquid feed pump P. The pipes 15 and 17 constitute an outflow fluid extraction system from the preparative column 22. Also valve V ₁ ~
Outflow direction changing means is constituted by _V3 and the pipes 11a to 13a. Furthermore, a valve mechanism 6,
The pipe 16 and the detector 7 constitute analysis means.

In the embodiment shown in FIG. 1, the switching valve 4
A sample solution, a strong solvent, and a weak solvent are supplied to the preparative column 5 by a liquid-feeding pump P, but instead of these, a liquid-feeding pump is installed in each of the pipes 1a, 2a, and 3a. , these pumps may be controlled by the controller 8, respectively. In addition, it is also possible to employ a step gradient device.

Further, the outflow direction of the effluent may be changed by using a switching valve that switches the outflow direction, instead of opening and closing the valves V ₁ to _{V 3} provided in each pipe.

Further, in the apparatus of FIG. 1, only three containers 11, 12, and 13 are shown for storing the fractionated effluent, but it is clear that the number of containers may be two or four or more.

Examples will be described below.

In the following, all "%" indicates "% by weight".

Experimental Example In the example apparatus shown in Fig. 1, the preparative column 5
A glass column with an inner diameter of 20 mm and a length of 60 mm was used as a packing material in the preparative column 5.
Filled with ODS30/60 mesh. In addition, 73% methanol is used as a strong solvent, and 67% methanol is used as a weak solvent.
Prepared methanol.

An organic multi-component sample was taken as a sample and separated using such an apparatus. First sample 1160
ml at 12ml/min, then strong solvent at 40ml/min.
The liquid was passed through at min. Next, after the target component began to be detected by the detector 7, the flow rate of the solvent was set to 29 ml/min,
The separation was continued by switching the solvent from strong to weak solvent. As a result, with sampling every 15 minutes, four points of analysis are performed until the target purity of 70% for recovery of the target component in the effluent is reached, and based on this data, analysis is performed at the point of expected purity of 80%. Valves V ₁ to _{V 3} were opened and closed to switch the sorting containers.

As a result, the recovery rate of the target component was an extremely high value of 75%.

On the other hand, when separation was performed using only 73% methanol without changing the solvent, the purity of the target component in the effluent was 78% at the second point after the detection of the target component with sampling every 15 minutes. reached. When switching the preparative container based on this data, the purity of the target component in the effluent was estimated to be around 90%.
The recovery rate of the target component was significantly lower than 75% in the case of the apparatus of the present invention.

As is clear from this result, according to the device of the present invention, after the target component is detected in the effluent, the elution rate is slowed down, so the rise in purity of the target component is delayed, and sufficient sampling is performed. be able to. Therefore, it is possible to sufficiently monitor the rise in purity of the target component, and it is possible to accurately change the flow direction of the effluent, greatly improving the recovery rate of the target component compared to conventional methods. becomes possible.

[Object of the Invention] As described above, according to the present invention, fractionation and analysis operations can be performed without using a fraction collector, a fractionation container, an autosampler, a sample bottle for an autosampler, or the like. Furthermore, since separation testing and analysis can be performed simultaneously, operation time can be shortened. Then, the analysis results can be fed back to and controlled the operation of the solvent supply system and the effluent recovery system.

Therefore, according to the present invention, it is possible to significantly save labor for complicated preparative separation and analysis operations, perform accurate fractionation operations, and efficiently recover target components with extremely high purity and recovery rate. It is considered possible.

[Brief explanation of drawings]

FIG. 1 is a system diagram showing the configuration of a chromatography apparatus according to an embodiment of the present invention. 4...Switching valve, 5...Preparative column, 7...Detector, 8...Controller.

Claims (1)

[Scope of Claims] 1. A preparative column filled with a packing material; a fluid supply means with a flow path switching mechanism for supplying a sample, a strong solvent, or a weak solvent to the preparative column; means for taking out the outflow fluid from the column; means for changing the outflow direction of the system for taking out the outflow fluid; analysis means for intermittently taking out and analyzing a portion of the outflow fluid; and based on a signal from the analysis means. A fluid chromatography device comprising: a controller for changing the outflow direction of the outflow direction changing means, wherein the flow path switching mechanism provided in the fluid supply means is configured such that a target component is first detected by the analysis means; to feed the weak solvent to the preparative column when
A liquid chromatography apparatus characterized in that it is controlled by the controller. 2. The liquid according to claim 1, wherein the controller is capable of controlling the fluid supply means to reduce the amount of fluid supplied to the preparative column when the target component is detected. Chromatography equipment. 3. Claims characterized in that the controller is capable of controlling the fluid supply means to supply the strong solvent to the preparative column when the concentration of the target component in the effluent reaches a predetermined value or higher. The liquid chromatography device according to item 1 or 2. 4. The controller according to claim 3, wherein the controller is capable of controlling the fluid supply means to supply the weak solvent to the preparative column when the concentration of the target component starts to decrease. Liquid chromatography equipment. 5. The liquid chromatography apparatus according to any one of claims 1 to 4, wherein the analysis means includes a chromatographic analysis column.