JP2780273B2 - Recycled liquid chromatograph - Google Patents

Description

The present invention relates to a recycle liquid chromatograph.

(Prior Art) A column of a liquid chromatograph becomes more expensive as its length becomes longer, and if it is longer, a liquid sending pressure becomes higher. Therefore, a column of a limited length is used. When separation between sample components cannot be achieved with a column having a limited length, recycle liquid chromatography has been performed in which an eluate containing an insufficiently separated component that has passed through the column is further returned to the column for subsequent separation. FIG. 7 is a flow chart showing an example of a conventional recycled liquid chromatograph. In FIG. 7, reference numeral 1 denotes a mobile phase container. At the time of sample introduction, the mobile phase 4 is sent by the pump 3 through the column 5, the detector 6, and the broken line flow path 8 of the recycle valve 7, and in this state, the sample introduction device 2 introduces the sample solution into the column. When the component band containing the insufficiently separated component elutes, the recycle valve 7 is switched to the solid line 9 to form a recycle channel.

When the target component is separated by repeating the recycling a plurality of times, the three-way valve 7 is switched to the flow path indicated by the broken line 8, and the sample component is discharged out of the liquid chromatograph system.

(Problems to be Solved by the Invention) In a conventional recycle liquid chromatograph, a plurality of components having a specific retention time relationship are eluted in an overlapping manner when a recycle separation is carried out, and cannot be separated at all. FIG. 8 is an explanatory view showing elution positions of peaks when performing recycle separation.

The retention time of component A is a, and the total volume of the detector cell, recycle valve, pump, sample introduction device and the flow path connecting these components is divided by the pump flow rate. Is assumed to be c. When A is recycled, the elution time of the second, third,... Becomes 2a + c, 3a + 2c,. On the other hand, assuming that the retention time of the B component is b, the elution times of the second, third, etc. are 2b + c, 3b + 2c.

If the retention time of the B component is equal to the second elution time of A, that is, if b = 2a + c, the second elution of the B component will also be 2b + c → 2 (2a + c) + c = 4a + 3c, which will coincide with the elution of the A component. . In the subsequent third and fourth elutions, the B component and the A component overlap and elute, and cannot be separated.

The present invention provides an apparatus capable of separating a plurality of components that have such a specific retention time relationship and are eluted and cannot be separated by recycle when they are separated by recycling.

(Means for Solving the Problems) In the recycled liquid chromatograph of the present invention,
A means for changing the volume of these channels is provided in the middle of a liquid chromatograph or a recycle channel in which a pump, a sample introduction device, a column, and a detector are sequentially connected. Preferably, the flow path volume changing means is configured by connecting a flow path switching mechanism having a loop of an appropriate capacity to the flow path.

(Operation) In the recycled liquid chromatograph of the present invention,
During recycling, the volume of the liquid chromatograph channel or the recycle channel can be increased or decreased. That is, it is possible to change the value of c for the component A and the value of c for the component B in FIG. 8, and it is possible to separate a plurality of components eluting in an overlapping manner by recycling chromatography.

(Embodiment) FIG. 1 is a flow chart of a recycled liquid chromatograph of the present invention.

The same reference numerals in the figure denote the same parts as in the conventional apparatus of FIG. At the time of the recycle analysis, the recycle valve 7 is connected to the position indicated by the solid line 9, and the sample component that has left the column 5 is again introduced into the column 5 through the detector 6, the recycle valve 9, the pump 3 and the sample introduction device 2. A control unit 10 controls switching of the flow path of the sample introduction device 2, switching of the recycle valve 7, and the like. 11 is a fraction collector. Second
The figure shows an example of a means for changing the flow channel volume.
It is an internal flow path diagram of the sample introduction device in a figure, and has shown the loading state. In the load state, the pump and the column are directly connected. FIG. 3 is an internal flow diagram of the sample introduction device in FIG. 1, showing an injection state. In the injection state, the sample holding coil is connected between the pump columns, and the volume of the flow channel is increased by the coil capacity. Glycyrrhizin in licorice was analyzed using such a recycled liquid chromatograph. The analysis conditions are as follows.

Column: Shim-pack PREP-ODS (H) (20 mm ID × 250 mm
L.) Mobile phase: mixture of 1% acetic acid water / acetonitrile 6: 4 Flow rate: 10 ml / min Temperature: room temperature Detection: UV light absorbance Wavelength 250 nm The sample solution is crushed with 0.1 g of licorice, 50 ml of mobile phase is added. And leave it for 1 hour to obtain the supernatant.
Was introduced into the column. FIG. 4 is a chromatogram obtained by analyzing the licorice extract without recycling. The peak indicated by G in the figure is a peak containing glycyrrhizin as a main component, and is subjected to recycle separation in order to further separate this peak and increase the purity of the cricyrrhizin fraction. I
The peak indicated by is a peak of an impurity, and when recycled and separated, it overlaps with crytyllithine and cannot be separated.

FIG. 5 is a chromatogram obtained by recycle separation of a licorice extract according to a conventional method.

First, the sample introduction device 2 is set in the loaded state, and the sample holding coil is filled with 1 ml of the licorice extract. Next, the sample introducing device 2 is set in an injection state, and the sample liquid is introduced into the column. This injection state switching time is the start of the analysis, which is time zero. Next, two minutes after the introduction of the sample liquid, the sample introduction device is returned to the loaded state. This is what is conventionally performed in order to minimize the volume of the flow path other than the column during the recycle analysis and to suppress the spread of the component band during the recycle.

Next, the elution time including the first glycyrrhizin peak indicated by G in FIG. 5, that is, 12 (9 minutes to 11
), The recycle valve is set to the solid line state 9 and the glycyrrhizin is recycled and separated. At 19 minutes (13 in the figure), the recycle valve was set to the solid line state 9 again, after which the recycle state was continued and the state of separation was observed.

In the chromatogram in FIG. 5, the impurity peak indicated by I in FIG. 4 completely overlapped with the glycyrrhizin peak eluted at the second, fourth and sixth times, and it was impossible to mutually separate the two components. .

FIG. 6 is a chromatogram obtained by separating a licorice extract by analysis using a recycled liquid chromatograph of the present invention. The analysis is started in the same manner as the analysis conditions for obtaining the chromatogram in FIG. At 9 minutes, the recycle valve was set to the solid line state 9 and the sample introduction device was changed from the load state to the injection state. Then, at 11 minutes, the recycle valve was set to the broken line state, and at 15 minutes, the sample introduction device was returned to the loaded state. The sample holding coil of the sample introduction device is 15
The glycyrrhizin peak at the second recycling was lower than that at the time of the analysis in FIG.
The sample was re-introduced into the column with a delay of 1.5 minutes [(volume inside the sample holding coil) ÷ (pump flow rate)]. As a result, the impurity peak indicated by I in the figure is separated from the glycyrrhizin peak and discharged out of the system through the dashed line 8 of the recycle valve 9. At 21 minutes (15 in the figure), the recycle valve 9 was switched to the solid line state, the recycle separation was continued as it was, and the appearance of the separation was observed. The impurity peak I did not overlap with the second and subsequent glycyrrhizin peaks.

In the above-described embodiment, the sample introduction device was once loaded immediately after sample introduction, and then injected immediately before recycling of the target component. May be made to be in a funnel state after passing through the holding coil at least once.

Further, in the recycle liquid chromatograph of the present invention, a flow channel volume changing means can be provided in a recycle channel from a column outlet to a pump inlet at the time of recycle separation.

(Effects) According to the present invention, even if there are a plurality of other components that strictly overlap with the second and subsequent recycling of the target component, the separation can be performed by a simple apparatus configuration without reconsidering the separation conditions. If the flow volume changing means of the apparatus of the present invention is provided in the middle of the recycle flow path between the column outlet and the pump inlet, there is an effect that the pressure resistance does not have to be particularly reserved.

[Brief description of the drawings]

FIG. 1 is a flow chart of a recycled liquid chromatograph of the present invention. FIG. 2 is an internal flow diagram of the sample introduction device in FIG. 1 and shows a load state, and FIG. 3 is also an internal flow diagram of the sample introduction device and shows an injection state. It is. FIG. 4 is a chromatogram obtained by analyzing the licorice extract without recycling. FIG. 5 is a chromatogram obtained by recycle separation of a licorice extract by a conventional method. FIG. 6 is a chromatogram obtained by separating a licorice extract by the recycled liquid chromatograph of the present invention.
FIG. 7 is a flow diagram showing an example of a conventional recycle liquid chromatograph, and FIG. 8 is an explanatory diagram showing peak elution positions when performing recycle separation.

Claims (1)

(57) [Claims] 1. A liquid chromatography flow path in which a pump, a sample introduction device, a column, and a detector are sequentially connected, and a recycle flow path for introducing a solution passing through the detector to a pump inlet via a flow path switching valve. A liquid chromatograph comprising: a means for changing the volume of the liquid chromatograph or the recycle flow path in the middle of the liquid chromatograph or the recycle flow path.