Dispensing method and dispensing device

Abstract

To provide a separation method that can suppress the inclusion of water in the separated target component. [Solution] The method for separating the target component from a sample to be measured comprises a separation step for separating the target component, a capture step for capturing the separated target component in a trap column, an elution step for leaching the target component out of the trap column, and a recovery step for recovering the target component leached out of the trap column. In the elution step, the water and target component leached out of the trap column are separated by adjusting the inflow rate of the elution organic solvent into the trap column.

Description

Technical Field

[0001] The present invention relates to a fractionation method and a fractionation apparatus.

Background Art

[0002] After separating a measurement sample by a liquid chromatograph, a preparative LC apparatus is provided that successively (online) purifies and recovers each of the separated components (see, for example, Patent Document 1). Specifically, in an LC unit (separation unit), a plurality of target components are separated from a measurement sample by passing the measurement sample together with a mobile phase through a separation column. Subsequently, in a purification unit, each target component is temporarily captured by a trap column, and then each target component is eluted with an eluent and recovered individually. By using this preparative LC apparatus, a plurality of target components mixed in a measurement sample can be individually purified and recovered, so that the target components can be subjected to an analyzer such as a nuclear magnetic resonance apparatus for each type, and further detailed analysis of the target components becomes possible.

Prior Art Documents

Non-Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] Incidentally, in preparative LC systems, the LC section used to separate the sample involves mixing a water-containing mobile phase with the sample in a reverse-phase chromatography separation mode and passing it through a separation column. In this separation mode, the target components, along with the water-containing mobile phase, then enter the trap column in the purification section. Therefore, when the target components captured in the trap column are eluted and recovered together with the eluate, a problem occurs where water is mixed into the recovered target components in addition to the eluate. Furthermore, regardless of the separation mode, the target components captured in the trap column may be washed with a water-containing liquid (such as a diluent), and in this case as well, water can enter the trap column and contaminate the eluate. Since water contamination negatively affects subsequent analysis, it is necessary to remove the water from the recovered target components (e.g., drying, distillation), which is time-consuming and labor-intensive.

[0005] This invention can suppress the inclusion of water in the separated target component. [Means for solving the problem]

[0006] A preparative method according to a first aspect of the present invention comprises, in order, a separation step of separating a target component in a sample solution by passing the sample solution through a separation column, a capture step of capturing the separated target component in a trap column, an elution step of eluting the target component from the trap column by introducing an elution organic solvent into the trap column, and a recovery step of recovering the target component eluted from the trap column, wherein in the elution step, the flow rate of the elution organic solvent into the trap column is controlled to separate the water discharged from the trap column from the target component.

[0007] A preparative apparatus according to a first aspect of the present invention is an apparatus for carrying out a preparative method according to a first aspect, comprising: a separation column for separating the target component; a trap column for capturing the separated target component; an elution means for introducing the elution organic solvent into the trap column; a recovery means for recovering the target component that has flowed out of the trap column; and a control means for adjusting the inflow rate of the elution organic solvent into the trap column. [Effects of the Invention]

[0008] According to the first aspect of the present invention, the target component can be separated while suppressing the inclusion of water. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 shows an image diagram of a sorting apparatus used to separate and capture the target component in the first embodiment. [Figure 2] Figure 2 shows an image of a sorting apparatus used to elute and recover the target component in the first embodiment. [Figure 3] Figure 3 shows the chromatogram output by the second detector in Example 1. The horizontal axis represents elution time, and the vertical axis represents signal intensity. [Figure 4] Figure 4 shows the chromatogram output by the second detector in Example 2. [Figure 5] Figure 5 shows the chromatogram output by the second detector in Example 3. [Figure 6] Figure 6 shows the chromatogram output by the second detector in Example 4. [Figure 7] Figure 7 shows the chromatogram output by the second detector in Comparative Example 1. [Figure 8] Figure 8 shows the chromatogram output by the second detector in Comparative Example 2. [Figure 9] Figure 9 shows the chromatogram output by the second detector in Comparative Example 3. [Modes for carrying out the invention]

[0010] 1. First Embodiment 1-1.Preparative separation device An example of a preparative apparatus of the first embodiment used in the first aspect of the present invention will be described with reference to Figures 1 and 2. This preparative apparatus 1 is a chromatographic preparative purification apparatus and, as shown in Figures 1 and 2, is equipped with a separation unit 2 and a purification unit 3 in succession.

[0011] The separation unit 2 is a means for performing separation by liquid chromatography and comprises a separation pump 4, a sample injection unit 5, a separation column 6, and a first detector 7. The separation pump 4 delivers the mobile phase contained in the mobile phase container 8 to the sample injection unit 5. The sample injection unit 5 is a unit for injecting the sample to be measured and introduces the sample to be measured into the flow path in the separation unit 2. The sample injection unit 5 is, for example, an autosampler. Various types of separation columns 6 can be used depending on the type of target component (compound), and preferably, reverse-phase columns such as C18 columns are used. The first detector 7 detects each target component separated by passing through the separation column 6 and outputs it as a chromatogram. Examples of the first detector 7 include ultraviolet-visible absorbance (UV) detectors, photodiode (PDA) detectors, and fluorescence detectors. Although not shown in the figures, the separation pump 4 and other components may be provided in multiple units depending on the type of liquid contained in the mobile phase.

[0012] The purification unit 3 is a means for purifying and recovering each target component separated from the separation unit 2, and comprises a first flow path switching valve 9, a second flow path switching valve 10, an elution pump 11, a dilution pump 12, a trap column 13, a second detector 14, and a recovery unit 15.

[0013] The first flow path switching valve 9 is a valve that can select any flow path from multiple flow paths, for example, a hexagonal valve. By switching, the first flow path switching valve 9 can guide the target component sent from the separation unit 2 to the trap column 13, or guide the discharge organic solvent sent from the elution pump 11 to the trap column 13.

[0014] The second flow path switching valve 10 is located downstream of the first flow path switching valve 9 and is a valve that can select any flow path from multiple flow paths, for example, a hexagonal valve. By switching, the second flow path switching valve 10 can direct the target component sent from the separation unit 2 via the first flow path switching valve 9 to the trap column 13, direct the diluent sent from the dilution pump 12 to the trap column 13, or direct the component sent from the separation unit 2 via the first flow path switching valve 9 to the outside of the separation device 1.

[0015] The elution pump 11 is an elution means that delivers the elution organic solvent contained in the elution solvent container 16 to the trap column 13, and elutes the target component into the organic solvent. The dilution pump 12 delivers the diluent contained in the diluent container 17 to the trap column 13. The trap column 13 is a column for purifying the target component, which temporarily captures the target component sent from the separation unit 2 and allows (removes) unwanted components other than the target component to pass through. The type of trap column 13 is appropriately determined according to the target component, and specific examples include the Shim-pack series manufactured by Shimadzu Corporation. The second detector 14 detects each target component from the trap column 13 and outputs it as a chromatogram. The second detector 14 is the same as that of the first detector 7. The recovery unit 15 is a recovery means that contains each target component in a separate recovery container, for example, a fraction collector. Although not shown in the figures, the elution pump 11 and the dilution pump 12, etc., may be provided in multiple units depending on the type of liquid contained in the elution organic solvent or diluent.

[0016] The fractionation device 1 is equipped with a control unit 18 such as a computer. The control unit 18 is connected to various pumps (separation pump 4, elution pump 11, dilution pump 12), sample injection unit 5, each flow path switching valve 9, 10, and recovery unit 15, and stores a program for controlling these. Specifically, the program of the control unit 18 controls various pumps (separation pump 4, elution pump 11, dilution pump 12) to adjust the inflow rate and flow rate of each liquid fed from each container (mobile phase container 8, elution solvent container 16, diluent container 17). It controls the sample injection unit 5 to adjust the amount of measurement sample introduced into the flow path. It controls each flow path switching valve 9, 10 to adjust the switching of a plurality of flow paths. It controls the recovery unit 15 to adjust each target component reaching the recovery unit 15 to be accommodated in each container for each target component and / or for each time.

[0017] 1-2. Fractionation method An example of the fractionation method of the first embodiment used in the first aspect of the present invention will be described. This fractionation method uses the fractionation device 1 and includes a preparation step and an implementation step in order. <opposite0000094"

[0018] (1) Preparation step In this step, the measurement sample, mobile phase, diluent, and elution organic solvent are set in the fractionation device 1.

[0019] Specifically, the measurement sample containing the target component is injected into the sample injection unit 5. Also, the mobile phase, elution organic solvent, and diluent are respectively injected into the mobile phase container 8, elution solvent container 16, and diluent container 17. Each container may be single or plural and is appropriately set according to the type of liquid used. Also, according to the number of those containers, each pump is prepared as single or plural.

[0020] <0000 \ 103>The mobile phase is appropriately determined depending on the type of target component, the type of column, the separation mode, etc., but it is preferable to use a liquid used in reverse-phase mode. Specifically, examples include water and organic solvents. As the water in the mobile phase, for example, a buffer solution to which acetic acid, ammonium acetate, formic acid, ammonium formate, ammonia, etc., may be used may be used. Examples of organic solvents include alcohols such as methanol and ethanol, and for example, acetonitrile and acetone. These may be used individually or as a mixture of two or more. When mixing, a mixed solvent of two or more may be injected into one mobile phase container beforehand, or multiple mobile phase containers 8 may be prepared, each containing water or an organic solvent, and these liquids may be mixed in the flow path. The mobile phase preferably contains water, and more preferably is a mixed solution of water and an organic solvent. This allows reverse-phase chromatography to be performed, and a wide variety of compounds can be separated more reliably.

[0021] The diluent is a liquid for purifying the target component and can be any solvent that is immiscible with the target component, and is appropriately determined depending on the target component. Examples of diluents include water and organic solvents as exemplified in the mobile phase. The diluent preferably contains water, and more preferably is water or a mixed solvent of water and an organic solvent. This allows for the removal of unwanted components attached to the target component captured in the trap column, thereby washing (purifying) the target component. In the first embodiment, preferably, at least one of the mobile phase and the diluent contains water.

[0022] Any organic solvent that dissolves the target component can be used, and is appropriately determined depending on the target component. Examples include acetone, acetonitrile, and dichloromethane. These may be used individually or in combination of two or more.

[0023] (2) Implementation process In this process, the pumps 4, 11, and 12, valves 9 and 10, detectors 7 and 14, and control unit 18 of the preparative apparatus 1 are operated to separate and purify the target component.

[0024] Specifically, in the separation unit 2, the mobile phase is delivered from the mobile phase container 8 by the operation of the separation pump 4. In the sample injection unit 5, the sample to be measured is mixed with the mobile phase and passes through the separation column 6. As the sample passes through, it flows into the separation column 6 and is separated according to each target component before flowing out of the separation column 6 (separation step). The separated liquid consisting of the sample to be measured and the mobile phase that has passed through the separation column 6 is detected by the first detector 7 and then reaches the purification unit 3. The first detector 7 outputs a chromatogram of the sample to be measured. By checking this chromatogram, the separation of the target components and the flow time of the target components can be confirmed.

[0025] The rate at which the mobile phase is delivered, and consequently the rate at which the mobile phase containing the sample to be measured enters the separation column 6, V1, is not limited and can be, for example, 1 mL / min or more, preferably 2 mL / min or more, or, for example, 20 mL / min or less, preferably 10 mL / min or less.

[0026] Next, in the purification section 3, as shown in Figure 1, the separated liquid flows into the trap column 13 via the first flow path switching valve 9 and the second flow path switching valve 10 in sequence. At this time, the dilution pump 12 is activated, and the diluent is delivered from the dilution container 1, which then flows through the second switching valve and merges with the separated liquid. The mixture of the separated liquid and the diluent flows into the trap column 13. At this point, the target component is captured in the trap column 13 (capture step). Meanwhile, the mobile phase and diluent flow out of the trap column 13 and are discharged outside the preparative device 1 via the first flow path switching valve 9. As a result, any unwanted components mixed in the separated liquid are discharged outside along with the diluent, etc.

[0027] Next, as shown in Figure 2, the first flow path switching valve 9 is switched (rotated), the elution pump 11 is activated, and the dilution pump 12 is stopped, causing the elution organic solvent to be delivered from the elution solvent container 16 and flow into the trap column 13 via the first flow path switching valve 9. The captured target component is eluted into the elution organic solvent (elution step) and flows out of the trap column 13 together with the elution organic solvent. Subsequently, the target component is detected by the second detector 14 via the second flow path switching valve 10 and the first flow path switching valve 9 in sequence, and is recovered in the recovery unit 15 (recovery step). The second detector 14 outputs a chromatogram of the target component.

[0028] In the purification section 3, the capture and elution processes are performed intermittently or continuously, divided into sections based on the type of target component or time intervals. For example, by referring to the chromatogram obtained from the first detector 7 and switching the first channel switching valve 9 and the second channel switching valve 10 as needed to correspond to the peaks shown by each target component, the desired target component is guided to the trap column 13 along with the mobile phase, and also guided to the recovery section 15 along with the elution organic solvent, as described above. On the other hand, the mobile phase that does not contain the target component (mobile phase with a retention time that does not show a peak) is directly discharged outside the preparative apparatus 1 without passing through the trap column 13 by switching the first channel switching valve 9 and / or the second channel switching valve 10. These operations are repeated for each target component. As a result, each type of target component is individually captured in the trap column 13 and discharged.

[0029] In this case, the inflow rate of the diluent into the trap column 13 is not limited; for example, it may be 1 mL / min or more, preferably 5 mL / min or more, or for example, 50 mL / min or less, preferably 40 mL / min or less.

[0030] In the first embodiment, during the elution process, the rate at which the elution organic solvent is delivered, and consequently the inflow rate V2 of the elution organic solvent into the trap column 13, is adjusted to the rate at which water and the target component are separated in the liquid flowing out of the trap column 13. Specifically, the inflow rate V2 is set to, for example, 1 mL / min or less, preferably 0.8 mL / min or less, more preferably 0.6 mL / min or less, and even more preferably 0.5 mL / min or less. The lower limit is not limited, but from the viewpoint of the time required to complete the separation, it should be, for example, 0.1 mL / min or more. Furthermore, the ratio (V2 / V1) of the inflow rate V2 of the elution organic solvent into the trap column 13 to the inflow rate V1 of the mobile phase into the separation column 6 is, for example, 0.3 or less, preferably 0.2 or less, more preferably 0.15 or less, and also, for example, 0.01 or more, preferably 0.05 or more. By setting the inflow rate V2 or the above ratio within the above range, water can be reliably excluded during the separation of the target component.

[0031] In the recovery process, recovery is carried out in the recovery unit 15, divided into sections for each target component or by time intervals. Specifically, based on the chromatogram obtained from the second detector 14, the peaks indicating the target components are fractionated, and each fractionated target component is collected in its own recovery container. This makes it possible to isolate only a desired portion of the target components from among the continuously arriving target components. In particular, it is possible to isolate the target components in a water-free state together with the elution organic solvent.

[0032] The target component recovered in the recovery process can then be analyzed using analytical equipment such as a nuclear magnetic resonance spectrometer (analysis process). This allows for a detailed understanding of the target component.

[0033] According to the first embodiment, the contamination of the separated target component with water can be suppressed. This is presumed to be because, conventionally, in the elution process, the components discharged from the trap column 13 include the target component and the elution organic solvent, as well as the mobile phase and diluent remaining from the capture process, and at least one of these mobile phase and diluent contains water. As a result, water is mixed into the target component that is ultimately recovered. In contrast, according to the first embodiment, the inflow rate of the elution organic solvent into the trap column 13 is controlled so that water is not mixed into the recovered target component. Specifically, the inflow rate is significantly reduced. This greatly delays the time it takes for the target component to come into contact with the elution organic solvent and be eluted. As a result, water begins to be discharged from the trap column 13 before the target component is discharged into the trap column 13. That is, water is sufficiently discharged first, followed by the discharge of the target component. Therefore, by separating the desired portion of the target component recovered in the recovery unit 15 based on the peaks of the chromatogram of the second detector 14, the contamination of the obtained target component with water can be suppressed. As a result, when analyzing the target component, there is no need to remove water by distillation or other means, and the separation and analysis of the target component can be carried out smoothly.

[0034] In the example of the first embodiment described above, the application of a nuclear magnetic resonance spectrometer is shown as an example of the analytical process. However, the present invention can also be applied to other applications where the presence of water affects measurements using other devices. Examples of such other devices include gas chromatographs, ultraviolet spectrometers, and Fourier transform infrared spectrometers.

[0035] 2. Appearance Those skilled in the art will understand that the exemplary embodiments described above are specific examples of the following embodiments.

[0036] (Section 1) A separation method according to one embodiment is a method for separating a target component from a measurement sample, comprising: a separation step of separating the target component by passing the measurement sample together with a mobile phase through a separation column; a capture step of capturing the separated target component in a trap column; an elution step of flowing an elution organic solvent into the trap column to cause the target component to flow out of the trap column; and a recovery step of recovering the target component that has flowed out of the trap column, wherein in the elution step, the flow rate of the elution organic solvent into the trap column may be adjusted to separate the water and the target component flowing out of the trap column.

[0037] (Paragraph 2) In the preparative method described in Paragraph 1, the inflow rate may be 1 mL / min or less.

[0038] (Clause 3) In the dispensing method described in paragraph 1 or 2, the inflow rate may be 0.6 mL / min or less.

[0039] (Article 4) In the separation method described in any one of paragraphs 1 to 3, the target component may be analyzed by a nuclear magnetic resonance spectrometer after the recovery step.

[0040] (Clause 4) A preparative apparatus according to one embodiment is an apparatus for carrying out the preparative method described in any one of paragraphs 1 to 4, and may include a separation column for separating the target component, a trap column for capturing the separated target component, an elution means for introducing the elution organic solvent into the trap column, a recovery means for recovering the target component that has flowed out of the trap column, and a control means for adjusting the rate at which the elution organic solvent flows into the trap column. [Examples]

[0041] The present invention will now be described in detail with reference to examples and comparative examples, but the scope of the present invention is not limited thereto.

[0042] (Example 1) As a chromatographic preparative purification device, the ultra-high-speed preparative trap purification system "Nexera UFPLC" (manufactured by Shimadzu Corporation) was used. As the measurement sample (target component), guaiazulene that exhibits a blue color was used. The separation step, capture step, elution step, and recovery step were carried out under the measurement conditions shown below (see Figures 1 and 2).

[0043] <LC Preparative Unit> Mobile phase: Methanol Flow rate of the mobile phase: 4 mL / min Separation column: Not used for the analysis of the standard product Injection volume of the measurement sample: 100 μL Detection wavelength of the UV detector: 230 nm ("SPD-20A", manufactured by Shimadzu Corporation)

[0044] <Purification Unit> Diluent: Water Flow rate of the diluent: 8 mL / min Elution organic solvent: Dichloromethane Flow rate of the elution organic solvent: 0.5 mL / min Trap column: Shim-Pack UFPLC 20×30 (35 mm×8 mm I.D., 20 - 30 μm) Detection wavelength of the UV detector: 254 nm ("SPD-20A", manufactured by Shimadzu Corporation)

[0045] The chromatogram obtained by the UV detector (second detector 14) in the purification unit is shown in Figure 3. Regarding this, the components fractionated (recovered) at 7.4 to 7.9 minutes were fraction 1 (Fr.1), the components fractionated at 7.9 to 8.4 minutes were fraction 2 (Fr.2), the components fractionated at 8.4 to 8.9 minutes were fraction 3 (Fr.3), the components fractionated at 8.9 to 9.4 minutes were fraction 4 (Fr.4), and the components fractionated at 9.4 to 9.9 minutes were fraction 5 (Fr.5), and they were recovered. When these were put into test tubes and observed, it was observed that the liquid recovered as fraction 2 did not cause layer separation and exhibited only blue color. That is, it was found that substantially no water was mixed in.

[0046] (Example 2) The procedure was carried out in the same manner as in Example 1, except that the inflow rate of the elution organic solvent was changed to 0.25 mL / min. The chromatogram obtained by the UV detector in the purification section is shown in Figure 4. Each fraction was collected by separating it as shown by the dashed line in Figure 4. When the liquid of fraction 2 was observed, it was found that the liquid had not undergone layer separation and consisted of a deep blue liquid (a mixture of guaiazulene and organic solvent). In other words, it was found that virtually no water was present.

[0047] (Example 3) The procedure was carried out in the same manner as in Example 1, except that the inflow rate of the elution organic solvent was changed to 0.75 mL / min. The chromatogram obtained by the UV detector in the purification section is shown in Figure 5. The fractions were collected by separating them as shown in Figure 5. When the liquid of fraction 3 was observed, a clear liquid was observed near the surface, indicating layer separation between the clear liquid (water) and the deep blue liquid. In other words, a small amount of water was mixed in. When the amount of water mixed in was measured, it was less than 5% by volume relative to the blue liquid.

[0048] (Example 4) The procedure was carried out in the same manner as in Example 1, except that the inflow rate of the elution organic solvent was changed to 1 mL / min. The chromatogram obtained by the UV detector in the purification section is shown in Figure 6. Each fraction was collected by separating the samples as shown in Figure 6. When the liquid of fraction 2 was observed, a clear liquid was observed near the surface, indicating layer separation between the clear liquid (water) and the deep blue liquid. In other words, a small amount of water was present. The amount of water present was measured to be less than 5% by volume relative to the deep blue liquid.

[0049] (Comparative Example 1) The procedure was carried out in the same manner as in Example 1, except that the inflow rate of the elution organic solvent was changed to 1.5 mL / min. The chromatogram obtained by the UV detector in the purification section is shown in Figure 7. Each fraction was collected by separating the samples as shown in Figure 7. When the liquid of fraction 2 was observed, a clear liquid was observed at the top, indicating layer separation between the clear liquid (water) and the deep blue liquid. In other words, a large amount of water was mixed in. When the amount of water mixed in was measured, it exceeded 10 volume percent relative to the deep blue liquid.

[0050] (Comparative Example 2) The procedure was carried out in the same manner as in Example 1, except that the inflow rate of the elution organic solvent was changed to 2 mL / min. The chromatogram obtained by the UV detector in the purification section is shown in Figure 8. Each fraction was collected by separating the samples as shown in Figure 8. When the liquid of fraction 2 was observed, a clear liquid was observed at the top, indicating layer separation between the clear liquid (water) and the deep blue liquid. In other words, a large amount of water was mixed in. When the amount of water mixed in was measured, it exceeded 10 volume percent relative to the deep blue liquid.

[0051] (Comparative Example 3) The procedure was carried out in the same manner as in Example 1, except that the inflow rate of the elution organic solvent was changed to 4 mL / min. The chromatogram obtained by the UV detector in the purification section is shown in Figure 9. Each fraction was collected by separating the samples as shown in Figure 9. When the liquid of fraction 2 was observed, a clear liquid was observed at the top, indicating layer separation between the clear liquid (water) and the deep blue liquid. In other words, a large amount of water was mixed in. When the amount of water mixed in was measured, it exceeded 10 volume percent relative to the deep blue liquid. [Explanation of Symbols]

[0052] 1 Preparation device 2 Separation section 3 Purification section 4 Separation pump 5 Sample injection unit 6 Separation column 7 First detection unit 8 Mobile phase container 9 First flow path switching valve 10 Second flow path switching valve 11 Elution pump 12 Dilution pump 13 Trap column 14 Second detector 15 Recovery unit 16 Solvent container for elution 17 Diluent container 18 Control unit

Claims

1. A method for separating a target component from a sample to be measured, A separation step in which the target component is separated by passing the measurement sample together with the mobile phase through a separation column. A capture step in which the separated target component is captured in a trap column, An elution step in which the target component is eluted from the trap column by introducing an organic solvent for elution into the trap column, and A recovery step to recover the target component that has leaked out from the trap column. Equipped with, A preparative method for separating water and the target component flowing out of the trap column by adjusting the inflow rate of the elution organic solvent into the trap column during the elution step.

2. The preparative method according to claim 1, wherein the inflow rate is 1 mL / min or less.

3. The preparative method according to claim 1, wherein the inflow rate is 0.6 mL / min or less.

4. The separation method according to claim 1, wherein, after the recovery step, the target component is analyzed by a nuclear magnetic resonance spectrometer.

5. An apparatus for carrying out the sorting method described in any one of claims 1 to 4, A separation column for separating the aforementioned target component, A trap column for capturing the separated target component, Elution means for introducing the elution organic solvent into the trap column, A recovery means for recovering the target component that has leaked out of the trap column, and Control means for adjusting the inflow rate of the elution organic solvent into the trap column. A sorting device equipped with the following features.

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