Fractionation method and fractionation device

Abstract

Provided is a fractionation method capable of inhibiting the mixing of water into a target component to be fractionated. A method for fractionating a target component from a sample includes a separation step of separating the target component, a trapping step of trapping the separated target component in a trapping column, an elution step of causing the target component to flow out of the trapping column, and a recovery step of recovering the target component that has flowed out of the trapping column. In the elution step, water and the target component that have flowed out of the trapping column are separated by adjusting the flow rate of an elution organic solvent into the trapping column.

Description

Technical Field

[0001] This invention relates to a separation method and a separation device. Background Technology

[0002] A separate LC apparatus has been provided (for example, see Patent Document 1), which continuously (online) purifies and recovers the separated components after separating and measuring a sample by liquid chromatography. Specifically, in the LC section (separation section), the sample to be measured is passed through a separation column together with the mobile phase, thereby separating multiple target components from the sample. Then, in the purification section, the various target components are temporarily captured in a collection column, eluted with an eluent, and recovered individually. Using this separate LC apparatus, multiple target components present mixed in the sample can be purified and recovered individually, thus allowing the target components to be provided by type to analytical devices such as nuclear magnetic resonance (NMR) devices, enabling more detailed analysis of the target components.

[0003] Existing technical documents Patent documents Patent Document 1: WO2017 / 033256 Summary of the Invention The technical problem that the invention aims to solve However, in the separation LC apparatus, the following is implemented: In the LC section for separating the sample, the aqueous mobile phase is mixed with the sample in reversed-phase chromatography separation mode and passed through the separation column. In this separation mode, subsequently, in the purification section, each target component enters the trapping column along with the aqueous mobile phase. Therefore, when the target components trapped in the trapping column are eluted into the eluent and recovered together with the eluent, water may be mixed into the recovered target components in addition to the eluent. Furthermore, regardless of the separation mode, there is a possibility of washing the target components trapped in the trapping column with an aqueous liquid (diluent, etc.), which also results in water intrusion into the trapping column and water mixing into the eluent. Water contamination can adversely affect subsequent analyses, therefore, water removal operations (e.g., drying, distillation) are required for the recovered target components, which is time-consuming and labor-intensive.

[0004] This invention can suppress the mixing of water into the separated target components.

[0005] Solution to the above technical problems The separation method according to a first aspect of the present invention comprises, in sequence: a separation step, passing a sample solution through a separation column to separate a target component from the sample solution; a capture step, capturing the separated target component in a capture column; an elution step, allowing an elution organic solvent to flow into the capture column to elute the target component from the capture column; and a recovery step, recovering the target component eluted from the capture column, wherein in the elution step, water discharged from the capture column and the target component are separated by controlling the flow rate of the elution organic solvent to the capture column.

[0006] The separation apparatus of the first aspect of the present invention is an apparatus for implementing the separation method of the first aspect, comprising: a separation column for separating the target component; a trapping column for capturing the separated target component; an elution unit for allowing the elution organic solvent to flow into the trapping column; a recovery unit for recovering the target component flowing out of the trapping column; and a control unit for adjusting the flow rate of the elution organic solvent into the trapping column.

[0007] Invention Effects According to a first aspect of the present invention, it is possible to extract the target component while suppressing the mixing of water. Attached Figure Description

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[0017] 1. First Implementation Method 1-1. Dispensing device use Figures 1-2 An example of a separation apparatus used in a first embodiment of the first aspect of the present invention will be described. This separation apparatus 1 is a chromatographic separation and purification apparatus, such as… Figures 1-2 As shown, it continuously includes a separation section 2 and a purification section 3.

[0018] Separation unit 2 is a unit for performing liquid chromatography separation, and includes 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 component that injects the sample to be measured, introducing the sample into the flow path within the separation unit 2. The sample injection unit 5 is, for example, an autosampler. As the separation column 6, various columns can be used depending on the type of target component (compound), and reversed-phase columns such as C18 columns are preferred. The first detector 7 detects each target component separated by the separation column 6 and outputs it as a chromatogram. Examples of first detectors 7 include, for example, ultraviolet-visible absorbance (UV) detectors, photodiode (PDA) detectors, and fluorescence detectors. Furthermore, although not shown, multiple separation pumps 4 can be provided depending on the type of liquid contained in the mobile phase.

[0019] Purification unit 3 is a unit that purifies and recovers each target component separated from separation unit 2, and includes a first flow path switching valve 9, a second flow path switching valve 10, an elution pump 11, a dilution pump 12, a collection column 13, a second detector 14, and a recovery unit 15.

[0020] The first flow path switching valve 9 is a valve capable of selecting any flow path from multiple flow paths, such as a six-way valve. By switching, the first flow path switching valve 9 can guide the target component delivered from the separation section 2 to the collection column 13, or guide the elution organic solvent delivered from the elution pump 11 to the collection column 13.

[0021] The second flow path switching valve 10 is located downstream of the first flow path switching valve 9 and is a valve capable of selecting any flow path from multiple flow paths, such as a six-way valve. By switching, the second flow path switching valve 10 can guide the target component delivered from the separation unit 2 via the first flow path switching valve 9 to the collection column 13, or guide the diluent delivered from the dilution pump 12 to the collection column 13, or guide the component delivered from the separation unit 2 via the first flow path switching valve 9 to the outside of the separation device 1.

[0022] Elution pump 11 is an elution unit that delivers the elution organic solvent contained in elution solvent container 16 to the trapping column 13, eluting the target component into the organic solvent. Dilution pump 12 delivers the diluent contained in diluent container 17 to the trapping column 13. The trapping column 13 is a column for purifying the target component, temporarily capturing the target component delivered from separation section 2 and allowing non-essential components other than the target component to pass through (remove). The type of trapping column 13 is appropriately determined according to the target component; specifically, the Shim-pack series manufactured by Shimadzu Corporation can be cited as an example. Second detector 14 detects each target component from the trapping column 13 and outputs a chromatogram. The second detector 14 can be the same as the first detector 7. Recovery section 15 is a recovery unit that contains each target component separately in different recovery containers, such as a fraction collector. Furthermore, although not shown, multiple elution pumps 11 and dilution pumps 12 may be provided depending on the type of liquid contained in the elution organic solvent or diluent.

[0023] The separation device 1 is equipped with a control unit 18, including a computer. The control unit 18 is connected to various pumps (separation pump 4, elution pump 11, dilution pump 12), the sample injection unit 5, the flow path switching valves 9 and 10, and the recovery unit 15, and stores programs for controlling these components. Specifically, the program in the control unit 18 controls the various pumps (separation pump 4, elution pump 11, dilution pump 12), adjusting the inflow rate and flow rate of each liquid delivered 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 sample to be measured in the introduced flow path. It controls the flow path switching valves 9 and 10 to adjust the switching between multiple flow paths. It controls the recovery unit 15 to adjust the collection of each target component arriving at the recovery unit 15 into each container according to the target component and / or according to time.

[0024] 1-2. Sorting Method An example of a dispensing method according to a first embodiment of the first aspect of the present invention will be described. This dispensing method uses a dispensing device 1 and includes a preparation step and an implementation step in sequence.

[0025] (1) Preparation steps In this step, the sample to be measured, the mobile phase, the diluent, and the organic solvent for elution are loaded into the dispensing device 1.

[0026] Specifically, the sample containing the target component is injected into the sample injection unit 5. Furthermore, the mobile phase, elution organic solvent, and diluent are injected into the mobile phase container 8, the elution solvent container 16, and the diluent container 17, respectively. Each container can be single or multiple, depending on the type of liquid used. In addition, depending on the number of containers, one or more pumps are also prepared.

[0027] The mobile phase is appropriately determined based on the type of target component, column type, separation mode, etc., but a liquid suitable for reversed-phase mode is preferred. Specifically, examples include water and organic solvents. Water as the mobile phase can be a buffer solution containing acetic acid, ammonium acetate, formic acid, ammonium formate, ammonia, etc. Organic solvents include alcohols such as methanol and ethanol, and solvents such as acetonitrile and acetone. These can be used individually or in mixtures of two or more. In the case of mixing, a mixture of two or more solvents can be pre-injected into a mobile phase container. Alternatively, multiple mobile phase containers 8 can be prepared, each containing water or an organic solvent, and these liquids can be mixed in the flow path. The mobile phase preferably contains water, and more preferably a mixture of water and an organic solvent. This allows for the implementation of reversed-phase chromatography, enabling more reliable separation of a wide variety of compounds.

[0028] The diluent is a liquid used to purify the target component; it can be any solvent immiscible with the target component, and the choice depends on the target component. Examples of diluents include water and organic solvents, which are exemplified in the mobile phase. The diluent preferably contains water, and more preferably water or a mixture of water and an organic solvent. This allows the removal of unnecessary components adhering to the target component captured by the collection column, thus cleaning (purifying) the target component. In the first embodiment, it is preferable that at least one of the mobile phase and the diluent contains water.

[0029] As an organic solvent for elution, any organic solvent that can dissolve the target component is acceptable, and the choice should be appropriate based on the target component. Examples include acetone, acetonitrile, and dichloromethane. These can be used individually or in mixtures of two or more.

[0030] (2) Implementation steps In this step, the pumps 4, 11, 12, valves 9, 10, detectors 7, 14 and control unit 18 of the separation device 1 are operated to separate and purify the target component.

[0031] Specifically, in separation section 2, the mobile phase is delivered from mobile phase container 8 by the operation of separation pump 4. In sample injection section 5, the sample to be measured is mixed with the mobile phase and passed through separation column 6. During passage, the sample to be measured flowing into separation column 6 is separated according to each target component, and simultaneously flows out of separation column 6 (separation step). The separation solution, consisting of the sample to be measured that has passed through separation column 6 and the mobile phase, is detected by first detector 7 and then reaches purification section 3. First detector 7 outputs a chromatogram of the sample to be measured. By confirming this chromatogram, the separation of the target components and the elution time of the target components can be confirmed.

[0032] The flow rate of the mobile phase, and more specifically, the inflow rate V1 of the mobile phase containing the sample to the separation column 6, is not limited, but is, for example, 1 mL / min or more, preferably 2 mL / min or more, and, for example, 20 mL / min or less, preferably 10 mL / min or less.

[0033] Next, in purification section 3, as... Figure 1 As shown, the separated liquid flows into the collection column 13 sequentially via the first flow path switching valve 9 and the second flow path switching valve 10. At this time, through the operation of the dilution pump 12, the diluent is delivered from the diluent container 1 and, via the second switching valve, merges with the separated liquid. The mixture of separated liquid and diluent flows into the collection column 13. At this point, the target component is captured in the collection column 13 (capture step). On the other hand, the mobile phase and diluent flow out of the collection column 13 and are discharged to the outside of the separation device 1 via the first flow path switching valve 9. Thus, unnecessary components mixed in with the separated liquid are discharged to the outside along with the diluent.

[0034] Next, as Figure 2 As shown, through the switching (rotation) of the first flow path switching valve 9, the operation of the elution pump 11, and the stopping of the dilution pump 12, the elution organic solvent is supplied from the elution solvent container 16, flows into the trapping 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 trapping column 13 together with the elution organic solvent. Then, the target component is sequentially detected by the second detector 14 via the second flow path switching valve 10 and the first flow path switching valve 9, and is recovered to the recovery unit 15 (recovery step). The second detector 14 outputs a chromatogram of the target component.

[0035] In the purification unit 3, capture and elution steps are performed intermittently or continuously, depending on the type of target component or by time. For example, referring to the chromatogram obtained by the first detector 7, the first flow path switching valve 9 and the second flow path switching valve 10 are switched at any time in a manner corresponding to the peaks shown for each target component. As described above, the desired target component is guided to the capture column 13 along with the mobile phase and to the recovery unit 15 along with the elution organic solvent. On the other hand, for the mobile phase that does not contain the target component (the mobile phase that does not show a retention time of the peak), by switching the first flow path switching valve 9 and / or the second flow path switching valve 10, it is discharged directly to the outside of the separation device 1 without passing through the capture column 13. These operations are repeated for each target component. Thus, the target components are individually captured in the capture column 13 according to their type and eluted.

[0036] At this time, the flow rate of the diluent into the collection column 13 is not limited. For example, it can be set to 1 mL / min or more, preferably 5 mL / min or more. Alternatively, it can be set to 50 mL / min or less, preferably 40 mL / min or less.

[0037] In the first embodiment, during the elution step, the delivery rate of the elution organic solvent, and consequently, the inflow rate V2 of the elution organic solvent into the collection column 13, is adjusted to the rate at which water and the target component are separated in the liquid flowing out of the collection 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 separation completion time, it is acceptable to set it to, 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 collection 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 furthermore, 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 removed during the separation of the target component.

[0038] In the recovery step, recovery is performed in the recovery unit 15 according to the target component or according to time. That is, based on the chromatogram obtained by the second detector 14, the peaks representing the target component are divided, and the divided target components are collected into individual recovery containers. As a result, it is possible to separate only the desired target component from the continuously arriving target components. In particular, it is possible to separate the anhydrous target component together with the elution organic solvent.

[0039] The target component recovered in the recovery step is then preferably analyzed using an analytical apparatus such as a nuclear magnetic resonance device (analysis step). This allows for the acquisition of detailed information about the target component.

[0040] According to the first embodiment, it is possible to suppress the contamination of water in the separated target component. This is presumably as follows. In the prior art, during the elution step, the component discharged from the trapping column 13 contains, in addition to the target component and the elution organic solvent, the mobile phase and diluent remaining from the trapping step, and at least one of the mobile phase and diluent contains water. Therefore, water is also contaminated in the ultimately recovered target component. In contrast, according to the first embodiment, the inflow rate of the elution organic solvent into the trapping column 13 is controlled to prevent water from contaminating the recovered target component. Specifically, the inflow rate is significantly reduced. This significantly delays the time for the target component to come into contact with and be eluted by the elution organic solvent. Therefore, water begins to be discharged from the trapping column 13 before the target component is discharged. That is, water is fully discharged first, followed by the target component. Therefore, the target component recovered in the recovery section 15 is separated based on the peaks of the chromatogram of the second detector 14, thereby suppressing the contamination of water in the obtained target component. As a result, when analyzing the target components, there is no need to remove water through distillation or other means, and the separation and analysis of the target components can be carried out smoothly.

[0041] In one example of the first embodiment described above, the application of a nuclear magnetic resonance (NMR) apparatus as an analytical step was illustrated. However, as other application examples, the present invention can also be applied to situations where the presence of water would have an impact when measured in other apparatuses. Examples of such other apparatuses include gas chromatographs, ultraviolet spectrophotometers, and Fourier transform infrared (FTIR) spectrometers.

[0042] 2. Aspects Those skilled in the art will understand that the above exemplary embodiments are specific examples of the following aspects.

[0043] (Item 1) One aspect of the separation method can be a method for separating a target component from a sample, comprising: a separation step in which the sample and a mobile phase are passed together through a separation column, thereby separating the target component; a capture step in which the separated target component is captured in a capture column; an elution step in which an elution organic solvent is introduced into the capture column, thereby causing the target component to elute from the capture column; and a recovery step in which the target component elutes from the capture column, wherein in the elution step, water and the target component elute from the capture column are separated by adjusting the inflow rate of the elution organic solvent into the capture column.

[0044] (Item 2) In the separation method described in Item 1, the inflow rate may be less than 1 mL / min.

[0045] (Item 3) In the fractionation method according to Item 1 or 2, the inflow rate may be 0.6 mL / min or less.

[0046] (Item 4)In the fractionation method according to any one of Items 1 to 3, after the recovery step, the target component may be analyzed by a nuclear magnetic resonance apparatus.

[0047] (Item 4)A fractionation device on one hand may be a device for implementing the fractionation method according to any one of Items 1 to 4, and includes: a separation column for separating the target component; a capture column for capturing the separated target component; an elution unit for flowing the elution organic solvent into the capture column; a recovery unit for recovering the target component flowing out from the capture column; and a control unit for adjusting the inflow rate of the elution organic solvent into the capture column.

[0048]

Examples

[0049] (Example 1) As a chromatographic fractionation and purification device, a super-high-speed fractionation and capture purification system "Nexera UFPLC" (manufactured by Shimadzu Corporation) was used. As a measurement sample (target component), azulene showing blue was used. The separation step, capture step, elution step, and recovery step were carried out according to the following measurement conditions (refer to Figure 1 and Figure 2 ).

[0050] <LC fractionation part> Mobile phase: methanol Inflow rate of the mobile phase: 4 mL / min Separation column: Not used because it is an analysis of a standard product Injection volume of the measurement sample: 100 μL Detection wavelength of the UV detector: 230 nm ("SPD-20A", manufactured by Shimadzu Corporation) <Purification part> Diluent: water Inflow rate of the diluent: 8 mL / min Elution organic solvent: dichloromethane Inflow rate of the elution organic solvent: 0.5 mL / min Capture 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) The chromatogram obtained by the UV detector (second detector 14) in the purification section is shown in Figure 3 For this diagram, the component collected (recovered) at 7.4–7.9 fractions is designated as fraction 1 (Fr.1), the component collected at 7.9–8.4 fractions is designated as fraction 2 (Fr.2), the component collected at 8.4–8.9 fractions is designated as fraction 3 (Fr.3), the component collected at 8.9–9.4 fractions is designated as fraction 4 (Fr.4), and the component collected at 9.4–9.9 fractions is designated as fraction 5 (Fr.5) for recovery. When these fractions are placed in test tubes for observation, it is observed that the liquid recovered as fraction 2 does not undergo layer separation and only appears blue. That is, it can be concluded that it is essentially not mixed with water.

[0051] (Example 2) Except for changing the flow rate of the elution organic solvent to 0.25 mL / min, the procedure was the same as in Example 1. The chromatogram obtained from the UV detector in the purification section is shown in [image / image / etc.]. Figure 4 .like Figure 4 The fractions were separated and recovered as indicated by the dotted lines. Observing fraction 2, it was found that no layer separation occurred; the liquid consisted of a deep blue liquid (a mixture of guaiazoline and an organic solvent). This indicates that water was not substantially mixed in.

[0052] (Example 3) Except for changing the flow rate of the elution organic solvent to 0.75 mL / min, the procedure was the same as in Example 1. The chromatogram obtained from the UV detector in the purification section is shown in [image / image / etc.]. Figure 5 .like Figure 5 The fractions were separated and recovered as shown. Observation of fraction 3 revealed a transparent liquid near the surface, indicating that the transparent liquid (water) separated from the dark blue liquid. This suggests the presence of trace amounts of water. The degree of water contamination was measured and found to be less than 5% by volume relative to the blue liquid.

[0053] (Example 4) Except for changing the inflow rate of the elution organic solvent to 1 mL / min, the procedure was the same as in Example 1. The chromatogram obtained from the UV detector in the purification section is shown in... Figure 6 .like Figure 6 The fractions were separated and recovered as shown. Observation of fraction 2 revealed a transparent liquid near the surface, indicating that the transparent liquid (water) separated from the dark blue liquid. This suggests the presence of trace amounts of water. The degree of water contamination was measured and found to be less than 5% by volume relative to the dark blue liquid.

[0054] (Comparative Example 1) Except for changing the flow rate of the elution organic solvent to 1.5 mL / min, the procedure was the same as in Example 1. The chromatogram obtained from the UV detector in the purification section is shown in [image / image / etc.]. Figure 7 .like Figure 7 The fractions were separated and recovered as shown. Observation of fraction 2 revealed a transparent liquid (water) at the top, indicating layer separation between the transparent liquid and the dark blue liquid. This suggests significant water contamination. The degree of water contamination was measured and found to exceed 10% by volume relative to the dark blue liquid.

[0055] (Comparative Example 2) Except for changing the flow rate of the elution organic solvent to 2 mL / min, the procedure was the same as in Example 1. The chromatogram obtained from the UV detector in the purification section is shown in... Figure 8 .like Figure 8 The fractions were separated and recovered as shown. Observation of fraction 2 revealed a transparent liquid (water) at the top, indicating layer separation between the transparent liquid and the dark blue liquid. This suggests significant water contamination. The degree of water contamination was measured and found to exceed 10% by volume relative to the dark blue liquid.

[0056] (Comparative Example 3) Except for changing the inflow rate of the elution organic solvent to 4 mL / min, the procedure was the same as in Example 1. The chromatogram obtained from the UV detector in the purification section is shown in... Figure 9 .like Figure 9 The fractions were separated and recovered as shown. Observation of fraction 2 revealed a transparent liquid (water) at the top, indicating layer separation between the transparent liquid and the dark blue liquid. This suggests significant water contamination. The degree of water contamination was measured and found to exceed 10% by volume relative to the dark blue liquid.

[0057] [Symbol Explanation] 1. Dispensing device 2 Separation section 3 Purification Department 4. Separation pump 5. Sample injection section 6 Separation Column 7. First Inspection Department 8. Mobile phase container 9. First flow path switching valve 10 Second Flow Path Switching Valve 11. Elution pump 12. Dilution pump 13 Trapping column 14. Second detector 15 Recycling Department 16 Elution solvent containers 17. Diluent container 18. Control Department.

Claims

1. A separation method for separating a target component from a sample, comprising: The separation step involves passing the test sample and the mobile phase together through a separation column, thereby separating the target component. The capture step involves capturing the separated target component in a capture column; The elution step involves flowing an elution organic solvent into the trapping column, thereby causing the target component to elute from the trapping column; and The recovery step involves recovering the target component flowing out of the trapping column. In the elution step, the water flowing out of the collection column and the target component are separated by adjusting the flow rate of the elution organic solvent into the collection column.

2. The extraction method as described in claim 1, wherein, The inflow rate is less than 1 mL / min.

3. The extraction method as described in claim 1, wherein, The inflow rate is less than 0.6 mL / min.

4. The extraction method as described in claim 1, wherein, Following the recovery step, the target component is analyzed using a nuclear magnetic resonance (NMR) device.

5. A dispensing apparatus for implementing the dispensing method according to any one of claims 1 to 4, comprising: A separation column for separating the target component; A collection column for capturing the separated target components; The elution organic solvent is allowed to flow into the elution unit of the collection column; A recovery unit for recovering the target component flowing out of the trapping column; and A control unit that adjusts the inflow rate of the elution organic solvent into the trapping column.

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