A supercritical fluid chromatography separation method and device

Abstract

The present application discloses a supercritical fluid chromatographic separation method and device, the supercritical fluid chromatographic separation method comprising: placing a sample in a pre-separation device; connecting one end of the pre-separation device to the pipeline in front of the chromatographic column, and connecting the other end of the pre-separation device to a supercritical fluid preparative chromatographic column; using supercritical carbon dioxide as an extraction solvent, pre-separating the sample, and loading the pre-separated target component into the supercritical fluid preparative chromatographic column; connecting the pipeline in front of the chromatographic column to the supercritical fluid preparative chromatographic column; using supercritical carbon dioxide and a modifier as a mobile phase to separate the target component. The supercritical fluid chromatographic separation method adopted in the present application can complete the extraction, pretreatment, loading and separation of the sample in one step, solve the problem that the strength and viscosity of the sample dissolving reagent in supercritical fluid chromatography do not match the mobile phase, and eliminate the interference of the loading solvent and impurities in the sample on the supercritical fluid chromatographic separation.

Description

Technical Field

[0001] The present application relates to the field of chromatography technology, and in particular to a supercritical fluid chromatography separation method and device. Background Art

[0002] In recent years, environmentally friendly subcritical / supercritical carbon dioxide (Sc-CO 2 Supercritical fluid chromatography (SFC) with ) as the mobile phase is considered to be a green, environmentally friendly, efficient and fast separation technology. Compared with liquid chromatography (LC), SFC has a faster flow rate (about 3 times that of LC), a higher separation flux and a lower organic solvent consumption. In particular, preparative SFC has certain advantages in sample purification due to its mild separation conditions, rapid separation and simple post-processing.

[0003] However, sample dissolution has always been a difficult problem in SFC separation. The mobile phase of SFC consists of supercritical CO 2 and a certain proportion of modifiers. Most of the samples are dissolved in the modifier, and its strength and viscosity do not match the SFC mobile phase, which often leads to band broadening, decreased separation, and poor repeatability. This problem is more prominent in preparative SFC, and the sample may even precipitate and re-dissolve after injection, resulting in serious distortion of the chromatographic peaks. In addition, when separating complex samples, interference from impurities often becomes an obstacle to efficient SFC separation. The polarity distribution range of complex samples is relatively wide, which increases the difficulty of sample dissolution, and some impurities are not easy to elute in SFC, which also causes problems for separation. In summary, there is an urgent need to develop a method to simultaneously solve the problems of injection solvent mismatch and impurity interference in preparative SFC. Summary of the invention

[0004] The present application provides a supercritical fluid chromatography separation method and device, which can realize sample extraction, pretreatment, sample loading and separation in one step.

[0005] The present application provides a supercritical fluid chromatographic separation method, comprising: placing a sample in a pre-separation device; connecting one end of the pre-separation device to a pre-column pipeline of a chromatographic column, and connecting the other end of the pre-separation device to a supercritical fluid preparative chromatographic column; using supercritical carbon dioxide as an extraction solvent to pre-separate the sample, and loading the pre-separated target component into the supercritical fluid preparative chromatographic column; connecting the pre-column pipeline of the chromatographic column to the supercritical fluid preparative chromatographic column; using supercritical carbon dioxide and a modifier as mobile phases to separate the target component.

[0006] Optionally, in some embodiments of the present application, pre-separation includes extraction, pre-treatment and loading.

[0007] Optionally, in some embodiments of the present application, the sample is mixed with the chromatographic filler and then placed in a pre-separation device.

[0008] Optionally, in some embodiments of the present application, the extraction solvent further includes a modifier, and the modifier accounts for 0 to 10% of the total flow of the supercritical carbon dioxide and the modifier.

[0009] Optionally, in some embodiments of the present application, the modifier includes at least one of methanol, ethanol, isopropanol or acetonitrile.

[0010] Optionally, in some embodiments of the present application, the chromatographic filler includes a silica matrix filler or a macroporous resin filler.

[0011] Optionally, in some embodiments of the present application, the particle size range of the chromatographic filler may be 10 to 60 μm, 20 to 50 μm, or 30 to 40 μm.

[0012] Optionally, in some embodiments of the present application, the sample includes Hedyotis diffusa or Alpinia galanga or an extract thereof.

[0013] Optionally, in some embodiments of the present application, the mass ratio of the sample to the chromatographic filler can be 1:(1.5-10), or 1:(2-9), or 1:(3-8).

[0014] Optionally, in some embodiments of the present application, under the pre-separation conditions, the flow rate range of supercritical carbon dioxide can be 5-50 mL / min, 10-40 mL / min, or 20-30 mL / min.

[0015] Optionally, in some embodiments of the present application, under the pre-separation conditions, the back pressure range may be 100 to 150 bar, 110 to 140 bar, or 120 to 130 bar.

[0016] Optionally, in some embodiments of the present application, under the pre-separation conditions, the extraction time range can be 10 to 20 minutes, 11 to 19 minutes, or 12 to 18 minutes.

[0017] Optionally, in some embodiments of the present application, the mixing step includes: dissolving the sample in a solvent, adding a chromatographic filler, and removing the solvent after grinding.

[0018] Optionally, in some embodiments of the present application, the step of separating the target component includes: eluting on a supercritical fluid preparative chromatography column under isocratic or gradient conditions to separate the target component.

[0019] Optionally, in some embodiments of the present application, the diameter of the supercritical fluid preparation chromatographic column ranges from 10 to 30 mm, and the diameter of the supercritical fluid preparation chromatographic column can be 10 mm, 20 mm, or 30 mm.

[0020] Optionally, in some embodiments of the present application, the length of the supercritical fluid preparation chromatography column ranges from 150 to 250 mm, and the length of the supercritical fluid preparation chromatography column can be 150 mm or 250 mm.

[0021] Optionally, in some embodiments of the present application, the flow rate range of the supercritical fluid preparation chromatography column can be 10-200 mL / min, or 20-150 mL / min, or 50-100 mL / min.

[0022] Optionally, in some embodiments of the present application, the sample loading amount of the supercritical fluid preparation chromatography column can be 1 to 1000 mg, or 10 to 500 mg, or 50 to 100 mg.

[0023] Optionally, in some embodiments of the present application, the retention capacity of the supercritical fluid preparation chromatography column for the target component is greater than the chromatography filler used for pre-separation.

[0024] Correspondingly, the present application also provides a supercritical fluid chromatography device, including a chromatographic column front pipeline, a supercritical fluid preparation chromatographic column and a detector, the supercritical fluid chromatography device also includes a pre-separation device; when the pre-separation device realizes the extraction, pretreatment and loading functions, one end of the pre-separation device is connected to the chromatographic column front pipeline, and the other end of the pre-separation device is connected to the supercritical fluid preparation chromatographic column; when the pre-separation device completes the extraction, pretreatment and loading functions, the connection of the pre-separation device is disconnected, the chromatographic column front pipeline is connected to the supercritical fluid preparation chromatographic column, and the separation of the target component begins.

[0025] Optionally, in some embodiments of the present application, connection holes are provided at both ends of the pre-separation device.

[0026] Optionally, in some embodiments of the present application, the pressure range of the pre-separation device may be 300 to 400 bar, 310 to 390 bar, or 320 to 380 bar.

[0027] Optionally, in some embodiments of the present application, the pre-separation device is a stainless steel high-sealing device.

[0028] This application has the following beneficial effects through one-step pretreatment and separation of SFC samples:

[0029] 1) CO 2It is not only the extraction solvent in the pre-separation device, but also the loading solvent of SFC, and matches the mobile phase of SFC. Therefore, the problem of peak broadening caused by the mismatch between the strength and viscosity of the sample dissolving reagent in the preparation of SFC and the SFC mobile phase is solved.

[0030] 2) Mixing the sample with the chromatographic filler is based on the adsorption characteristics of the chromatographic filler. During the extraction process, the non-target components are retained on the chromatographic filler, thereby achieving the selective extraction of the target components and eliminating the interference of impurities on the subsequent SFC separation. In addition, the addition of the chromatographic filler disperses the sample, improves the contact between the sample and the solvent, and avoids CO 2 The flow through the mixture creates a channeling effect.

[0031] 3) Continuous loading of target components can be achieved during extraction and pretreatment, and sample extraction, pretreatment, loading and separation can be achieved in one step, which is convenient to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0033] Figure 1 is a schematic diagram of a supercritical fluid chromatography apparatus;

[0034] Figure 2 It is a schematic diagram of supercritical fluid chromatography separation method;

[0035] Figure 3 is the SFC chromatogram of the sample in Example 1;

[0036] Figure 4 is the SFC chromatogram of the sample injected as liquid in the comparative example; DETAILED DESCRIPTION

[0037] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of this application.

[0038] The present application provides a supercritical fluid chromatography separation method and device. The following are detailed descriptions. It should be noted that the description order of the following embodiments is not intended to limit the preferred order of the embodiments.

[0039] In the present application, supercritical carbon dioxide refers to a type of carbon dioxide between liquid and gas. When it is at the critical temperature (31.06°C) and the critical pressure (7.39MPa), that is, at the critical point and above, the interface between the liquid and gas states suddenly disappears, forming a new state that has both the high permeability of gas and the high solubility of liquid, and also has new properties, such as low viscosity and large self-diffusion coefficient of supercritical carbon dioxide.

[0040] The present application embodiment provides a supercritical fluid chromatography device, such as Figure 1 As shown, it includes a chromatographic column front pipeline 1, a pre-separation device 2, a supercritical fluid preparation chromatographic column 3 and a detector 4; the inlet end of the pre-separation device 2 is connected to the chromatographic column front pipeline 1, the outlet end of the pre-separation device 2 is connected to the front end of the supercritical fluid preparation chromatographic column 3, and the rear end of the supercritical fluid preparation chromatographic column 3 is connected to the pipeline in front of the detector 4.

[0041] In some embodiments of the present application, the supercritical fluid preparation chromatographic column 3 can be a semi-preparative or preparative chromatographic column, the diameter of the chromatographic column is 10 to 30 mm, the flow rate range is 10 to 200 mL / min, and the sample load is 1 to 1000 mg; while the conventionally used analytical SFC column has a diameter of 4.6 mm, a flow rate range of 2 to 4 mL / min, and a sample load of less than 1 mg. The supercritical fluid preparation chromatographic column 3 used in the present application is significantly higher than the conventional analytical SFC column in terms of flow rate range and sample load. The present application uses a supercritical fluid preparation chromatographic column, which can not only achieve sample extraction, loading, enrichment and separation in one step, but also has a large scale of separated samples.

[0042] In the present application, the sample is placed in the pre-separation device 2 for pre-treatment, and the pre-treatment can be extraction or other sample pre-separation methods. The pre-separation device 2 is a highly sealed, high-pressure resistant device. Its specifications are 5 to 25 mL, and a sample amount of up to 10 g can be added to the pre-separation device 2, and the flow rate of the pre-separation device 2 is 5 to 50 mL / min. The pre-separation device in the present application can handle a large amount of samples, and the present application can select different sample pre-treatment methods, which can be used in conjunction with the device of the present application to achieve large-scale pre-treatment and solid loading of samples before supercritical fluid preparation chromatography column separation, thereby achieving selective extraction of target components and eliminating the interference of impurities on subsequent SFC separation.

[0043] The present application also provides a supercritical fluid chromatography separation method, such as Figure 2 As shown, including:

[0044] S1: Mix the sample 5 with the chromatographic filler 6, wherein the mass ratio of the sample 5 to the chromatographic filler 6 is 1:(1.5-10), and load the sample 5 or the mixture of the sample 5 and the chromatographic filler 6 into the pre-separation device 2, and fill the two ends of the pre-separation device 2 with appropriate amounts of absorbent cotton;

[0045] S2: Connect the inlet end of the pre-separation device 2 to the front pipeline 1 of the chromatographic column, connect the outlet end of the pre-separation device 2 to the front end of the supercritical fluid preparation chromatographic column 3, and connect the rear end of the supercritical fluid preparation chromatographic column 3 to the pipeline in front of the detector 4;

[0046] S3: Extraction and sample addition: Extraction under the following conditions, supercritical CO 2 The flow rate range is 5 to 50 mL / min, the back pressure range is 100 to 150 bar, and the extraction time range is 10 to 20 min. The target components in the pre-separation device 2 are continuously extracted and loaded into the supercritical fluid preparation chromatography column 3;

[0047] S4: After the extraction is completed, switch to the SFC path, that is, connect the front pipeline of the chromatographic column 1 to the front end of the supercritical fluid preparation chromatographic column 3, add a modifier, and separate the target components on the supercritical fluid preparation chromatographic column 3.

[0048] In some embodiments of the present application, the chromatographic filler includes a silica matrix filler and a macroporous resin filler, including but not limited to any specifications of silica gel bonded octadecyl (C18), silica gel bonded octyl (C8), silica gel, cation exchange filler (SCX), anion exchange filler (SAX), SCX-C18 mixed filler, and SAX-C18 mixed filler; utilizing the adsorption characteristics of the chromatographic filler, non-target components are firmly retained on the chromatographic filler and are not easily extracted, thereby achieving selective extraction of target components.

[0049] In some embodiments of the present application, the sample and the chromatographic filler are mixed uniformly and prepared as follows: weigh an appropriate amount of the sample in a mortar, add a small amount of ethanol to dissolve the sample, then add the chromatographic filler to the solution, the mass ratio of the two is in the range of 1:(1.5-10), grind uniformly, and evaporate the solvent.

[0050] In some embodiments of the present application, the retention capacity of the supercritical fluid preparative chromatographic column for the target component is stronger than the chromatographic filler mixed with the sample in the pre-separation device. The screening of the supercritical fluid preparative chromatographic column is carried out as follows: the extraction conditions are simulated on the SFC analytical chromatographic column to ensure that the target component is not eluted under these conditions.

[0051] In some embodiments of the present application, after the extraction step is completed, the SFC path is switched, a modifier is added, and elution is performed on a supercritical fluid preparative chromatographic column under isocratic or gradient conditions to complete the separation of the target components. Isocratic elution refers to an elution method in which the composition ratio and flow rate of the mobile phase remain constant during the analysis cycle of the sample components; gradient elution refers to the concentration ratio of the mobile phase being continuously changed to a certain extent during the same analysis cycle.

[0052] In some embodiments of the present application, the modifier includes any one of methanol, ethanol, isopropanol, acetonitrile or a mixed solvent thereof.

[0053] The following describes the invention in conjunction with specific embodiments.

[0054] Embodiment 1

[0055] The materials and material specifications used in this embodiment are as follows:

[0056] Sample: The precipitate after Hedyotis diffusa was extracted with 95% ethanol and then precipitated with water;

[0057] Chromatographic packing: C18 (40 μm, 100A);

[0058] Specifications of pre-separation device: 5mL;

[0059] Specifications of supercritical fluid preparative chromatography column: 250×10mm id, 10μm.

[0060] The supercritical fluid chromatography separation method of this embodiment comprises the following steps:

[0061] S1: Preparation of sample and chromatographic medium mixture

[0062] Weigh the dried powder (100 g) of Hedyotis diffusa, add 95% ethanol (800 mL) with a solid-liquid ratio of 8 times, and reflux extract twice, each time for 1 hour. Combine the two extracts, and use a rotary evaporator to reduce pressure and concentrate to 20 mL to obtain a dark green extract. Slowly add 100 mL of water to the extract under stirring conditions, and let it stand at 4 ° C for 24 hours. The precipitate is the Hedyotis diffusa sample.

[0063] Weigh 1.0 g of Hedyotis diffusa sample into a mortar, add a small amount of ethanol to dissolve the sample, then add 2.0 g of C18 (40 μm, 100A) chromatographic filler to the solution, grind evenly, evaporate the solvent, and obtain a mixture of the sample and chromatographic filler.

[0064] S2: Device connection: Weigh 330 mg of sample and chromatographic filler mixture and load them into a 5 mL pre-separation device. Fill both ends of the pre-separation device with appropriate amounts of absorbent cotton. The inlet of the pre-separation device is connected to the pipeline in front of the chromatographic column, and the outlet of the pre-separation device is connected to the front end of the XAmide semi-preparative column (250×10mm id, 10μm). The rear end of the XAmide semi-preparative column is connected to the pipeline in front of the detector.

[0065] S3: Extraction and loading: In CO 2Under the conditions of flow rate 15 mL / min, back pressure 150 bar, and extraction time 10 min, the target components in the pre-separation device were continuously loaded onto the XAmide semi-preparative column.

[0066] S4: SFC separation: After extraction and loading, switch to the SFC line to start separation of the target components. The chromatographic conditions are as follows: XAmide semi-preparative column (250×10mm id, 10μm), mobile phase is food grade CO 2 (A), modifier is preparative grade methanol (B), 0-5min, 0-4% B, 5-15min, 4-10% B, 15-21min, 10-15% B. Flow rate: 15mL / min, back pressure: 140bar, detection wavelength: 254nm, SFC separation results are shown as follows Figure 3 As shown. Figure 3 It can be seen that the target component has a moderate retention time on the XAmide semi-preparative column and exhibits good separation ability and chromatographic peak shape.

[0067] Embodiment 2

[0068] The materials and material specifications used in this embodiment are as follows:

[0069] Sample: The precipitate after Hedyotis diffusa was extracted with 95% ethanol and then precipitated with water;

[0070] Chromatographic packing: C18 (40 μm, 100A);

[0071] Specifications of pre-separation device: 5mL;

[0072] Specifications of supercritical fluid preparative chromatography column: 250×20mm id, 10μm.

[0073] The supercritical fluid chromatography separation method of this embodiment comprises the following steps:

[0074] S1: Preparation of sample and chromatographic medium mixture

[0075] The preparation method of Hedyotis diffusa sample is the same as that in Example 1.

[0076] 1.0 g of Hedyotis diffusa sample was weighed into a mortar, and a small amount of ethanol was added to dissolve the sample. Subsequently, 3.0 g of C18 (40 μm, 100 A) chromatographic filler was added to the solution, and after grinding evenly, the solvent was evaporated to prepare a sample and chromatographic filler mixture.

[0077] S2: Device connection: Weigh 1.0g of sample and chromatographic filler mixture and load it into a 5mL pre-separation device. Fill both ends of the pre-separation device with appropriate amount of absorbent cotton. The inlet end of the pre-separation device is connected to the pipeline before the chromatographic column, and the outlet end of the pre-separation device is connected to the front end of the XAmide preparative column (250×20mm id, 10μm). The rear end of the XAmide preparative column is connected to the pipeline before the detector.

[0078] S3: Extraction and loading: In CO 2 Under the conditions of flow rate 30 mL / min, back pressure 150 bar, and extraction time 15 min, the target components in the pre-separation device were continuously loaded onto the XAmide preparative column.

[0079] S4: SFC separation: After extraction and loading, switch to the SFC pipeline to separate the target components. The chromatographic conditions include: XAmide preparative column (250×20mm id, 10μm), mobile phase is food grade CO 2 (A), modifier is preparative grade methanol (B), 0-5 min, 0-4% B, 5-15 min, 4-10% B, 15-21 min, 10-15% B. Flow rate: 53 mL / min, back pressure: 150 bar, detection wavelength: 254 nm.

[0080] The target component separated in this example has a moderate retention time on the XAmide semi-preparative column and exhibits good separation ability and chromatographic peak shape.

[0081] Embodiment 3

[0082] The materials and material specifications used in this embodiment are as follows:

[0083] Sample: The precipitate after Hedyotis diffusa was extracted with 95% ethanol and then precipitated with water;

[0084] Chromatographic packing: C8 (40μm, 100A);

[0085] Pre-separation device specifications: 25mL;

[0086] Specifications of supercritical fluid preparative chromatography column: 250×30mm id, 10μm.

[0087] The supercritical fluid chromatography separation method of this embodiment comprises the following steps:

[0088] S1: Preparation of sample and chromatographic medium mixture

[0089] The preparation method of Hedyotis diffusa sample is the same as that in Example 1.

[0090] 2.0 g of Hedyotis diffusa sample was weighed into a mortar, and a small amount of ethanol was added to dissolve the sample. Subsequently, 8.0 g of C8 (40 μm, 100 A) chromatographic filler was added to the solution, and after grinding evenly, the solvent was evaporated to prepare a mixture of the sample and the chromatographic filler.

[0091] S2: Device connection: Weigh 10.0g of sample and chromatographic filler mixture into a 25mL pre-separation device, and fill both ends of the pre-separation device with appropriate amounts of absorbent cotton. The inlet end of the pre-separation device is connected to the pipeline before the chromatographic column, and the outlet end of the pre-separation device is connected to the front end of the XAmide preparative column (250×30mm id, 10μm), and the rear end of the XAmide preparative column is connected to the pipeline before the detector.

[0092] S3: Extraction and loading: In CO 2 Under the conditions of flow rate 50 mL / min, back pressure 120 bar, and extraction time 15 min, the target components in the pre-separation device were continuously loaded onto the XAmide preparative column.

[0093] S4: SFC separation: After extraction and loading, switch to the SFC pipeline to separate the target components. SFC chromatographic conditions include: XAmide preparative column (250×30mm id, 10μm), mobile phase is food grade CO 2 (A), modifier is preparative grade methanol (B), 0-5min, 0-4% B, 5-15min, 4-11% B, 15-21min, 11-16% B. Flow rate: 100mL / min, back pressure: 140bar, detection wavelength: 254nm. The target component separated in this example has a moderate retention time on the XAmide semi-preparative column and exhibits good separation ability and chromatographic peak shape.

[0094] Embodiment 4:

[0095] The materials and material specifications used in this embodiment are as follows:

[0096] Sample: Galangal dried powder;

[0097] Chromatographic packing: silica gel (40μm, 100A);

[0098] Specifications of pre-separation device: 5mL;

[0099] Specifications of supercritical fluid preparative chromatography column: 150×20mm id, 5μm.

[0100] The supercritical fluid chromatography separation method of this embodiment comprises the following steps:

[0101] S1: Preparation of sample and chromatographic medium mixture

[0102] 2.0 g of dried galangal powder was weighed into a mortar, and a small amount of ethanol was added to dissolve the sample. Subsequently, 3.0 g of silica gel (40 μm, 100 A) chromatographic filler was added to the solution, and after grinding evenly, the solvent was evaporated to prepare a mixture of the sample and the chromatographic filler.

[0103] S2: Device connection: Weigh 300 mg of sample and chromatographic filler mixture and load them into a 5 mL pre-separation device. Fill both ends of the pre-separation device with appropriate amounts of absorbent cotton. The inlet of the pre-separation device is connected to the pipeline before the chromatographic column, and the outlet of the pre-separation device is connected to the front end of the XAmide preparative column (150×20mm id, 5μm). The rear end of the XAmide preparative column is connected to the pipeline before the detector of the preparative SFC system.

[0104] S3: Extraction and loading: In CO 2 Under the conditions of flow rate 30 mL / min, back pressure 150 bar, and extraction time 10 min, the target components in the pre-separation device were continuously loaded onto the Diol preparative column.

[0105] S4: SFC separation: After extraction and loading, switch to the SFC pipeline to separate the target components. The SFC preparative chromatographic conditions are: Diol preparative column (150×20mm id, 5μm), mobile phase is food grade CO 2 (A), modifier is preparative grade ethanol (B), 0-10 min, 0-15% B, 10-15 min, 15-20% B. Flow rate: 50 mL / min, back pressure: 150 bar, detection wavelength: 254 nm.

[0106] The target component separated in this example has moderate retention capacity on the Diol preparative column, showing good separation ability and chromatographic peak shape.

[0107] The comparative example adopts the traditional liquid injection method to carry out supercritical fluid chromatography, but does not adopt the one-step sample pretreatment and separation method of the present application.

[0108] The materials and material specifications used in the comparative examples are as follows:

[0109] Sample: The precipitate after Hedyotis diffusa was extracted with 95% ethanol and then precipitated with water;

[0110] Chromatographic packing: C18 (40 μm, 100A);

[0111] Specifications of pre-separation device: 5mL;

[0112] Specifications of supercritical fluid preparative chromatography column: 250×10mm id, 10μm.

[0113] The supercritical fluid chromatography separation method of the comparative example comprises the following steps:

[0114] S1: Preparation of sample and chromatographic filler mixture: The preparation of Hedyotis diffusa sample and chromatographic filler mixture is the same as in Example 1;

[0115] S2: Device connection: Weigh 330 mg of sample and chromatographic filler mixture and put them into a 5 mL pre-separation device. Fill both ends of the pre-separation device with appropriate amounts of absorbent cotton. Connect the inlet end of the pre-separation device to the pipeline before the chromatographic column, and connect the outlet end of the pre-separation device to the pipeline before the detector;

[0116] S3: Extraction: in CO 2 Under the conditions of flow rate of 15 mL / min, back pressure of 150 bar, extraction time of 10 min, compensation pump flow rate of 10 mL / min, and compensation liquid of preparative-grade ethanol, the target components in the pre-separation device were continuously collected in the fraction tank;

[0117] S4: Sample loading: The collected target components are concentrated until there is no liquid, and 3 mL of ethanol-n-hexane mixed solution (n-hexane / ethanol = 4 / 1 (v / v)) is added to redissolve. The purpose of adding n-hexane is to reduce the solvent strength as much as possible; the traditional liquid method is used for sample injection;

[0118] S5: SFC separation: Switch to the SFC line and start separation of the target components. Chromatographic conditions are as follows: XAmide semi-preparative column (250×10mm id, 10μm), mobile phase is food grade CO 2 (A), modifier is preparative grade methanol (B), 0-5min, 6-6% B, 5-15min, 6-10% B, 15-21min, 10-15% B. Flow rate: 15mL / min, back pressure: 140bar, detection wavelength: 254nm, SFC separation results are shown as follows Figure 4 As shown. Figure 4 It can be seen that the chromatographic peaks are seriously distorted and the separation effect is poor.

[0119] The present application provides a supercritical fluid chromatography separation method and device for one-step sample pretreatment and separation, wherein the method adds a pre-separation device to the front end of a supercritical fluid preparation chromatography column, places the sample or the sample mixed with the chromatography filler into the pre-separation device, performs continuous pre-separation of the target component under supercritical conditions and loads it into the supercritical fluid preparation chromatography column, and after the pre-separation and loading are completed, switches to the SFC path for separation. It can solve the problem that the strength and viscosity of the sample dissolving reagent in supercritical fluid chromatography do not match the mobile phase, and eliminate the interference of impurities on supercritical fluid chromatography separation.

[0120] The above is a detailed introduction to a supercritical fluid chromatography separation method and device provided by the present application. Specific examples are used in this article to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method of the present application and its core idea. At the same time, for technicians in this field, according to the idea of ​​the present application, there will be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as a limitation on the present application.

Claims

1. A supercritical fluid chromatography separation method, characterized in that: include: The sample is mixed with a chromatographic filler and placed in a pre-separation device. The sample includes Hedyotis diffusa, Alpinia galanga, Hedyotis diffusa extract or Alpinia galanga extract. The chromatographic filler includes any specification of fillers selected from silica gel bonded octadecyl C18, silica gel bonded octyl C8, silica gel, cation exchange filler SCX, anion exchange filler SAX, SCX-C18 mixed filler and SAX-C18 mixed filler. The mass ratio of the sample to the chromatographic filler is 1:(1.5-10). The steps of mixing the sample with the chromatographic filler include: dissolving the sample in a solvent, adding the chromatographic filler, and removing the solvent after grinding; connecting one end of the pre-separation device to a pipeline before a chromatographic column, and connecting the other end of the pre-separation device to a supercritical fluid preparation chromatographic column; using supercritical carbon dioxide as an extraction solvent to pre-separate the sample. The pre-separation includes sample extraction, pre-treatment and sample loading, wherein the pre-treatment is extraction, and the extraction conditions include: the flow rate range of the supercritical carbon dioxide is 5-50 mL / min; the back pressure range is 100-150 bar; the extraction time range is 10-20 minutes, and the target component after pre-separation is loaded into the supercritical fluid preparation chromatographic column, wherein the supercritical fluid preparation chromatographic column is a XAmide semi-preparative column or a XAmide preparation column, and the retention capacity of the supercritical fluid preparation chromatographic column for the target component is greater than that of the chromatographic filler; the front pipeline of the chromatographic column is connected to the supercritical fluid preparation chromatographic column; the supercritical carbon dioxide and the modifier are used as mobile phases to separate the target component, and the adsorption characteristics of the chromatographic filler are utilized to firmly retain the non-target components on the chromatographic filler and are not easily extracted, thereby realizing the selective extraction of the target component.

2. The supercritical fluid chromatography separation method according to claim 1, characterized in that: The extraction solvent also includes the modifier, and the modifier accounts for 0-10% of the total flow of the supercritical carbon dioxide and the modifier.

3. The supercritical fluid chromatography separation method according to claim 1, characterized in that: The modifier includes at least one of methanol, ethanol, isopropanol or acetonitrile.

4. The supercritical fluid chromatography separation method according to claim 1, characterized in that: The step of separating the target component comprises: eluting on the supercritical fluid preparation chromatographic column under isocratic or gradient conditions to separate the target component.

5. The supercritical fluid chromatography separation method according to claim 1, characterized in that: The diameter of the supercritical fluid preparation chromatographic column is 10 to 30 mm; and / or the length of the supercritical fluid preparation chromatographic column is 150 to 250 mm.

6. The supercritical fluid chromatography separation method according to claim 1, characterized in that: The flow rate range of the supercritical fluid preparation chromatographic column is 10 to 200 mL / min; and / or the sample loading amount of the supercritical fluid preparation chromatographic column is 1 to 1000 mg.

7. A supercritical fluid chromatography device, comprising a chromatographic column front pipeline, a supercritical fluid preparative chromatographic column and a detector, characterized in that: The supercritical fluid chromatography separation method according to any one of claims 1 to 6, wherein the supercritical fluid chromatography device further comprises a pre-separation device; when the pre-separation device realizes the extraction, pre-treatment and sample loading functions, one end of the pre-separation device is connected to the front pipeline of the chromatographic column, and the other end of the pre-separation device is connected to the supercritical fluid preparative chromatographic column; after the pre-separation device completes the extraction, pre-treatment and sample loading functions, the connection of the pre-separation device is disconnected, the front pipeline of the chromatographic column is connected to the supercritical fluid preparative chromatographic column, and the separation of the target component is started.

8. The supercritical fluid chromatography device according to claim 7, characterized in that: Both ends of the pre-separation device are provided with connection holes, and the pressure resistance range of the pre-separation device is 300-400 bar.

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