CN101637665B - Accelerated solvent extraction-high-speed countercurrent chromatography combined method and device - Google Patents

Abstract

The invention discloses an accelerated solvent extraction-high-speed countercurrent chromatography combined method and a device thereof. Use an accelerated solvent extraction device to extract the material to obtain the extract, which is collected in a liquid storage bottle; pump the extract into the quantitative loop through a peristaltic pump; pump the stationary phase in the liquid storage bottle into the high-speed countercurrent chromatograph with a plunger pump, and start the high-speed countercurrent At the same time, use a plunger pump to pump the mobile phase in the liquid storage bottle into the high-speed countercurrent chromatograph to balance the solvent system; pump the sample solution in the quantitative loop into the high-speed countercurrent chromatograph, and use the preparative high-speed countercurrent chromatography to purify and prepare Obtain the target components, or use analytical high-speed countercurrent chromatography to analyze the target components in the natural product extract; the above steps integrate accelerated solvent extraction and high-speed countercurrent chromatography through switching valves to realize online extraction, separation and purification of natural product active ingredients The preparation has the characteristics of rapidity, high efficiency and high degree of automation, and has broad application prospects in the fields of food, medicine and biology.

Description

Accelerated solvent extraction-high-speed countercurrent chromatography combined method and device

technical field

The invention relates to an accelerated solvent extraction-high-speed countercurrent chromatography (ASE-HSCCC) combined method, and an accelerated solvent extraction-high-speed countercurrent chromatography combined device for implementing the method, which is suitable for the detection of target components in the fields of food, medicine, biology, etc. Extraction separation, purification preparation or analysis.

Background technique

The traditional off-line natural product extraction, separation and purification preparation process is cumbersome and time-consuming, which easily leads to the loss of target components, and the extraction rate and recovery rate are low. Accelerated solvent extraction (ASE) is an extraction technique that increases the solubility of substances and the diffusion rate of solutes by increasing temperature and pressure, thereby improving extraction efficiency. It has the advantages of time saving, less solvent consumption, high extraction rate and easy automation. It is suitable for the extraction and separation of active ingredients in natural substances.

High-speed countercurrent chromatography (HSCCC) is a continuous and efficient liquid-liquid partition chromatographic separation preparation technology. It realizes separation based on the different distribution coefficients of substances in two immiscible liquids. It does not require a solid carrier and avoids irreversible adsorption. The problems caused by the loss, inactivation, denaturation and other problems of the separated substances can not only make all the samples recovered, but the recovered samples can better reflect their original characteristics; moreover, due to the sufficient contact between the separated substances and the liquid stationary phase, the The amount of preparation is greatly improved. According to the difference in effective separation column volume, high-efficiency countercurrent chromatography mainly includes two types: analytical type and preparative type. Among them, the analytical type has the characteristics of fast separation of target components and high separation purity; the preparative type has the advantages of strong purification ability, large preparation volume, and wide application range.

Although accelerated solvent extraction and high-speed countercurrent chromatography are widely used in the fields of natural products, food, and medicine, the defect of accelerated solvent extraction is that the utilization rate of samples is low, and high-speed countercurrent chromatography alone also has certain defects. And the two are still in the state of independent research.

Contents of the invention

The purpose of the present invention is to overcome the defects of existing off-line natural product extraction separation and purification preparation, and provide a novel accelerated solvent extraction-high-speed countercurrent chromatography method, the technical problem to be solved is to make it effective The operation steps are reduced, the extraction time is shortened, the sample loss is reduced, and the product yield is increased, so that it is more suitable for practical use.

Another object of the present invention is to provide a fast and efficient processing method integrating extraction, separation, purification, preparation or analysis.

The purpose of the present invention and the solution to its technical problems are achieved by adopting the following technical solutions. According to an accelerated solvent extraction-high-speed countercurrent chromatography method proposed in the present invention, it includes the following steps: using accelerated solvent extraction to extract materials to obtain an extract; using preparative high-speed countercurrent chromatography to purify the extract to obtain a target component, or Analysis of target components in natural product extracts by analytical high-speed countercurrent chromatography.

The purpose of the present invention and its technical problems can also be further realized by adopting the following technical measures.

According to the combined method of the embodiment of the present invention, it also includes the steps: 1) the extraction solution obtained by extracting the material by accelerated solvent extraction is collected in a liquid storage bottle; 2) pump the extraction solution in the liquid storage bottle into the quantitative loop through a peristaltic pump ; 3) adopt the plunger pump to pump the stationary phase in the liquid storage bottle into the high-speed countercurrent chromatograph, start the high-speed countercurrent chromatograph, and use the plunger pump to pump the mobile phase in the liquid storage bottle into the high-speed countercurrent chromatograph at the same time, carry out solvent System balance; 4) When the high-speed countercurrent chromatography solvent system balance is established, the sample solution in the quantitative loop is pumped into the high-speed countercurrent chromatography for separation and purification preparation or analysis; the above steps integrate accelerated solvent extraction and high-speed countercurrent chromatography through switching valves, Realize the online extraction, separation and purification preparation or analysis of active ingredients of natural products.

According to the combined method of the embodiment of the present invention, the solvent system of high-speed countercurrent chromatography adopts n-hexane with a volume ratio of 1:2:1:2: ethyl acetate: methanol: water, the upper phase is the stationary phase, and the lower phase is the mobile phase. Mutually.

According to the combined method of the embodiment of the present invention, the extraction conditions in the accelerated solvent extraction process are as follows: use the mobile phase of high-speed countercurrent chromatography as the extraction solution, extract at 90-120°C and a pressure of 85-120bar for 10-20min, and circulate Extract twice or more to obtain an extract.

According to the combined method of the embodiment of the present invention, wherein the switching valve is a six-way valve; after the equilibrium of the high-speed countercurrent chromatography solvent system is established, the six-way valve is rotated so that the position a and position f of the six-way valve are connected, The b position is connected with the c position and the d position is connected with the e position.

The purpose of the present invention and the solution to its technical problem also adopt the following technical solutions to achieve. An accelerated solvent extraction-high-speed countercurrent chromatography combined device proposed according to the present invention includes: an accelerated solvent extraction device for extracting materials to obtain an extract; a high-speed countercurrent chromatograph for purifying, preparing or analyzing the target in the extract Components; solvent pump, used to pump the extract from the accelerated solvent extraction device into the high-speed countercurrent chromatograph; switching valve, used to connect the accelerated solvent extraction device and the high-speed countercurrent chromatograph, to realize the switch between the injection state and the separation state .

The purpose of the present invention and its technical problems can also be further realized by adopting the following technical measures.

According to the combined device of the embodiment of the present invention, the volume of the extraction tank used in the accelerated solvent extraction device is 1-100 mL.

In the combined device according to the embodiment of the present invention, the high-speed countercurrent chromatograph is a preparative high-speed countercurrent chromatograph or an analytical high-speed countercurrent chromatograph.

According to the combination device of the embodiment of the present invention, the switching valve is a six-way valve.

According to the combined device of the embodiment of the present invention, the connecting pipe between the accelerated solvent extraction device and the high-speed countercurrent chromatograph is a polytetrafluoroethylene pipe or a polypropylene pipe, and the diameter of the connecting pipe is 0.8-3.0 mm.

By virtue of the above technical scheme, the accelerated solvent extraction-high-speed countercurrent chromatography method and device of the present invention have at least the following advantages:

Combining the advantages of accelerated solvent extraction with less solvent consumption, rapid and efficient extraction, and the advantages of high-speed counter-current chromatography for large-scale preparation and high-purity purification, a combined method of accelerated solvent extraction and high-speed counter-current chromatography was developed, which can effectively reduce natural products, etc. The target component in the material is extracted and separated, the loss of the sample in the whole process of purification preparation or analysis shortens the extraction time, simplifies the operation steps, and improves the product yield.

The above description is only an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, it can be implemented according to the contents of the description, and in order to make the above and other purposes, features and advantages of the present invention more obvious and understandable , now particularly cited preferred embodiments, and with the accompanying drawings, detailed description is as follows.

Description of drawings

Fig. 1 is a schematic diagram of the sampling process of the ASE-HSCCC combined device of the present invention;

Fig. 2 is a schematic diagram of the separation process of the ASE-HSCCC combined device of the present invention;

Fig. 3 is the high-performance liquid chromatogram of Sargassum extract;

Fig. 4 is the high-speed countercurrent chromatogram of ASE-preparative HSCCC of the present invention separating Sargassum;

Fig. 5 is the high-efficiency liquid chromatogram of Isoferidin separated by ASE-preparative HSCCC of the present invention;

Fig. 6 is the high-speed countercurrent chromatogram of ASE-analysis type HSCCC of the present invention separating sargassum;

Fig. 7 is a high performance liquid chromatogram of isoferidin separated by ASE-analytical HSCCC of the present invention.

1: Accelerated solvent extraction device 2: Liquid storage bottle

3: Peristaltic pump 4: Quantitative loop

5: Six-way valve 6: Three-way valve

7: Liquid storage bottle 8: Liquid storage bottle

9: Plunger pump 10: High-speed countercurrent chromatograph

11: Waste liquid bottle I: Isoferidin.

a, b, c, d, e, f: toggle bits

Detailed ways

In order to further elaborate the technical means and effects that the present invention adopts to achieve the predetermined invention purpose, below in conjunction with accompanying drawing and preferred embodiment, to the accelerated solvent extraction-high speed countercurrent chromatography method and its device that propose according to the present invention and its specific Embodiments, structures, features and effects thereof are described in detail below.

Please refer to FIG. 1 and FIG. 2 for further details on the combined accelerated solvent extraction-high-speed countercurrent chromatography method and device of the present invention. The extraction, separation and purification preparation process of the accelerated solvent extraction-high-speed countercurrent chromatography method of the present invention includes: an accelerated solvent extraction device 1 for extracting materials; a high-speed countercurrent chromatography solvent system balance process for establishing a high-speed countercurrent chromatography hydrodynamic balance system ; High-speed countercurrent chromatograph 10, used for separation and purification of materials. The extraction conditions of accelerated solvent extraction (such as extraction temperature, pressure, extraction time and number of cycles, etc.) and the conditions of high-speed countercurrent chromatography (such as injection volume, different solvent systems) can be determined according to different targets, and are not limited here. Accelerated solvent extraction-high-speed countercurrent chromatographic coupling device of the present invention passes polytetrafluoroethylene or polypropylene connecting pipe (not shown in the figure), and the diameter of connecting pipe can be 0.8-3.0mm, switching valve (in this embodiment is Six-way valve 5), solvent pump (in this embodiment, peristaltic pump 3 and plunger pump 9), etc. realize the full-automatic or semi-automatic operation of the whole machine.

Please refer to FIG. 1 , during the process, position a of the six-way valve 5 communicates with position b, position c communicates with position d, position e communicates with position f. First extract material by accelerated solvent extraction device 1, the used extraction tank volume of this accelerated solvent extraction device 1 can be 1-100mL; Adopt liquid storage bottle 2 to collect after extraction finishes; Adopt plunger pump 9 to remove the stationary phase in liquid storage bottle 8 Pump into the high-speed countercurrent chromatograph 10; start the high-speed countercurrent chromatograph 10, and simultaneously use the plunger pump 9 to pump the mobile phase in the liquid storage bottle 7 into the high-speed countercurrent chromatograph to balance the solvent system.

Pump the extract in the liquid storage bottle 2 into the quantitative loop 4 through the peristaltic pump 3; when the high-speed countercurrent chromatography solvent system balance is established, please refer to Figure 2 and rotate the six-way valve 5 so that the number a of the six-way valve 5 Position is connected to position f, position b is connected to position c, and position d is connected to position e; the sample solution in the quantitative loop 4 is pumped into the high-speed countercurrent chromatography 10, and the sample solution is purified, prepared or analyzed.

Example 1

(Using ASE-preparative HSCCC to separate and purify isoferidin in Sargassum)

The specific operation is: weigh 2.0g of 20-40mesh Sargassum sample into a 22mL ASE extraction tank, use HSCCC mobile phase as the extraction solution, extract at 100°C and 100bar pressure for 10min, cycle extraction twice, collect and extract (see Figure 3 for the high performance liquid chromatogram of the Sargassum extract), the extract was directly pumped into the preparative HSCCC for purification and preparation, and the waste liquid during the sampling process was collected into the waste liquid bottle 11. The conditions of preparative HSCCC: injection volume 10mL, solvent system is n-hexane: ethyl acetate: methanol: water=1:2:1:2 (volume ratio), column volume is 240mL, rotating speed 800rpm, above phase n-hexane/ Ethyl acetate was used as the stationary phase, methanol/water as the lower phase was used as the mobile phase, and the flow rate was 2.0mL/min; the retention rate of the stationary phase was about 54%, and the ultraviolet detection wavelength was 344nm. Fig. 4 is the countercurrent chromatogram obtained by ASE-preparative HSCCC separation of Sargonopsis; Fig. 5 is the high-performance liquid chromatogram of the Isoferidin separated by ASE-preparative HSCCC, and the liquid chromatographic detection purity of Isoferidine is greater than 99% %.

Example 2

(Using ASE-analytical HSCCC to separate and purify Isoferidin in Sargassum)

The specific operation is: Weigh 2.0g of 20-40mesh Sargassum sample into a 22mL extraction tank, use HSCCC mobile phase as the extraction solution, extract at 100°C and 100bar pressure for 10min, cycle extraction twice, and collect the extraction solution (The high-performance liquid chromatogram of the Sargassum extract is shown in FIG. 3 ), the extract is directly pumped into the analytical HSCCC for purification and separation, and the waste liquid during the sampling process is collected into the waste liquid bottle 11 . Analytical high-speed countercurrent chromatography conditions: injection volume 2mL, solvent system is n-hexane: ethyl acetate: methanol: water = 1:2:1:2 (volume ratio), column volume is 43mL, rotation speed is 1600rpm, the above phase is n-hexane / ethyl acetate as the stationary phase, the lower phase methanol/water as the mobile phase, the flow rate is 2.0mL/min; the retention rate of the stationary phase is about 51%, and the ultraviolet detection wavelength is 344nm. Fig. 6 is the countercurrent chromatogram obtained by ASE-analytical HSCCC separation of sarcocarp; Fig. 7 is the high performance liquid chromatogram of Isoferidin separated by ASE-analytic HSCCC; the liquid chromatographic detection purity of isoferidine is greater than 99% %.

Obviously, by adopting the accelerated solvent extraction-high-speed countercurrent chromatography method and device of the present invention, the active ingredient isoferidin with a purity greater than 99% can be directly obtained from Sargassum, and the rapid extraction of Isoferidin from Sargassum can be realized online. Extraction separation and purification preparation. The combined device combines the advantages of the accelerated solvent extraction device for fast and efficient extraction and separation and the high-efficiency purification and preparation of high-speed countercurrent chromatography, which effectively reduces the extraction, separation, purification and preparation or analysis of target components in natural products and other samples. Sample loss, shorten extraction time, reduce intermediate links, and improve separation efficiency.

In addition, in the above examples, Isoferidin in Sargassum is used as the object to illustrate, but the present invention is not only applicable to the extraction and separation, purification and preparation or analysis of active ingredients in natural products, but also can be applied to food, medicine, biological Rapid online extraction and separation, purification preparation or analysis of target components in other complex objects.

According to the above description, it can be seen that the accelerated solvent extraction-high-speed countercurrent chromatography instrument of the present invention has the advantages of wide adaptability, high speed and high efficiency, and high recovery rate, and can be widely used in the extraction and separation of target components in complex objects such as food, medicine, and biology. , purification, preparation and analysis.

The above description is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this field Those skilled in the art, without departing from the scope of the technical solution of the present invention, can use the technical content disclosed above to make changes or modify them into equivalent embodiments with equivalent changes, but if they do not depart from the technical solution of the present invention, the Technical Essence Any simple modifications, equivalent changes and modifications made to the above embodiments still fall within the scope of the technical solution of the present invention.

Claims (9)

1. an accelerated solvent extraction-high-speed countercurrent chromatography method, is characterized in that it comprises steps: 1) The extract obtained by extracting the material by accelerated solvent extraction is collected in a liquid storage bottle; 2) Pump the extract in the liquid storage bottle into the quantitative loop through a peristaltic pump; 3) Use a plunger pump to pump the stationary phase in the liquid storage bottle into the preparative or analytical high-speed countercurrent chromatograph, start the high-speed countercurrent chromatograph, and use a plunger pump to pump the mobile phase in the liquid storage bottle into the preparative or analytical high-speed countercurrent chromatograph. Analytical high-speed countercurrent chromatograph for solvent system balance; 4) After the equilibrium of the high-speed countercurrent chromatography solvent system is established, the sample solution in the quantitative loop is pumped into the preparative or analytical high-speed countercurrent chromatography for separation and purification preparation or analysis; The above steps integrate accelerated solvent extraction and high-speed countercurrent chromatography through switching valves to realize online extraction, separation, purification, preparation or analysis of active ingredients of natural products. 2. the accelerated solvent extraction-high-speed countercurrent chromatography method according to claim 1, is characterized in that, the solvent system of high-speed countercurrent chromatography adopts the normal hexane that volume ratio is 1: 2: 1: 2: ethyl acetate: methyl alcohol : water, the upper phase is the stationary phase, and the lower phase is the mobile phase. 3. The combined method of accelerated solvent extraction-high-speed countercurrent chromatography according to claim 1, characterized in that, the extraction conditions of accelerated solvent extraction are: the mobile phase of high-speed countercurrent chromatography is used as the extracting solution, at 90 to 120 ° C and 85 Extract under a pressure of ~120 bar for 10 to 20 minutes, and extract twice or more in a cycle to obtain an extract. 4. The accelerated solvent extraction-high-speed countercurrent chromatography method according to claim 1, characterized in that: the switching valve is a six-way valve; when the equilibrium of the high-speed countercurrent chromatography solvent system is established, the six-way valve is rotated to make the six-way valve Position a of the through valve communicates with position f, position b communicates with position c, and position d communicates with position e. 5. An accelerated solvent extraction-high-speed countercurrent chromatography coupled device, characterized in that it comprises: Accelerated solvent extraction unit for extracting material to obtain extract; High-speed countercurrent chromatography, used for purification preparation or analysis of target components in the extract; A solvent pump, used to pump the extract from the accelerated solvent extraction device into the high-speed countercurrent chromatograph; The switching valve is used to connect the accelerated solvent extraction device and the high-speed countercurrent chromatograph to realize the switching between the sample injection state and the separation state. 6. The combined accelerated solvent extraction-high-speed countercurrent chromatography device according to claim 5, characterized in that: the volume of the extraction tank used in the accelerated solvent extraction device is 1-100 mL. 7. The combined accelerated solvent extraction-high-speed countercurrent chromatography device according to claim 5, wherein the high-speed countercurrent chromatograph is a preparative high-speed countercurrent chromatograph or an analytical high-speed countercurrent chromatograph. 8. The combined accelerated solvent extraction-high-speed countercurrent chromatography device according to claim 5, characterized in that: the switching valve is a six-way valve. 9. The accelerated solvent extraction-high-speed countercurrent chromatography combined device according to claim 5, characterized in that: the connecting pipe between the described accelerated solvent extraction device and the high-speed countercurrent chromatograph is a polytetrafluoroethylene tube or a polypropylene tube pipe, the diameter of the connecting pipe is 0.8-3.0mm.

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