WO2018188184A1

WO2018188184A1 - Multifunctional on-line combination device for temperature and pressue increase-assisted extraction, trapping, and chromatographic separation

Info

Publication number: WO2018188184A1
Application number: PCT/CN2017/087736
Authority: WO
Inventors: 钟启升; 刘佳琪; 申玲玲; 姚劲挺; 黄涛宏
Original assignee: 岛津企业管理（中国）有限公司
Priority date: 2017-04-12
Filing date: 2017-06-09
Publication date: 2018-10-18
Also published as: CN107121504B; CN107121504A

Abstract

A multifunctional on-line combination device for temperature and pressure increase-assisted extraction, trapping, and chromatographic separation, comprising four chromatographic pumps (16, 21, 22, 23), three three-way mixers (13, 14, 28), two six-way switching valves (11, 12), a temperature and pressure increase-assisted extracting unit (24), a trapping column (15), a chromatographic column (32), detectors (33, 34), etc. By on-line combining a trapping-desorption-switching device and an extraction-chromatographic separation device to control the switching of the two high pressure six-way switching valves (11, 12) and the pressure adjustment of back pressure regulators (26, 27), it is possible to automatically realize free combinations of two extraction forms, i.e. supercritical fluid extraction and accelerated solvent extraction, and two chromatography technologies, i.e. supercritical fluid chromatography and high-performance liquid chromatography, increasing the functional diversification of the device, being able to be effectively used in on-line extraction, trapping, desorption, separation and detection of trace targets in complex solid or semi-solid samples in the fields of environment, food, medicine, biology, etc.

Description

Multifunctional heating and pressure extraction-trapping-chromatography separation online combined equipment

Technical field

The invention belongs to the field of analytical chemical sample pretreatment, and particularly relates to the design, analysis and application of a multi-functional heating and pressure extraction-trapping-chromatography separation online combination device, and is suitable for environment, food, medicine, biological solids or the like. On-line extraction, capture, desorption, separation and detection of trace organics in semi-solid samples.

Background technique

Heating and pressurizing during the extraction process can greatly improve the extraction speed and recovery rate. Supercritical fluid extraction (SFE) and accelerated solvent extraction (ASE) are two common pretreatment extractions. technology. Specifically, SFE usually uses carbon dioxide as a fluid, and can be converted into a supercritical fluid by heating and pressurizing to achieve supercritical conditions, which has the advantages of green, safety, and high extraction efficiency; ASE is rapidly extracted by an organic solvent by increasing temperature and pressure. Solid or semi-solid samples have the advantages of less organic solvent, rapid, less matrix influence, and high extraction efficiency.

Supercritical fluid chromatography (SFC) and high performance liquid chromatography (HPLC) are used to separate samples. SFC combines the characteristics of gas chromatography and liquid chromatography. High-boiling, low-volatility samples have faster analysis speeds and conditions than high-performance liquid chromatography. HPLC is currently the most widely used chromatographic method and is widely used in almost every field of quantitative and qualitative analysis. Among them, Hydrophilic interaction chromatography (HILIC) is also called "anti-reverse phase" chromatography. In HILIC, the stationary phase is polar. Usually, acetonitrile and water are used as mobile phases, wherein the aqueous phase is a strong solvent. This is exactly the opposite of traditional reversed phase chromatography. HILIC is currently the most successful method for retaining and isolating polar compounds. HILIC has become increasingly popular over the past decade, driven by the development of polar drugs and metabolomics in the pharmaceutical industry.

Mass spectrometry (MS) provides abundant structural information in one analysis and is considered to be a universal method with high specificity and sensitivity and widely used.

At present, various pre-processing methods and separation methods are mostly used independently or offline, and the combination of online pre-processing and separation cannot be realized, and conversion between methods cannot be realized.

Summary of the invention

Based on this, the object of the present invention is to provide an on-line combined device, which has both supercritical fluid extraction and accelerated solvent extraction, and requires pre-treatment technology for heating and pressurization, as well as supercritical fluid chromatography and high performance liquid chromatography. Separation system, The in-line capture-desorption-switching device is embedded in the supercritical fluid extraction-supercritical fluid chromatography system to realize the free combination of pretreatment technology and chromatographic separation system.

The invention is realized by the following technical solutions: multi-functional heating and pressure extraction-trapping-chromatography separation on-line equipment, including online trapping-desorption-switching device and extraction-chromatography separating device;

The online trapping-desorption-switching device includes a first high-pressure six-way switching valve and a second high-pressure six-way switching valve, a first three-way mixer, a second three-way mixer, and a collecting column for flow path switching a first chromatographic pump and a connecting pipe for connecting the components; the first high-pressure six-way switching valve and the second high-pressure six-way switching valve are respectively provided with six interfaces in a clockwise order; the first chromatographic pump a first interface connected to the first high-pressure six-way switching valve; the first inlet and the second inlet of the first three-way mixer are respectively connected to the sixth interface and the second interface of the first high-pressure six-way switching valve The outlet of the first three-way mixer is connected to one end of the collecting column, the other end of the collecting column is connected to the first interface of the second high-pressure six-way switching valve, and the second interface of the second high-pressure six-way switching valve is connected Entering the first inlet of the second three-way mixer;

The extraction-chromatography separation device includes a third chromatography pump for pushing a solvent, a warming pressure extraction unit, a first three-way interface, a chromatography column, and a communication tube for connecting the components; the third chromatographic pumping Into one end of the extraction unit, the other end of the warming and pressure extraction unit is respectively connected to the fifth interface of the first high-pressure six-way switching valve and the second inlet of the first three-way mixer through the first three-way interface; The inlet of the column is connected to the outlet of the second three-way mixer.

Further, the extraction-chromatography separation device further includes a second chromatography pump and a third three-way mixer for pushing liquid carbon dioxide, and the second chromatography pump and the third chromatography pump are respectively connected to the third three-way mixer The first inlet and the second inlet, the outlet of the third three-way mixer is connected to one end of the warming pressure extraction unit. The warming and pressure extraction unit has the functions of simultaneous heating and pressurization, and the supercritical extraction function or the accelerated solvent extraction function can be realized according to the introduction of the solvent; when the liquid carbon dioxide is pushed, the temperature is greater than 31 ° C, the pressure When it is greater than 7.4Mpa, it will be converted into supercritical state, and supercritical extraction will be carried out. At this time, the third chromatographic pump can change the polarity of the extraction solution by increasing the polarity of the extraction solution, and if the carbon dioxide is not introduced, The two-chromat pump stops the pump, and only the third chromatographic pump pushes the organic solvent, such as acetonitrile, and accelerates the solvent extraction after heating and pressurizing.

Further, the multifunctional warming pressure extraction-trap-chromatography separation line-in-line apparatus further includes a first back pressure regulator and a second back pressure regulator, wherein the first back pressure regulator is disposed on the column Downstream of the flow path, the second back pressure regulator is disposed between the downstream of the flow path of the warming and pressure extraction unit and the upstream of the flow path of the column.

Further, the extraction-chromatography separation device further includes a fourth chromatographic pump, a mass spectrometer detector, and a second three-way interface, wherein the fourth chromatographic pump and the mass spectrometer detector are respectively connected to the outlet of the column through a second three-way interface . The fourth chromatographic pump is used to push a solvent that promotes ionization, and the sample is flowed into the mass spectrometer for detection.

Further, the first back pressure regulator is disposed between the column and the detector, and the second back pressure regulator is disposed between the first three-way port and the first high-pressure six-way switching valve.

Further, the extraction-chromatography separation device further includes a high pressure liquid phase detector disposed between the chromatography column and the first back pressure regulator. The flow direction of the flow path is automatically controlled by cooperatively adjusting the first back pressure regulator and the second back pressure regulator.

Further, the second back pressure regulator outlet is provided with a one-way valve having a filter. It is used to filter the extract and control the extract to not reflux.

Further, the multifunctional warming pressure extraction-capture-chromatography separation online combination device further includes an autosampler disposed on the warming pressure extraction unit and the first three-way interface The multi-functional warming pressure extraction-capture-chromatography separation online combination device further includes a column oven, the online trapping-desorption-switching device and the column are disposed at the column temperature Inside the box. The autosampler can quantitatively add the substance to be analyzed to the device, thereby improving the detection accuracy of the device; and setting the online trapping-desorption-switching device and the column in the column oven to effectively adjust the column temperature Improve the sensitivity of detection.

Further, the first chromatographic pump incorporates a low pressure gradient proportional valve having at least two solvents of different polarities. The two solvents with different polarities are relatively weak in polarity and the polarity of the other solvent is strong. The first chromatographic pump has a low-pressure gradient proportional valve, which can directly perform gradient when the target enters the column. Elution, to achieve high performance liquid chromatography mode; and, the weakly polar solvent can be used as a dilution solvent before the target enters the trap column, and the extract flowing out of the extraction device is effectively mixed in the first three-way mixer, The target is transferred to a weakly polar solvent to complete the dilution; the strong polar solvent is a strong polar solvent of the target on the capture column and can be used as a desorbent for the target.

Further, the third interface of the first high-pressure six-way switching valve and the third interface of the second high-pressure six-way switching valve are respectively sealed.

Further, the trap column is a reverse phase extraction column or a hydrophilic interaction chromatography column, and the column is a reverse phase chromatography column or a hydrophilic interaction chromatography column. Different analysis modes can be achieved by using different trap columns and columns.

The invention has the following beneficial effects:

(1) The multifunctional heating and pressure extraction-trapping-chromatography separation on-line device of the present invention controls the switching of two high-pressure six-way switching valves and the pressure adjustment of two back pressure regulators, and can automatically realize supercritical Fluid extraction, accelerated solvent extraction two kinds of extraction forms combined with supercritical fluid chromatography, high performance liquid chromatography two kinds of chromatographic techniques, such as supercritical fluid extraction - capture - supercritical fluid chromatography online combination mode, supercritical Fluid extraction-trapping-high performance liquid chromatography on-line mode, accelerated solvent extraction-trapping-supercritical fluid chromatography on-line mode, accelerated solvent extraction-trapping-high performance liquid chromatography on-line mode, common supercritical Fluid extraction - supercritical fluid chromatography mode, high performance liquid chromatography mode, etc.

(2) Place the trap column and the three-way mixer between the two high-pressure six-way switching valves, according to the valve state of the two high-pressure six-way switching valves and the delivery of the two inlet solvents of the three-way mixer. To achieve multiple flow path selection and improve the diversification of system functions.

(3) The existing supercritical fluid extraction-supercritical fluid chromatography system needs to be separated and then enters the chromatographic separation column. The splitting will result in poor reproducibility. The present invention uses supercritical fluid extraction-trapping-supercritical fluid chromatography. The online combined analysis mode can fully enrich the target in the trap column, which can greatly improve the sensitivity and reproducibility of the method.

(4) On-line conversion of the extract or vapor-liquid mixture to transfer the target to a weak solvent facilitates enrichment of the target in the trap column, further improving the sensitivity of the method and reducing the matrix effect of the method.

For a better understanding and implementation, the invention will be described in detail below with reference to the drawings.

DRAWINGS

1 is a schematic view of a multi-functional heating and pressure extraction-trapping-chromatography separation online combination device of the present invention;

2 is a schematic diagram of flow path communication of step S11 in Embodiment 1;

3 is a schematic diagram of flow path communication of step S12 in Embodiment 1;

4 is a schematic diagram of flow path communication of step S13 in Embodiment 1;

Figure 5 is a schematic diagram showing the flow path communication of step S14 in the first embodiment;

6 is a schematic diagram of flow path communication in step S15 in the first embodiment;

7 is a schematic diagram of flow path communication of step S21 in Embodiment 2;

8 is a schematic diagram of flow path communication of step S22 in Embodiment 2;

9 is a schematic diagram of flow path communication of step S23 in Embodiment 2;

10 is a schematic diagram of flow path communication of step S24 in Embodiment 2;

Figure 11 is a schematic diagram showing the flow path of the step S25 in the second embodiment;

12 is a schematic diagram of flow path communication in Embodiment 3;

Figure 13 is a schematic view showing the flow path communication in the fourth embodiment;

Figure 14 is a multi-functional warming pressure extraction-trapping-chromatography separation line of the present invention. The six-fold analysis of vanillin, p-hydroxybenzaldehyde, vanillic acid and p-hydroxybenzoic acid in the vanilla bean pods. Chromatogram; 1a, 1b, 1c, and 1d are chromatograms of vanillin, p-hydroxybenzaldehyde, vanillic acid, and p-hydroxybenzoic acid, respectively, for the first analysis; 2a, 2b, 2c, and 2d, respectively Chromatogram of four compounds of vanillin, p-hydroxybenzaldehyde, vanillic acid and p-hydroxybenzoic acid for the second iteration; and so on, 6a, 6b, 6c and 6d are vanilla aldehydes for the sixth repeated analysis, respectively. Chromatograms of four compounds of p-hydroxybenzaldehyde, vanillic acid and p-hydroxybenzoic acid.

detailed description

In order to further illustrate the technical means and technical effects of the present invention in order to achieve the intended purpose of the invention, the multi-functional warming pressure extraction-trapping-chromatography separation online combination device proposed by the present invention will be described below with reference to the embodiments and the accompanying drawings. Structure, The features and specific embodiments thereof will be described in detail as follows.

Please refer to FIG. 1 , which is a schematic diagram of the multi-functional warming pressure extraction-capture-chromatography separation online combination device of the present invention, which comprises an online trapping-desorption-switching device and an extraction-chromatography separation device.

The on-line trap-desorption-switching device includes a first high-pressure six-way switching valve 11 and a second high-pressure six-way switching valve 12, a first three-way mixer 13, and a second three-way mixer 14 for flow path switching. The collecting column 15, the first chromatographic pump 16, and a connecting pipe for connecting the components. In this embodiment, the first three-way mixer 13 and the second three-way mixer 14 are all three-way micro-mixers, and the three-way micro-mixer can be any T-type three-way interface or have a mixing function. Mixer for connecting the tubing and pooling the solvent for mixing. The first high-voltage six-way switching valve 11 is sequentially provided with a first interface a, a second interface b, a third interface c, a fourth interface d, a fifth interface e, and a sixth interface f in a clockwise order. The second high-pressure six-way switching valve 12 sequentially sets the first interface A, the second interface B, the third interface C, the fourth interface D, the fifth interface E, and the sixth interface F in a clockwise order. The first chromatographic pump 16 is connected to the first interface a of the first high-pressure six-way switching valve 11. The first three-way mixer 13 is provided with a first inlet 13a, a second inlet 13b, and an outlet 13c. The second three-way mixer 14 is also provided with a first inlet 14a, a second inlet 14b and an outlet 14c. The first inlet 13a and the second inlet 13b of the first three-way mixer 13 are respectively connected to the sixth interface f and the second interface b of the first high-pressure six-way switching valve 11, the first three-way mixer 13 The outlet 13c is connected to one end of the trap column 15, the other end of the trap column is connected to the first port A of the second high-pressure six-way switching valve 12, and the second port B of the second high-pressure six-way switching valve 12 is connected. The first inlet 14a of the second three-way mixer 14. The fifth interface e of the first high-pressure six-way switching valve 11 and the sixth interface F of the second high-pressure six-way switching valve 12 are respectively connected to an external receiving container for receiving waste liquid; the first high-pressure six-way switching valve The third interface c of the second high-voltage six-way switching valve 12 and the fifth interface E of the second high-pressure six-way switching valve 12 are sealed. The first chromatographic pump 16 has a low pressure gradient proportional valve built therein, and the first chromatographic pump 16 has at least two solvents, respectively a weakly polar solvent and a strong polar solvent. The low-pressure gradient proportional valve can directly perform gradient elution when the target enters the column 32 to realize a high-performance liquid chromatography mode; and the weakly polar solvent can be used as a dilution solvent before the target enters the trap column. The extract flowing out of the extraction device is effectively mixed in the first three-way mixer 13, and the target is transferred to a weakly polar solvent to complete the dilution; the strong polar solvent is a strong polar solvent of the target on the trap column. It can be used as a desorbent for the target.

The extraction-chromatography separation device includes a second chromatographic pump 21 for pumping liquid carbon dioxide, a third chromatographic pump 22 and a fourth chromatographic pump 23 for pushing solvent, a warming and pressure extraction unit 24, and an autosampler 25 a first back pressure regulator 26, a second back pressure regulator 27, a third three-way mixer 28, a first three-way interface 29, a second three-way interface 31, a column 32, a high pressure liquid phase detector 33, A mass spectrometer detector 34, a column oven 35, and a communication tube for connecting the components. In the present embodiment, the third three-way mixer 28 is a three-way micromixer. The third three-way mixer 28 is provided with a first inlet 28a, a second inlet 28b, and an outlet 28c. The second chromatographic pump 21 and the third chromatographic pump 22 are respectively connected to the third three-way hybrid The first inlet 28a and the second inlet 28b of the combiner 28, the outlet 28c of the third three-way mixer 28 communicates with one end of the warming and pressurizing extraction unit 24, and the other end of the warming and pressurizing extraction unit 24 passes The first three-way interface 29 is respectively connected to the fifth interface e of the first high-pressure six-way switching valve 11 and the second inlet 14b of the second three-way mixer 14. One end of the chromatography column 32 is connected to the outlet 14c of the second three-way mixer 14, and the other end is connected to the high-pressure liquid phase detector 33, and the high-pressure liquid phase detector 33 is respectively connected to the fourth through the second three-way interface 31. The chromatographic pump 23 and the mass spectrometer detector 34 are used for chromatographic separation and mass spectrometry detection; the built-in flow cell of the high pressure liquid phase detector 33 can withstand a pressure exceeding 40 MPa. The first back pressure regulator 26 is disposed between the column 32 and the detector 34, and the second back pressure regulator 27 is disposed between the first three-way port 29 and the first high-pressure six-way switching valve 11. The outlet of the second back pressure regulator 27 is provided with a check valve having a filter for filtering the extract and controlling the extract to not flow back. The flow direction of the flow path is automatically controlled by cooperatively adjusting the first back pressure regulator 26 and the second back pressure regulator 27. The autosampler 25 is disposed between the warming and pressure extraction unit 24 and the first three-way interface 29. The autosampler 25 can quantitatively add the substance to be analyzed into the device, thereby improving the detection accuracy of the device. The on-line trapping-desorption-switching device and the column 32 are disposed in the column oven 35, which can effectively adjust the column temperature and improve the sensitivity of detection. The warming and pressing extraction unit 24 has the functions of simultaneous heating and pressurization, and the supercritical extraction function or the accelerated solvent extraction function can be realized according to the introduction of the solvent; when the liquid carbon dioxide is pushed, the temperature is greater than 31 ° C, When the pressure is greater than 7.4Mpa, it is converted into a supercritical state, and supercritical extraction is performed. At this time, the third chromatographic pump 22 is introduced into the organic solvent to change the polarity of the extraction solution and increase the polarity range of the extract; if carbon dioxide is not introduced, That is, the second chromatographic pump 21 stops the pump, and only the third chromatographic pump 22 pushes the organic solvent, such as acetonitrile, and accelerates the solvent extraction after heating and pressurizing.

In this embodiment, the first high-pressure six-way switching valve 11 and the second high-pressure six-way switching valve each include two connected states, which are a “0” bit state and a “1” bit state, respectively.

When the first high-pressure six-way switching valve 11 is in the "0" state, the first interface a of the first high-pressure six-way switching valve 11 is connected to the second interface b of the first high-voltage six-way switching valve 11, first The third port c of the high-pressure six-way switching valve 11 is connected to the fourth port d of the first high-pressure six-way switching valve 11, and the fifth port e of the first high-pressure six-way switching valve 11 is connected to the first high-pressure six-way switching valve 11 The sixth interface f. At this time, the warming and pressurizing extraction unit 24 extracts through the fifth interface e and the sixth interface f of the first high-pressure six-way switching valve 11 to communicate with the collecting column; the first chromatographic pump 16 passes the first high voltage six The first interface a and the second interface b of the switching valve 11 are in communication with the collecting column. When the first high-pressure six-way switching valve 11 is in the "1" position state, the first interface a of the first high-voltage six-way switching valve 11 is connected to the sixth interface f of the first high-voltage six-way switching valve 11, first The second port b of the high-pressure six-way switching valve 11 is connected to the third port c of the first high-pressure six-way switching valve 11, and the fourth port d of the first high-pressure six-way switching valve 11 is connected to the first high-pressure six-way switching valve 11 The fifth interface e. At this time, the warming and pressing extraction unit 24 extracts through the fifth interface e and the fourth interface d of the first high-pressure six-way switching valve 11 to communicate with the external receiving container; the first chromatographic pump 16 passes the first high voltage six The first interface a and the sixth interface f of the switching valve 11 communicate with the trap column.

When the second high-pressure six-way switching valve 12 is in the "0" state, the first interface A of the second high-pressure six-way switching valve 12 is connected to the second interface B of the second high-pressure six-way switching valve 12, and second The third port C of the high-pressure six-way switching valve 12 is connected to the fourth port D of the second high-pressure six-way switching valve 12, and the fifth port E of the second high-pressure six-way switching valve 12 is connected to the second high-pressure six-way switching valve 12 The sixth interface. At this time, the trap column communicates with the column 32 through the first port A and the second port B of the second high-pressure six-way switching valve 12. When the second high-pressure six-way switching valve 12 is in the "1" position state, the first interface A of the second high-pressure six-way switching valve 12 is connected to the sixth interface of the second high-pressure six-way switching valve 12, and the second high voltage The second interface B of the six-way switching valve 12 is connected to the third interface C of the second high-pressure six-way switching valve 12, and the fourth interface D of the second high-pressure six-way switching valve 12 is connected to the second high-pressure six-way switching valve 12 The fifth interface E. At this time, the trap column is connected to the outer container through the first interface A and the sixth interface of the second high-pressure six-way switching valve 12.

Compared with the prior art, the multifunctional heating and pressure extraction-collection-chromatography separation on-line device of the present invention controls the switching of two high-pressure six-way switching valves and the pressure adjustment of the back pressure regulator, and can automatically realize the super The combination of critical fluid extraction and accelerated solvent extraction is freely combined with supercritical fluid chromatography and high performance liquid chromatography. For example, supercritical fluid extraction-capture-supercritical fluid chromatography online combination mode, super Critical fluid extraction-trapping-high performance liquid chromatography on-line mode, accelerated solvent extraction-trapping-supercritical fluid chromatography on-line mode, accelerated solvent extraction-trapping-high performance liquid chromatography on-line mode, common super Critical fluid extraction-supercritical fluid chromatography mode, high-performance liquid chromatography mode, etc., solves the problem that the existing various analytical methods are complicated in operation steps and the analysis conditions are different, and the online combination of various analysis methods is realized.

Example 1

Supercritical fluid extraction-reverse phase extraction column trapping-supercritical fluid chromatography online combined analysis mode

The embodiment provides the supercritical fluid extraction-reverse phase extraction column trapping-supercritical fluid chromatography on-line combined analysis mode of the multifunctional heating and pressure extraction-collection-chromatography separation on-line equipment. Extraction, enrichment and separation of medium and low polar compounds. In this embodiment, the trap column is a reverse phase extraction column, and the column 32 is a supercritical fluid dedicated reverse phase chromatography column. The warming and pressure extraction unit 24 is heated and pressurized by introducing liquid carbon dioxide. That is to achieve carbon dioxide supercritical fluid extraction, the specific analysis steps of the analysis mode are as follows:

S11: Static extraction-SPE balance: Please refer to FIG. 2, which is a schematic diagram of the flow path communication in step S11 of the embodiment. The first high-pressure six-way switching valve 11 is in a "1" position state, and the second high-voltage six-way The switching valve 12 is in the "1" position state, the pressure of the first back pressure regulator 26 is set to 40 MPa, and the pressure of the second back pressure regulator 27 is set to 10 MPa; at this time, the second chromatography pump 21 pushes liquid carbon dioxide The third chromatographic pump 22 pushes the modifier, and the liquid carbon dioxide is mixed with the modifier in the third three-way mixer 28, and then enters the warming and pressure extraction unit 24 to perform static supercritical extraction on the sample. Since the pressure of the first back pressure regulator 26 is set to 40 MPa, the pressure of the second back pressure regulator 27 is set to 10 MPa, The flow path of the first back pressure regulator 26 is blocked, and the flow path of the second back pressure regulator 27 is connected, and the warming and pressure extraction unit 24 performs static extraction through the first high-pressure six-way switching valve 11 The external receiving container receives the waste liquid. The first chromatographic pump 16 pushes the weakly polar solvent into the trapping column through the first high-pressure six-way switching valve 11 and the first three-way mixer 13 and flows into the outside through the sixth interface of the second high-pressure six-way switching valve 12. The receiving container receives the waste liquid and balances the trap column.

S12: Dynamic extraction-SPE enrichment: Please refer to FIG. 3, which is a schematic diagram of flow path communication in step S12 of the embodiment. The first high-pressure six-way switching valve 11 is in a “0” position state, and the second high voltage is six. The switching valve 12 is in the "1" position state, the pressure of the first back pressure regulator 26 is set to 40 MPa, and the pressure of the second back pressure regulator 27 is set to 10 MPa; at this time, the second chromatography pump 21 pushes the liquid state The carbon dioxide, third chromatography pump 22 pushes the modifier, and the liquid carbon dioxide and the modifier are mixed in the third three-way mixer 28 and then enter the warming and pressure extraction unit 24 to perform dynamic supercritical extraction on the sample. The extraction solution enters the first three-way mixer 13 through the fifth interface e and the sixth interface f of the first high-pressure six-way switching valve 11, and the first chromatography pump 16 pushes the weak solvent through the first of the first high-pressure six-way switching valve 11 The interface a and the second interface b enter the first three-way mixer 13, mix with the extraction solution, and dilute the extraction solution into a weak solvent, enter the trap column, and complete the enrichment of the target on the trap column.

S13: SPE de-mixing-column balance: Please refer to FIG. 4, which is a flow path communication diagram of step S13 of the embodiment, the first high-pressure six-way switching valve 11 is in a "1" position state, and the second high voltage The six-way switching valve 12 is in the "1" position state, the pressure of the first back pressure regulator 26 is set to 10 MPa, and the pressure of the second back pressure regulator 27 is set to 40 MPa; at this time, the first chromatography pump 16 pushes The solvent is cleaned, and the first interface a and the sixth interface f of the first high-pressure six-way switching valve 11 enter the trap column 15 to remove the target in the trap column 15, and the impurity solution is switched by the second high-pressure six-way switch. The first interface A and the sixth interface of the valve 12 flow into the external receiving container. The second chromatographic pump 21 pushes the liquid carbon dioxide, the third chromatographic pump 22 pushes the modifier, enters the chromatographic column 32 through the first three-way interface 29 and the second three-way mixer 14, and the column 32 is pre-equilibrated.

S14: SPE elution-chromatographic injection: Please refer to FIG. 5, which is a flow path communication diagram of step S14 of the embodiment, the first high-pressure six-way switching valve 11 is in a "1" position state, and the second high voltage The six-way switching valve 12 is in the "0" position state, the pressure of the first back pressure regulator 26 is set to 10 MPa, and the pressure of the second back pressure regulator 27 is set to 40 MPa; at this time, the first chromatography pump 16 pushes The solvent is analyzed, and the first interface a and the sixth interface f of the first high-pressure six-way switching valve 11 enter the collecting column 15 to desorb the target on the collecting column 15, and the desorbed liquid passes through the second high-pressure six-way switching valve 12 The first interface A and the second interface B flow into the second three-way mixer 14. The second chromatographic pump 21 pushes the liquid carbon dioxide into the second three-way mixer 14, mixes and dilutes with the desorbed liquid, and enters the column 32.

S15: SFC separation-SPE balance: Please refer to FIG. 6, which is a schematic diagram of the flow path communication in step S15 of the embodiment. The first high-pressure six-way switching valve 11 is in a "1" position state, and the second high-voltage six-way The switching valve 12 is in the "1" position state, the pressure of the first back pressure regulator 26 is set to 10 MPa, and the pressure of the second back pressure regulator 27 is set to 40 MPa; At this time, the second chromatographic pump 21 pushes the liquid carbon dioxide, and the third chromatographic pump 22 pushes the modifier, mixes it in the third three-way mixer 28, and enters the column 32 to separate the target in the column 32. After the separation is completed, The target is analyzed by the mass spectrometer detector 34. The first chromatographic pump 16 pushes the solvent into the trapping column 15 through the first port a and the sixth port f of the first high-pressure six-way switching valve 11, and cleans or balances the trapping column 15.

By the switching of the first high-pressure six-way switching valve 11, the second high-pressure six-way switching valve 12, and the pressure adjustment of the two back pressure regulators, the supercritical fluid extraction-reverse phase extraction column of the present embodiment can be automatically switched. Set-supercritical fluid chromatography online combined analysis mode; this mode is suitable for on-line extraction, enrichment and separation of weakly polar compounds; and can convert solvent on-line to effectively transfer target to weakly polar solvent, which is beneficial to the target The enrichment of the object in the trap column 15 improves the recovery rate of the target; and the online combined device of the present invention can be realized without hardware modification.

Example 2

Accelerated solvent extraction-hydrophilic interaction extraction column trapping-hydrophilic interaction chromatography separation online combined analysis mode

The present embodiment provides an accelerated on-line extraction-hydrophilic interaction extraction column-hydrophilic interaction chromatography separation online combination analysis mode of the multifunctional heating and pressure extraction-trapping-chromatography separation on-line equipment. This mode is suitable for the extraction, enrichment and separation of medium and high polar compounds. In this embodiment, the trap column 15 is a hydrophilic interaction short column, the column 32 is a hydrophilic interaction chromatography column, and the warming and pressure extraction unit 24 only introduces acetonitrile, and is heated and added. Accelerated solvent extraction is achieved after pressing, and the specific analysis steps of the analysis mode are as follows:

S21: Accelerated solvent extraction-solvent balance: Please refer to FIG. 7, which is a schematic diagram of the flow path communication in step S21 of the embodiment. The first high-pressure six-way switching valve 11 is in a "1" position state, and the second high voltage is six. The switching valve 12 is in the "1" position state, the pressure of the first back pressure regulator 26 is set to 40 MPa, and the pressure of the second back pressure regulator 27 is set to 40 MPa; at this time, the third chromatography pump 22 pushes acetonitrile. The heated pressurization extraction unit is introduced to accelerate the solvent extraction of the target. The first chromatographic pump 16 pushes the equilibrium solvent into the trap column 15 through the first high-pressure six-way switching valve 11 and the first three-way mixer 13, and flows into the external receiving through the sixth interface of the second high-pressure six-way switching valve 12. The container receives the waste liquid and balances the trap column 15. Among them, the solvent pushed by the third chromatographic pump 22 is not limited to acetonitrile, but the effect of acetonitrile is best when a hydrophilic column is used.

S22: SPE enrichment: Please refer to FIG. 8 , which is a schematic diagram of the flow path communication in step S22 of the embodiment. The first high-pressure six-way switching valve 11 is in the “0” position state, and the second high-pressure six-way switching valve is in operation. 12 is in the "1" position state, the pressure of the first back pressure regulator 26 is set to 40 MPa, and the pressure of the second back pressure regulator 27 is set to 10 MPa; at this time, the third chromatography pump 22 pushes acetonitrile into the heating. The pressurized extraction unit 24 pushes the target into the collecting column 15 to enrich the target on the collecting column 15.

S23: SPE de-mixing-column balance: Please refer to FIG. 9 , which is a schematic diagram of the flow path connection in step S23 of the embodiment. The first high-pressure six-way switching valve 11 is in a "1" position state, the second high-pressure six-way switching valve 12 is in a "1" position state, and the pressure of the first back pressure regulator 26 is set to 10 MPa. The pressure of the second back pressure regulator 27 is set to 40 MPa; at this time, the first chromatographic pump 16 pushes the cleaning solvent, and enters the trapping column 15 through the first interface a and the sixth interface f of the first high-pressure six-way switching valve 11, The target in the trap column 15 is subjected to impurity removal, and the impurity solution flows into the external receiving container through the first port A and the sixth port of the second high-pressure six-way switching valve 12. The third chromatographic pump 22 pushes acetonitrile through the first three-way interface 29 and the second three-way mixer 14 into the column 32 to pre-equilibrate the column 32.

S24: SPE elution-chromatographic injection: Please refer to FIG. 10, which is a flow path communication diagram of step S24 of the embodiment, the first high-pressure six-way switching valve 11 is in a "1" position state, and the second high voltage The six-way switching valve 12 is in the "0" position, the pressure of the first back pressure regulator 26 is set to 10 MPa, and the pressure of the second back pressure regulator 27 is set to 40 MPa; at this time, the first chromatography pump 16 The analytical solvent is pushed, and the first interface a and the sixth interface f of the first high-pressure six-way switching valve 11 enter the trap column 15, and the target on the trap column 15 is eluted, and the eluate passes through the second high-pressure six-way. The first interface A and the second interface B of the switching valve 12 flow into the second three-way mixer 14. The third chromatographic pump 22 pushes the acetonitrile into the second three-way mixer 14, mixes and dilutes with the eluent, and enters the column 32.

S25: HILIC separation-SPE balance: Please refer to FIG. 11, which is a flow path communication diagram of step S25 of the embodiment. The first high-pressure six-way switching valve 11 is in a "1" position state, and the second high-voltage six-way The switching valve 12 is in the "1" position state, the pressure of the first back pressure regulator 26 is set to 10 MPa, the pressure of the second back pressure regulator 27 is set to 40 MPa; at this time, the low pressure in the third chromatography pump 22 The gradient proportional valve pushes the mixed solvent as a mobile phase to enter the column 32 for separation, and after the separation is completed, the target is analyzed by the mass spectrometer detector 34. The first chromatographic pump 16 pushes the solvent into the trapping column 15 through the first port a and the sixth port f of the first high-pressure six-way switching valve 11, and cleans or balances the trapping column 15.

By the switching of the first high-pressure six-way switching valve 11, the second high-pressure six-way switching valve 12, and the pressure adjustment of the two back pressure regulators, the accelerated solvent extraction-hydrophilic interaction extraction column of the present embodiment can be automatically switched. The capture-hydrophilic interaction chromatography separation on-line analysis mode can be used for extraction, enrichment and separation of medium and high polarity compounds; and the online combination device of the present invention can be realized without hardware modification.

Example 3

Supercritical fluid extraction-supercritical fluid chromatography separation analysis mode

The embodiment provides a supercritical fluid extraction-supercritical fluid chromatography separation analysis mode of the multifunctional heating and pressure extraction-trapping-chromatography separation line-in-line device, and the warming and pressure extraction unit 24 is introduced into the liquid state. Carbon dioxide is heated and pressurized to achieve supercritical fluid extraction of carbon dioxide. Please refer to FIG. 12 , which is a schematic diagram of the flow path communication of the embodiment. The specific analysis step of the analysis mode is: the first high-pressure six-way switching valve 11 is in a “1” state, the first The pressure of the back pressure regulator 26 is set to 10 MPa, the pressure of the second back pressure regulator 27 is set to 40 MPa; at this time, the second chromatography pump 21 pushes liquid carbon dioxide, and the third chromatography pump 22 pushes the modifier, the liquid state The carbon dioxide and the modifier are mixed in the third three-way mixer 28 and then introduced into the warming and pressure extraction unit 24, and the sample is subjected to static supercritical extraction. The extracted extract is subjected to a pressure difference (determining a split ratio) according to the pressure of the first back pressure regulator 26 and the pressure of the second back pressure regulator 27, thereby achieving the split: when the inner diameter is 4.6 mm C18 column with a length of 250mm and a particle size of 5μm, the back pressure regulator A pressure is equal to 15Mpa, the back pressure regulator B is equal to 14.8Mpa, and the flow ratio is 5mL/min, the separation ratio is about 3:97, of which 3 % enters the column 32; the second chromatographic pump 21 pushes the liquid carbon dioxide, the third chromatographic pump 22 pushes the modifier, mixes in the third three-way mixer 28, and enters the column 32 to separate the target in the column 32. After the separation is completed, the target is analyzed by the mass spectrometer detector 34.

By the switching of the first high-pressure six-way switching valve 11, the second high-pressure six-way switching valve 12, and the pressure adjustment of the two back pressure regulators, the supercritical fluid extraction-supercritical fluid chromatography separation of the present embodiment can be automatically switched. The analysis mode; and the online combined device of the present invention can be implemented without hardware modification.

Example 4

High performance liquid chromatography

This embodiment provides a high-performance liquid chromatography analysis mode of the multi-functional heating and pressure extraction-trapping-chromatography separation on-line device, and the mode can be realized without hardware modification of the system. Please refer to FIG. 13 , which is a schematic diagram of the flow path communication of the embodiment. The specific analysis step of the analysis mode is: the online trapping-desorption-switching device and the second chromatographic pump 21 are not activated, and the third chromatogram is The low pressure gradient proportional valve in the pump 22 pushes the mixed solvent as a mobile phase to enter the column 32 for separation. After the separation is completed, the target is analyzed by the mass spectrometer detector 34.

The multifunctional heating and pressurizing extraction-collection-chromatography separation on-line device of the present invention passes through the switching of the first high-pressure six-way switching valve 11, the second high-pressure six-way switching valve 12, and the two back pressure regulators The pressure adjustment can automatically switch to the high performance liquid chromatography analysis mode of the embodiment, and can be realized without the hardware modification of the online combination device of the present invention.

Example 5

In this embodiment, four kinds of compounds of vanillin, p-hydroxybenzaldehyde, vanillic acid and p-hydroxybenzoic acid in vanilla bean pods were analyzed by the multi-functional heating and pressure extraction-capture-chromatography separation on-line equipment. In this embodiment, the trap column 15 is Shimadzu Inertsil ODS-4, and the specification is

The column 32 is Shimadzu Shim-pack UC-X RP, and the specification is

(5 μm particle size).

The warming and pressurizing extraction unit 24 realizes a supercritical extraction function by introducing liquid carbon dioxide and heating and pressurizing, and has a built-in extraction tank filled with 100 mg of vanilla bean pods. The analysis process includes the following steps:

(1) In the initial state, the pressure of the first back pressure regulator 26 is set to 40 MPa, the pressure of the second back pressure regulator 27 is set to 10 MPa, and the first high pressure six-way switching valve 11 is at "1" In the position state, the second high-pressure six-way switching valve 12 is in the "1" position; at this time, the second chromatographic pump 21 pushes the liquid carbon dioxide, and the third chromatographic pump 22 pushes the modifier methanol, and the liquid carbon dioxide is changed. After mixing in the third three-way mixer 28, the agent enters the warming and pressure extraction unit 24 to perform static extraction on the sample vanilla bean pod. The static extraction device maintains the foregoing state, adjusts the parameters of the supercritical extraction unit, causes the pressure and temperature in the extraction tank to rise, converts the carbon dioxide into a supercritical fluid state, and performs supercritical static extraction.

(2) After the static extraction is completed, the state of the first high-pressure six-way switching valve 11 is switched to be in the state of “0” position. At this time, the gas-liquid mixture discharged through the second back pressure regulator 27 passes through the first high voltage six. The switching valve 11 flows into the first three-way mixer 13, and the first chromatographic pump 16 pushes the diluent water into the first three-way mixer 13 through the first high-pressure six-way switching valve 11, and mixes and dilutes with the gas-liquid mixture to enter the trap. The column 15 completes the enrichment of the target on the trap column 15. The effluent passing through the trap column 15 is discharged through the sixth port of the second high-pressure six-way switching valve 12, and waste liquid is collected.

(3) switching the state of the first high-pressure six-way switching valve 11 to be in the "1" position state, and the pressure of the first back pressure regulator 26 is set to 10 MPa, and the pressure of the second back pressure regulator 27 It is set to 40 MPa; at this time, the first chromatographic pump 16 pushes a methanol aqueous solution having a volume fraction of 10% as a cleaning solvent, and enters the collecting column 15 through the first interface a and the sixth interface f of the first high-pressure six-way switching valve 11 . The target in the trap column 15 is subjected to impurity removal, and the impurity solution flows into the external receiving container through the first port A and the sixth port of the second high-pressure six-way switching valve 12. The second chromatographic pump 21 pushes the liquid carbon dioxide, the third chromatographic pump 22 pushes the modifier, enters the chromatographic column 32 through the first three-way interface 29 and the second three-way mixer 14, and the column 32 is pre-equilibrated.

(4) After the end of the impurity removal and the equilibration of the column 32, the state of the second high-pressure six-way switching valve 12 is switched to be in the “0” state. At this time, the first chromatographic pump 16 pushes the analytical solvent methanol into the trap column. 15. The target on the trap column 15 is desorbed, and the desorbed liquid flows into the second three-way mixer 14 through the first port A and the second port B of the second high-pressure six-way switching valve 12. The second chromatographic pump 21 pushes the liquid carbon dioxide into the second three-way mixer 14, mixes and dilutes with the desorbed liquid, and enters the column 32. Since the flow rate of the carbon dioxide supercritical fluid is much larger than the flow rate of the trap column 15, the target in the desorbent will be displaced into a high proportion of the carbon dioxide supercritical fluid, and finally the column head of the column 32 will be focused to complete the column 32. Injection.

(5) After the injection of the column 32 is completed, the state of the second high-pressure six-way switching valve 12 is switched to be in the "1" position state. At this time, the second chromatography pump 21 pushes the liquid carbon dioxide, and the third chromatography pump 22 The modifier methanol is pushed, mixed in the third three-way mixer 28, and then enters the column 32 to separate the target in the column 32. After the separation is completed, the target is analyzed by the mass spectrometer detector 34.

Please refer to FIG. 14 , which is a six-time analysis of 100 mg of vanillin a vanillin a, p-hydroxybenzaldehyde b, vanillic acid by the multi-functional warming pressure extraction-capture-chromatography on-line equipment of the present invention. c and p-hydroxybenzoic acid d four A chromatogram of the compound. In the figure, vanillin, p-hydroxybenzaldehyde, vanillic acid and p-hydroxybenzoic acid are respectively from left to right in the order of peaks. It can be seen from the figure that the extraction-collection-separation by the apparatus of the present invention provides a stable baseline of the chromatographic line, sharp peaks and good resolution, indicating the multifunctional warming pressure extraction-trapping of the present invention. Chromatographic separation on-line equipment can be effectively used for on-line extraction, capture, desorption, separation and detection of trace targets in complex solid or semi-solid samples such as environment, food, medicine, and biology. And the analysis mode described in the above various embodiments, the apparatus of the present invention can automatically realize supercritical fluid extraction and acceleration by controlling valve switching of two high-pressure six-way switching valves and pressure adjustment of two back pressure regulators. The solvent extraction two extraction forms are combined with the free combination of supercritical fluid chromatography and high performance liquid chromatography to improve the efficiency, sensitivity and accuracy of sample analysis.

The above-described embodiments are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention.

Claims

Multifunctional heating and pressure extraction-trapping-chromatography separation on-line equipment, characterized in that it comprises an online trapping-desorption-switching device and an extraction-chromatography separation device;

The online trapping-desorption-switching device includes a first high-pressure six-way switching valve and a second high-pressure six-way switching valve, a first three-way mixer, a second three-way mixer, and a collecting column for flow path switching a first chromatographic pump and a connecting pipe for connecting the components; the first high-pressure six-way switching valve and the second high-pressure six-way switching valve are respectively provided with six interfaces in a clockwise order; the first chromatographic pump a first interface connected to the first high-pressure six-way switching valve; the first inlet and the second inlet of the first three-way mixer are respectively connected to the sixth interface and the second interface of the first high-pressure six-way switching valve The outlet of the first three-way mixer is connected to one end of the collecting column, the other end of the collecting column is connected to the first interface of the second high-pressure six-way switching valve, and the second interface of the second high-pressure six-way switching valve is connected Entering the first inlet of the second three-way mixer;

The extraction-chromatography separation device includes a third chromatography pump for pushing a solvent, a warming pressure extraction unit, a first three-way interface, a chromatography column, and a communication tube for connecting the components; the third chromatographic pumping Entering one end of the warming pressure extraction unit, the other end of the warming and pressure extraction unit is respectively connected to the fifth interface of the first high-pressure six-way switching valve and the first three-way mixer through the first three-way interface a second inlet; the inlet of the column is connected to the outlet of the second three-way mixer.
The multi-functional warming pressure extraction-capture-chromatography separation in-line apparatus according to claim 1, wherein said extraction-chromatography separation device further comprises a second chromatography pump for pushing liquid carbon dioxide and a three-way mixer, the second chromatographic pump and the third chromatographic pump are respectively connected to the first inlet and the second inlet of the third three-way mixer, and the outlet of the third three-way mixer is connected to the temperature and pressure One end of the extraction unit.
The multi-functional heating and pressure extraction-trapping-chromatography separation online combination device according to claim 1, wherein the multifunctional heating and pressure extraction-trapping-chromatography separation online combination device further comprises a first back pressure regulator and a second back pressure regulator, the first back pressure regulator is disposed downstream of a flow path of the chromatography column, and the second back pressure regulator is disposed at a flow path of the warming and pressure extraction unit Downstream and upstream of the flow path of the column.
The multi-functional warming pressure extraction-capture-chromatography separation line-in-line apparatus according to claim 1, wherein the extraction-chromatography separation device further comprises a fourth chromatography pump, a mass spectrometer detector, and a second three The fourth interface pump and the mass spectrometer detector are respectively connected to the outlet of the column through a second three-way interface.
The multi-functional heating and pressure extraction-collection-chromatography separation line-connecting device according to claim 3, wherein the first back pressure regulator is disposed between the column and the detector, The second back pressure regulator is disposed between the first three-way port and the first high-pressure six-way switching valve.
The multi-functional warming pressure extraction-capture-chromatography separation line-in-line apparatus according to claim 3, wherein the extraction-chromatography separation device further comprises a high-pressure liquid phase detector, and the high-pressure liquid phase detection Set on the column and the first A back pressure regulator between.
The multi-functional warming pressure extraction-trapping-chromatography separation on-line device according to claim 3, characterized in that: the second back pressure regulator outlet is provided with a one-way filter plate valve.
The multi-functional heating and pressure extraction-trapping-chromatography separation online combination device according to claim 1, wherein the multifunctional heating and pressure extraction-trapping-chromatography separation online combination device further comprises An autosampler disposed between the warming pressure extraction unit and the first three-way interface; the multifunctional heating and pressure extraction-trapping-chromatography separation online combination device further includes A column oven, the on-line trap-desorption-switching device and the column are disposed in the oven.
The multi-functional warming pressure extraction-capture-chromatography separation line-connecting apparatus according to claim 1, wherein said first chromatographic pump has a low-pressure gradient proportional valve built therein, and said first chromatographic pump has at least a pole Two solvents with different properties.
The multi-functional warming pressure extraction-trapping-chromatography separation on-line combination device according to claim 1, wherein: the third interface of the first high-pressure six-way switching valve and the second high-pressure six-way switching valve The third interface is sealed separately.