A device and method for online solid phase extraction of all types of plant hormones

Abstract

The present invention relates to a solid phase extraction method, and more specifically, to an online solid phase extraction device and method for all types of plant hormones. The method uses a fully automatic online solid phase extraction device with three sets of pressure heads, and adopts three solid phase extraction columns filled with different solid adsorbents to adsorb acidic, alkaline and neutral plant hormones respectively, and performs chromatographic separation and detection by sequential online elution and sampling. The method can achieve one-time sample pretreatment to obtain ten types of plant hormones, and is used in conjunction with liquid chromatography online to avoid losses caused by sample transfer and nitrogen blowing concentration, improve the sensitivity of analysis, reduce manual operations, and has the advantages of being fast and simple.

Description

Technical Field

[0001] The invention belongs to the field of analytical chemistry, and in particular relates to a plant sample pretreatment device and method. The method is suitable for sample pretreatment when analyzing all types of plant hormones in plant samples. Background Art

[0002] Endogenous plant hormones are a series of small molecule organic compounds synthesized in plants and involved in regulating the entire life process of plants. Endogenous plant hormones mainly include ten categories: auxin, abscisic acid, gibberellins, cytokinins, jasmonic acid, salicylic acid, ethylene, brassinosteroids, strigolactones and polypeptide hormones. Different types of plant hormones interact with each other in regulating plant physiological processes. In order to better understand the molecular mechanism of plant hormones and accurately describe their role in complex regulatory networks, it is necessary to simultaneously detect all categories in a plant tissue sample, that is, the concentration of all categories of plant hormones.

[0003] However, the matrix of plant samples is complex, and the content of plant hormones is extremely low, usually in the order of ng / g or even pg / g. Some hormones, such as brassinosteroids, are as low as 0.01-0.1 ng / g fresh weight. At the same time, the chemical properties of all categories of plant hormones vary greatly, including acidic plant hormones (auxin, abscisic acid, gibberellin, jasmonic acid, salicylic acid), weakly alkaline plant hormones (cytokinins, ethylene precursor 1-aminocyclopropanecarboxylic acid), and neutral brassinosteroids and strigolactones. The concentrations of various plant hormones in different plant tissues also vary greatly, which brings great difficulties to the quantitative analysis of all categories of plant hormones.

[0004] In the prior art, solid phase extraction is generally used to pre-treat plant samples, and then separation and detection are carried out through high performance liquid chromatography-tandem mass spectrometry. For the simultaneous analysis and detection of multiple plant hormones, multiple solid adsorbents are generally required for multi-step solid phase extraction. This off-line operation requires nitrogen blowing and concentration of the solid phase extraction eluent before it can be injected into the chromatograph for separation. The nitrogen blowing and concentration process is prone to cause the loss of low-boiling point compounds; the concentrated sample cannot all enter the chromatographic column, and the sample utilization rate is limited. At the same time, the combined use of multiple solid phase extraction columns, or the combined use of solid phase extraction and other extraction methods, is usually accompanied by multi-step liquid transfer operations, which inevitably leads to transfer losses.

[0005] Fully automatic online solid phase extraction technology is an effective means to solve the above problems. It relies on double-sided sealed solid phase extraction columns and mechanically controlled column movement to achieve fully automatic online solid phase extraction. Because it can be directly loaded with high pressure, it can be directly eluted in front of the liquid chromatography column, and the eluent enters the chromatographic column. The sample utilization rate of this step can reach 100%. However, the current fully automatic online solid phase extraction technology adopts a single-column operation mode, with only two pairs of chucks, which are used for loading-extraction and elution-chromatographic injection respectively. It is impossible to realize the series extraction of different SPE columns, and the instrument does not have an online derivatization function, which makes it difficult to meet the needs of all categories of plant hormone analysis. Summary of the invention

[0006] The purpose of the present invention is to provide an apparatus and method for online solid-phase extraction of all types of plant hormones. By improving the structure of the existing fully automatic online solid-phase extraction device, three different solid-phase extraction columns are connected in series for solid-phase extraction and eluted and injected in sequence, thereby achieving simultaneous purification and enrichment of all types of plant hormones in complex plant samples, namely, acidic, neutral and alkaline plant hormones, thereby improving the selectivity and sensitivity of subsequent chromatographic analysis, and solving the problem that the existing online solid-phase extraction cannot simultaneously perform pre-treatment on all types of plant hormones.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0008] On one hand, the present invention provides an online solid phase extraction device for all types of plant hormones, the device comprising a first pressure head group, a second pressure head group, a third pressure head group, a solid phase extraction column A, a solid phase extraction column B, a solid phase extraction column C, a six-way valve and a four-way valve;

[0009] Each set of press heads includes an upper press head and a lower press head, which are used to clamp the solid phase extraction column in a vertical direction;

[0010] The inlet of the first upper pressure head is the inlet of the sample solution, the outlet of the first lower pressure head is connected to the second position of the four-way valve, the inlet of the second upper pressure head is connected to the first position of the four-way valve, the outlet of the second lower pressure head is connected to the fourth position of the six-way valve, the inlet of the third upper pressure head is connected to the third position of the six-way valve, and the outlet of the third lower pressure head is connected to the sixth position of the six-way valve;

[0011] The fourth position of the four-way valve is the liquid inlet, and the third position of the four-way valve is connected to the waste liquid bottle one;

[0012] The second position of the six-way valve is the inlet of the chromatographic mobile phase, the first position is connected to the chromatographic column, and the fifth position is connected to the second waste liquid bottle;

[0013] Each set of pressure heads is provided with a liquid channel running through the upper and lower ends along the central axis;

[0014] The solid phase extraction column A, solid phase extraction column B and solid phase extraction column C are all cylindrical structures, with a circular through hole penetrating the upper and lower ends along the central axis of the column, the through hole is coaxial with the liquid channel inside the pressure head group, and the through hole is filled with a solid adsorbent with a cation exchange function, a solid adsorbent with anion exchange function, and a non-polar solid adsorbent or a solid adsorbent with a derivative functional group, respectively.

[0015] The device also includes a column tray, on which the solid phase extraction columns are placed, and the movement of the solid phase extraction columns is achieved through the horizontal movement of the column tray.

[0016] Each solid phase extraction column has sieve plates tightly integrated with the column at the upper and lower ends to encapsulate the filler in the through hole; the two end surfaces of the solid phase extraction column match the size of the contact surface of the pressure head group.

[0017] The through-hole direct diameters of the solid phase extraction column A, solid phase extraction column B and solid phase extraction column C are 1-3 mm, the volume of the solid adsorbent filled inside is 6.3-70 μL, and the filling volume is 80-100% of the through-hole volume.

[0018] Another aspect of the present invention provides a method for solid phase extraction using the above-mentioned online solid phase extraction device, characterized in that the method comprises the following steps:

[0019] (a) Activation: Before solid phase extraction, the first and second positions of the four-way valve are connected, the third position of the six-way valve is connected with the fourth, fifth and sixth positions, and the activation solution enters from the first upper pressure head and flows through the solid phase extraction column A, the solid phase extraction column B and the solid phase extraction column C in sequence, so as to activate the three solid phase extraction columns;

[0020] (b) Loading: The valve positions of the four-way valve and the six-way valve are the same as those in step (a); a liquid chromatography automatic sampler is used to quantitatively absorb a plant sample extract containing all types of plant hormones, and the plant sample extract is loaded onto solid phase extraction column A under the push of the loading solution, at which time the alkaline plant hormones in the extract are adsorbed by the solid adsorbent in solid phase extraction column A; the extract flowing out of solid phase extraction column A enters solid phase extraction column B through the four-way valve, and the acidic plant hormones in the extract are adsorbed by the solid adsorbent in solid phase extraction column B; the outflowing extract then enters solid phase extraction column C through the six-way valve, at which time the neutral plant hormones in the extract are adsorbed;

[0021] (c) Elution and separation: switch the six-way valve, so that positions 2 and 3, 4 and 5, and 1 and 6 of the six-way valve are connected. The liquid chromatography mobile phase enters the chromatographic column through the six-way valve and the solid phase extraction column C. At this time, the neutral plant hormones adsorbed on the solid phase extraction column C are eluted and directly enter the chromatographic column for separation and analysis; after the elution is completed, switch the six-way valve, so that positions 1 and 2 are connected; after the chromatographic analysis of the neutral plant hormones is completed, release the three sets of pressure heads, move the solid phase extraction column B between the third upper pressure head and the third lower pressure head, and clamp it; at this time, the solid phase extraction column A is moved between the second upper pressure head and the second lower pressure head and is clamped by them; switch again The six-way valve is switched to connect its second position with the third position, and the mobile phase enters the chromatographic column through the six-way valve and the solid phase extraction column B. At this time, the acidic plant hormones adsorbed on the solid phase extraction column B are eluted and directly enter the chromatographic column for separation and analysis; then the six-way valve is switched to connect its first position with the second position; after the analysis of the acidic plant hormones is completed, the three sets of pressure heads are released again, and the solid phase extraction column A is moved between the third upper pressure head and the third lower pressure head, and clamped; the six-way valve is switched to connect the second position with the third position, and the mobile phase enters the chromatographic column through the six-way valve and the solid phase extraction column A. At this time, the alkaline plant hormones adsorbed on the solid phase extraction column A are eluted and directly enter the chromatographic column for separation and analysis.

[0022] In the step (c), when the solid phase extraction column B moves between the third upper pressure head and the third lower pressure head, and the solid phase extraction column A is moved between the second upper pressure head and the second lower pressure head, the first position of the six-way valve is connected to the second position, the third position is connected to the fourth position, and the fifth position is connected to the sixth position; at this time, the four-way valve is first switched to connect the fourth position with the first position, and the eluent enters from the fourth position of the four-way valve, flows through the solid phase extraction column A and the solid phase extraction column B, and is finally discharged from the fifth position of the six-way valve to achieve the elution of the solid phase extraction column A and the solid phase extraction column B; after the elution is completed, the six-way valve is switched again, and the mobile phase elutes the solid phase extraction column B.

[0023] The solid phase extraction column C is filled with a solid adsorbent with a derivatization functional group, wherein the derivatization functional group is phenylboronic acid, which is used to derive the neutral plant hormone brassinosteroid. The compound containing the phenylboronic acid group is modified on the surface of the solid adsorbent through electrostatic interaction, and during elution, the electrostatic interaction is destroyed to cause the derivatized brassinosteroid to fall off the surface of the solid adsorbent.

[0024] The composition of the mobile phase is varied according to the elution of different solid phase extraction cartridges.

[0025] The activation solution is acetonitrile or methanol; the loading solution is a pure water solution; the eluent is methanol-water or acetonitrile-water solution, wherein the volume fraction of methanol or acetonitrile is not greater than 10%; the eluent volume is 20-200 μL.

[0026] The flow rate of the activation solution is 0.5-1 mL / min; the flow rates of the loading solution and the eluent are 0.1-0.5 mL / min; and the flow rate of the mobile phase during elution is 0.2-1 mL / min.

[0027] The full range of plant hormones includes auxin, abscisic acid, gibberellin, cytokinin, jasmonic acid, salicylic acid, ethylene precursors, brassinosteroids, strigolactones and polypeptide hormones.

[0028] The main differences between the present invention and the prior art are:

[0029] The online solid phase extraction method in the prior art can usually only be used for sample pretreatment of a class of plant hormones. For example, patent ZL201810332157.2 (a method for fully automatic serial solid phase extraction of brassinosteroids in plants) uses two serially connected solid phase extraction columns to extract and derive brassinosteroids in plant samples. This method is only applicable to brassinosteroids. Even if the column material is improved, it can only be used for a certain class of plant hormones and cannot simultaneously process other classes of plant hormones. The method disclosed in the literature (Analytical Chemistry, 2016, 44, 409-415) can also only be used for acidic plant hormones. Other methods that use capillary solid phase extraction on a valve in conjunction with online liquid chromatography can also only process one class of plant hormones at a time, and each time the capillary solid phase extraction column connected to the valve needs to be manually removed, and automatic column replacement cannot be achieved, which is completely different from the fully automatic solid phase extraction device used in the present invention. The online solid phase extraction method used in the present invention classifies the current ten types of plant hormones into three categories (acidic, alkaline and neutral), uses a fully automatic online solid phase extraction device with three sets of pressure heads, and uses three extraction columns filled with different solid adsorbents to adsorb acidic, alkaline and neutral plant hormones respectively. Then, according to the chemical properties of each major type of plant hormone, it is eluted online in sequence for chromatographic separation and analysis.

[0030] The beneficial effects of the present invention are:

[0031] (1) The volume of the adsorbent in the solid phase extraction column used is 6.3 to 70 μL, and the eluent can all enter the liquid chromatography column, avoiding the nitrogen blowing concentration step of traditional solid phase extraction, reducing the operation steps, avoiding sample loss during the nitrogen blowing process, greatly improving sample utilization, and realizing the online combination of solid phase extraction and chromatographic separation;

[0032] (2) Three sets of press heads and three solid adsorbents are used to treat acidic, alkaline and neutral plant hormones respectively. One sample pretreatment can achieve the extraction and enrichment of all categories (ten categories) of plant hormones, eliminating the multi-step sample transfer process. The method is simpler and faster, and reduces sample loss;

[0033] (3) Using a solid adsorbent with a derivatization functional group to derivatize brassinosteroids on a column, since on-column derivatization can increase the local reaction concentration and facilitate the control of the derivatization reaction time, compared with the method of eluting with a derivatization solution, on-column derivatization has a higher derivatization efficiency, which is very beneficial for improving the detection sensitivity of trace amounts of brassinosteroids;

[0034] (4) In this method, three solid phase extraction columns are used to retain acidic, alkaline and neutral plant hormones respectively, and each column is then eluted, chromatographically separated and detected in turn, thereby realizing the sequential chromatographic injection analysis of acidic, alkaline and neutral plant hormones, avoiding the problem of incompatible chromatographic separation conditions caused by large differences in chemical properties of different types of plant hormones, and is conducive to improving the resolution of chromatographic separation;

[0035] (5) The pretreatment method proposed in the present invention can perform classified solid phase extraction on all types of plant hormones according to their chemical properties. For example, acidic and alkaline plant hormones can be enriched on different solid phase extraction columns, and the conditions can be optimized separately, which is conducive to obtaining the best extraction effect for each type of plant hormone.

[0036] (6) The elution process can be increased or decreased according to the needs of the actual sample. The method is highly flexible and suitable for various plant samples.

[0037] (7) Compared with the existing fully automatic online solid phase extraction method, the technical solution of the present invention not only realizes the pretreatment of all types of plant hormones, but also realizes the series use of three solid phase extraction columns. This series connection can not only capture plant hormones of different properties separately, but also facilitates the acquisition of cleaner eluents, reduces the matrix effect of chromatographic analysis, and improves analytical selectivity.

[0038] There is no precedent for a fully automatic online solid phase extraction device with three sets of pressure heads. The present invention proposes a technical solution of three sets of pressure heads for the first time. By designing solid phase extraction columns and extraction processes, simultaneous sample pretreatment of all categories of plant hormones is achieved; the online combination of solid phase extraction and liquid chromatography, as well as the full automation of the method, avoids errors and sample losses caused by offline manual operation, saves time and labor, improves the throughput and speed of sample processing, and has the advantages of high extraction efficiency and simple operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a schematic diagram of three groups of pressure heads of the online solid phase extraction device of the present invention;

[0040] Figure 2 It is a schematic structural diagram of the online solid phase extraction device of the present invention;

[0041] In the figure, 11-first upper pressure head, 12-first lower pressure head, 21-second upper pressure head, 22-second lower pressure head, 31-third upper pressure head, 32-third lower pressure head, 40-column tray, 10-solid phase extraction column A, 20-solid phase extraction column B, 30-solid phase extraction column C, 50-chromatographic column. DETAILED DESCRIPTION

[0042] A fully automatic online solid phase extraction device such as Figure 1-2 As shown, it includes a first pressure head group, a second pressure head group, a third pressure head group, a solid phase extraction column A10, a solid phase extraction column B20, a solid phase extraction column C30, a column tray 40, a six-way valve and a four-way valve; each group of pressure heads has an upper pressure head and a lower pressure head, which are used to clamp the solid phase extraction column from a vertical direction; a liquid channel running through the upper and lower ends is provided inside the pressure head along the central axis; the solid phase extraction column is a cylindrical structure, and a circular through hole running through the upper and lower ends is opened along the central axis of the column, the through hole is coaxial with the liquid channel inside the pressure head group, the diameter of the through hole is 1 to 3 mm, and the through hole is filled with a solid adsorbent, the volume of the solid adsorbent is 6.3 to 70 μL, and the filling volume is 8 to 100% of the through hole volume; the upper and lower ends of the column are provided with sieve plates tightly combined with the column to encapsulate the filler in the through hole; the two end surfaces of the solid phase extraction column match the size of the contact surface of the pressure head group of the full-automatic online solid phase extraction device.

[0043] The inlet of the first upper pressure head 11 is the sample solution inlet, and the outlet of the first lower pressure head 12 is connected to the second position of the four-way valve; the first position of the four-way valve is connected to the inlet of the second upper pressure head 21, the third position of the four-way valve is connected to the waste liquid bottle one, and the fourth position of the four-way valve is the liquid inlet; the outlet of the second lower pressure head 22 is connected to the fourth position of the six-way valve, the inlet of the third upper pressure head 31 is connected to the third position of the six-way valve, and the outlet of the third lower pressure head 32 is connected to the sixth position of the six-way valve; the fifth position of the six-way valve is connected to the waste liquid bottle two, the first position is connected to the chromatographic column 50, and the second position is the chromatographic mobile phase inlet.

[0044] The first upper press head 11 and 12 clamp the solid phase extraction column A10, and the solid phase extraction column A10 is filled with a solid adsorbent with a cation exchange function; the second upper press head 21 and the second lower press head 22 clamp the solid phase extraction column B20, and the solid phase extraction column B20 is filled with a solid adsorbent with an anion exchange function; the third upper press head 31 and the third lower press head 32 clamp the solid phase extraction column C30, and the solid phase extraction column C30 is filled with a non-polar solid adsorbent or a solid adsorbent with a derivatization functional group, and the derivatization functional group is phenylboronic acid, which is used to derive the neutral plant hormone brassinosteroid; the compound containing the phenylboronic acid group is modified on the surface of the solid adsorbent through electrostatic interaction, and during elution, the electrostatic interaction is destroyed to cause the derivatized brassinosteroid to fall off the surface of the solid adsorbent.

[0045] The solid phase extraction columns are all placed on the column tray 40 , and the movement of the solid phase extraction columns is achieved through the horizontal movement of the column tray 40 .

[0046] The online solid phase extraction using the above device comprises the following steps:

[0047] (a) Activation: Before solid phase extraction, the first and second positions of the four-way valve are connected, the third position of the six-way valve is connected with the fourth, fifth and sixth positions, and the activation liquid enters from the first upper pressure head 11, flows through the solid phase extraction column A10, the solid phase extraction column B20 and the solid phase extraction column C30 in sequence, and activates the three solid phase extraction columns; the activation liquid is any one of acetonitrile or methanol, and the flow rate is 0.5-1 mL / min;

[0048] (b) Loading: The valve positions of the four-way valve and the six-way valve are the same as those in step a; a plant sample extract containing all types of plant hormones (auxin, abscisic acid, gibberellin, cytokinin, jasmonic acid, salicylic acid, ethylene precursor, brassinosteroid, strigolactone and polypeptide hormone) is quantitatively drawn by a liquid chromatography automatic sample injector, and the plant sample extract is loaded onto a solid phase extraction column A10 under the impetus of a pure aqueous solution, at which time the alkaline plant hormones in the extract are adsorbed by the solid adsorbent in the solid phase extraction column A10; the extract flowing out of the solid phase extraction column A10 enters a solid phase extraction column B20 through a four-way valve, and the acidic plant hormones in the extract are adsorbed by the solid adsorbent in the solid phase extraction column B20; the outflowing extract enters a solid phase extraction column C30 through a six-way valve, at which time the neutral plant hormones in the extract are adsorbed; the flow rate of the loading solution is 0.1 to 0.5 mL / min;

[0049] (c) Elution and separation: switch the six-way valve, the second position and the third position, the fourth position and the fifth position, and the first position and the sixth position of the six-way valve are connected, and the liquid chromatography mobile phase enters the chromatographic column 50 through the six-way valve and the solid phase extraction column C30. At this time, the neutral plant hormones adsorbed on the solid phase extraction column C30 are eluted and directly enter the chromatographic column 50 for separation and analysis; after the elution is completed, switch the six-way valve, and its first position is connected to the second position; after the chromatographic analysis and detection of the neutral plant hormones are completed, release the three groups of pressure heads, and rely on the movement of the column tray to move the solid phase extraction column B20 to between the third upper pressure head 31 and the third lower pressure head 32, and clamp it; at this time, the solid phase extraction column A10 is moved to between the second upper pressure head 21 and the second lower pressure head 22, and is clamped by them; switch the six-way valve again, so that its second position is connected to The third position is connected, and the mobile phase enters the chromatographic column 50 through the six-way valve and the solid phase extraction column B20. At this time, the acidic plant hormones adsorbed on the solid phase extraction column B20 are eluted and directly enter the chromatographic column 50 for separation and analysis; then the six-way valve is switched, and after the analysis of the acidic plant hormones is completed, the three groups of pressure heads are released again, and the solid phase extraction column A10 is moved to between the third upper pressure head 31 and the third lower pressure head 32, and clamped; the six-way valve is switched, and the mobile phase enters the chromatographic column 50 through the six-way valve and the solid phase extraction column A10. At this time, the alkaline plant hormones adsorbed on the solid phase extraction column A10 are eluted and directly enter the chromatographic column 50 for separation and analysis; the flow rate of the mobile phase during elution is 0.2-1mL / min, and the composition of the mobile phase changes according to the different solid phase extraction columns eluted.

[0050] In one embodiment of the present invention, in step (c), when the solid phase extraction column B20 is moved between the third upper pressure head 31 and the third lower pressure head 32, and the solid phase extraction column A10 is moved between the second upper pressure head 21 and the second lower pressure head 22, the first position of the six-way valve is connected to the second position, the third position is connected to the fourth position, and the fifth position is connected to the sixth position; at this time, the four-way valve is switched first, so that the fourth position is connected to the first position, and the eluent enters from the fourth position of the four-way valve and flows through the solid phase extraction column A10. Column A10 and solid phase extraction column B20 are finally discharged from position 5 of the six-way valve to achieve elution of the solid phase extraction column A10 and the solid phase extraction column B20; after the elution is completed, the six-way valve is switched again, and the mobile phase elutes the solid phase extraction column B20; the eluent is methanol-water or acetonitrile-water solution, wherein the volume fraction of methanol or acetonitrile is not greater than 10%; the volume of the eluent is 20 to 200 μL; the flow rate of the eluent is 0.1 to 0.5 mL / min.

[0051] The present invention is described below in conjunction with specific embodiments. The illustrative embodiments and descriptions of the present invention are used to explain the present invention but are not intended to limit the present invention.

[0052] Example 1

[0053] like Figure 2 As shown, a fully automatic online solid phase extraction device is used for solid phase extraction, and the device includes a first pressure head group, a second pressure head group, a third pressure head group, a solid phase extraction column A10, a solid phase extraction column B20, a solid phase extraction column C30, a column tray 40, a six-way valve and a four-way valve; each group of pressure heads has an upper pressure head and a lower pressure head, which are used to clamp the solid phase extraction column from a vertical direction; a liquid channel running through the upper and lower ends is provided inside the pressure head along the central axis; the solid phase extraction column is a cylindrical structure, and a circular through hole running through the upper and lower ends is opened along the central axis of the column, the through hole is coaxial with the liquid channel inside the pressure head group, the through hole has a diameter of 2mm, and the through hole is filled with a solid adsorbent, the volume of the solid adsorbent is 20μL, and the filling volume is 90% of the through hole volume; a sieve plate tightly combined with the column is provided at the upper and lower ends of the column to encapsulate the filler in the through hole; the two end surfaces of the solid phase extraction column match the contact surface size of the pressure head group of the fully automatic online solid phase extraction device.

[0054] The inlet of the first upper pressure head 11 is the sample solution inlet, and the outlet of the first lower pressure head 12 is connected to the second position of the four-way valve; the first position of the four-way valve is connected to the inlet of the second upper pressure head 21, the third position of the four-way valve is connected to the waste liquid bottle one, and the fourth position of the four-way valve is the liquid inlet; the outlet of the second lower pressure head 22 is connected to the fourth position of the six-way valve, the inlet of the third upper pressure head 31 is connected to the third position of the six-way valve, and the outlet of the third lower pressure head 32 is connected to the sixth position of the six-way valve; the fifth position of the six-way valve is connected to the waste liquid bottle two, the first position is connected to the chromatographic column 50, and the second position is the chromatographic mobile phase inlet.

[0055] The first upper pressure head 11 and the first lower pressure head 12 clamp the solid phase extraction column A10, which is a mixed cation exchange column; the second upper pressure head 21 and the second lower pressure head 22 clamp the solid phase extraction column B20, which is a mixed anion exchange column; the third upper pressure head 31 and the third lower pressure head 32 clamp the solid phase extraction column C30, which is filled with a silica gel solid adsorbent with a phenylboronic acid derivative functional group for deriving the neutral plant hormone brassinosteroid; the phenylboronic acid group is modified on the surface of the silica gel solid adsorbent through electrostatic interaction, and during elution, the electrostatic interaction is destroyed to cause the derived brassinosteroid to fall off the surface of the solid adsorbent.

[0056] The solid phase extraction columns are all placed on the column tray 40 , and the movement of the solid phase extraction columns is achieved through the horizontal movement of the column tray 40 .

[0057] The steps of online solid phase extraction are as follows:

[0058] (a) Activation: Before solid phase extraction, the first and second positions of the four-way valve are connected, the third and fourth positions, and the fifth and sixth positions of the six-way valve are connected, and the activation liquid acetonitrile enters from the first upper pressure head 11 at a flow rate of 1.0 mL / min, and flows through the solid phase extraction column A10, the solid phase extraction column B20 and the solid phase extraction column C30 in sequence, and the three solid phase extraction columns are activated;

[0059] (b) Loading: The valve positions of the four-way valve and the six-way valve are the same as those in step (a); a plant sample extract containing all types of plant hormones (auxin, abscisic acid, gibberellin, cytokinin, jasmonic acid, salicylic acid, ethylene precursor, brassinosteroids, strigolactones and polypeptide hormones) is quantitatively drawn by a liquid chromatography autosampler, and the plant sample extract is loaded onto a solid phase extraction cartridge A10 under the impetus of a pure aqueous solution. At this time, alkaline plant hormones in the extract, such as cytokinin and ethylene precursor 1-aminobutyric acid, are The extract from the solid phase extraction column A10 enters the solid phase extraction column B20 through a four-way valve, and the acidic plant hormones such as auxin, abscisic acid, gibberellin, jasmonic acid, salicylic acid, and the polypeptide hormone phytothiopeptide in the extract are adsorbed by the solid phase extraction column B20; the extract then enters the solid phase extraction column C30 through a six-way valve, and the neutral plant hormones brassinosteroid and strigolactone in the extract are adsorbed; the flow rate of the loading solution is 0.2 mL / min;

[0060] (c) Elution and separation: switch the six-way valve, and the second and third, fourth and fifth, and fifth and sixth positions of the six-way valve are connected. The liquid chromatography mobile phase enters the chromatographic column 50 through the six-way valve and the solid phase extraction column C30. At this time, the neutral plant hormones adsorbed on the solid phase extraction column C30 are eluted and directly enter the chromatographic column 50 for separation and analysis; after the elution is completed, switch the six-way valve, and its first and second positions are connected; after the chromatographic analysis of the neutral plant hormones is completed, release the three groups of pressure heads, and rely on the movement of the column tray 40 to move the solid phase extraction column B20 to between the third upper pressure head 31 and the third lower pressure head 32, and clamp it; at this time, the solid phase extraction column A10 is moved to between the second upper pressure head 21 and the second lower pressure head 22, and is clamped by them; switch again The six-way valve is used to connect its second position with the third position, and the mobile phase enters the chromatographic column through the six-way valve and the solid phase extraction column B20. At this time, the acidic plant hormones adsorbed on the solid phase extraction column B20 are eluted and directly enter the chromatographic column for separation and analysis; then the six-way valve is switched, and after the analysis of the acidic plant hormones is completed, the three sets of pressure heads are released again, and the solid phase extraction column A10 is moved between the third upper pressure head 31 and the third lower pressure head 32, and clamped; the six-way valve is switched, and the mobile phase enters the chromatographic column 50 through the six-way valve and the solid phase extraction column A. At this time, the alkaline plant hormones adsorbed on the solid phase extraction column A10 are eluted and directly enter the chromatographic column 50 for separation and analysis; the mobile phase during elution is 60% (v / v) acetonitrile-water solution, and the flow rate is 0.8mL / min.

[0061] Example 2

[0062] As described in Example 1, the difference is that during elution separation, when the solid phase extraction column B20 moves between the third upper pressure head 31 and the third lower pressure head 32, and the solid phase extraction column A is moved between the second upper pressure head 21 and the second lower pressure head 22, the first position of the six-way valve is connected to the second position, the third position is connected to the fourth position, and the fifth position is connected to the sixth position; at this time, the four-way valve is first switched to connect its fourth position to the first position, and the eluent enters from the fourth position of the four-way valve, flows through the solid phase extraction column A10 and the solid phase extraction column B20, and is finally discharged from the fifth position of the six-way valve to achieve the elution of the solid phase extraction column A10 and the solid phase extraction column B20; after the elution is completed, the six-way valve is switched again, and the mobile phase elutes the solid phase extraction column B; the eluent is 5% (v / v) methanol-water solution, the eluent volume is 60μL, and the flow rate is 0.1mL / min.

[0063] Example 3

[0064] The method described in Example 1 is different in that the through-hole diameter of the solid phase extraction column is 1 mm; the volume of the solid adsorbent filled inside is 8 μL; the flow rate of the loading solution is 0.5 mL / min, and the activation solution is methanol.

Claims

1. An online solid phase extraction device for all types of plant hormones, characterized in that: The device comprises a first pressure head group, a second pressure head group, a third pressure head group, a solid phase extraction column A (10), a solid phase extraction column B (20), a solid phase extraction column C (30), a column tray (40), a six-way valve and a four-way valve; Each set of press heads includes an upper press head and a lower press head, which are used to clamp the solid phase extraction column in a vertical direction; The inlet of the first upper pressure head (11) is the inlet of the sample solution, the outlet of the first lower pressure head (12) is connected to the second position of the four-way valve, the inlet of the second upper pressure head (21) is connected to the first position of the four-way valve, the outlet of the second lower pressure head (22) is connected to the fourth position of the six-way valve, the inlet of the third upper pressure head (31) is connected to the third position of the six-way valve, and the outlet of the third lower pressure head (32) is connected to the sixth position of the six-way valve; The fourth position of the four-way valve is the eluent inlet, and the third position of the four-way valve is connected to the waste liquid bottle one; The second position of the six-way valve is the chromatographic mobile phase inlet, the first position is connected to the chromatographic column (50), and the fifth position is connected to the waste liquid bottle 2; Each set of pressure heads is provided with a liquid channel running through the upper and lower ends along the central axis; The solid phase extraction column A (10), solid phase extraction column B (20), and solid phase extraction column C (30) are all cylindrical structures, with a circular through hole penetrating the upper and lower ends along the central axis of the column, the through hole being coaxial with the liquid channel inside the pressure head group, and the through hole being filled with a solid adsorbent with a cation exchange function, a solid adsorbent with an anion exchange function, and a non-polar solid adsorbent or a solid adsorbent with a derivatized functional group, respectively; The solid phase extraction columns are all placed on the column tray (40), and the movement of the solid phase extraction columns is achieved through the horizontal movement of the column tray (40).

2. The device according to claim 1, characterized in that: Each solid phase extraction column has sieve plates tightly integrated with the column at the upper and lower ends to encapsulate the filler in the through hole; the two end surfaces of the solid phase extraction column match the size of the contact surface of the pressure head group.

3. The device according to claim 1, characterized in that: The through-hole direct diameters of the solid phase extraction column A (10), solid phase extraction column B (20) and solid phase extraction column C (30) are 1 to 3 mm, the volume of the solid adsorbent filled inside is 6.3 to 70 μL, and the filling volume is 80 to 100% of the through-hole volume.

4. A method for solid phase extraction using the online solid phase extraction device according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: (a) Activation: Before solid phase extraction, the first and second positions of the four-way valve are connected, the third position of the six-way valve is connected with the fourth, fifth and sixth positions, and the activation liquid enters from the first upper pressure head (11), flows through the solid phase extraction column A (10), the solid phase extraction column B (20) and the solid phase extraction column C (30) in sequence, and activates the three solid phase extraction columns; (b) Loading: The valve positions of the four-way valve and the six-way valve are the same as those in step (a); a liquid chromatography automatic sampler is used to quantitatively absorb a plant sample extract containing all types of plant hormones, and the plant sample extract is loaded onto a solid phase extraction column A (10) under the push of the loading solution. At this time, the alkaline plant hormones in the extract are adsorbed by the solid adsorbent in the solid phase extraction column A (10); the extract flowing out of the solid phase extraction column A (10) enters the solid phase extraction column B (20) through the four-way valve, and the acidic plant hormones in the extract are adsorbed by the solid adsorbent in the solid phase extraction column B (20); the extracted extract then enters the solid phase extraction column C (30) through the six-way valve, and at this time, the neutral plant hormones in the extract are adsorbed; (c) Elution and separation: switch the six-way valve, so that the second and third positions, the fourth and fifth positions, and the first and sixth positions of the six-way valve are connected. The liquid chromatography mobile phase enters the chromatographic column (50) through the six-way valve and the solid phase extraction column C (30). At this time, the neutral plant hormone adsorbed on the solid phase extraction column C (30) is eluted and directly enters the chromatographic column (50) for separation and analysis; after the elution is completed, switch the six-way valve, so that its first position is connected to the second position; after the chromatographic analysis of the neutral plant hormone is completed, release the three sets of pressure heads, move the solid phase extraction column B (20) between the third upper pressure head (31) and the third lower pressure head (32), and clamp them; at this time, the solid phase extraction column A (10) is moved between the second upper pressure head (21) and the second lower pressure head (22), and is clamped by them; switch the six-way valve again The six-way valve is switched to connect the second position with the third position, and the mobile phase enters the chromatographic column (50) through the six-way valve and the solid phase extraction column B (20). At this time, the acidic plant hormones adsorbed on the solid phase extraction column B (20) are eluted and directly enter the chromatographic column (50) for separation and analysis; the six-way valve is then switched to connect the first position with the second position; after the acidic plant hormone analysis is completed, the three sets of pressure heads are released again, and the solid phase extraction column A (10) is moved between the third upper pressure head (31) and the third lower pressure head (32), and clamped; the six-way valve is switched to connect the second position with the third position, and the mobile phase enters the chromatographic column (50) through the six-way valve and the solid phase extraction column A (10). At this time, the alkaline plant hormones adsorbed on the solid phase extraction column A (10) are eluted and directly enter the chromatographic column (50) for separation and analysis.

5. The method according to claim 4, characterized in that: In the step (c), when the solid phase extraction column B (20) moves between the third upper pressure head (31) and the third lower pressure head (32), and the solid phase extraction column A (10) is moved between the second upper pressure head (21) and the second lower pressure head (22), the first position of the six-way valve is connected to the second position, the third position is connected to the fourth position, and the fifth position is connected to the sixth position; at this time, the four-way valve is first switched to connect the fourth position with the first position, and the eluent enters from the fourth position of the four-way valve, flows through the solid phase extraction column A (10) and the solid phase extraction column B (20), and finally is discharged from the fifth position of the six-way valve, so as to achieve the elution of the solid phase extraction column A (10) and the solid phase extraction column B (20); after the elution is completed, the six-way valve is switched again, and the mobile phase elutes the solid phase extraction column B (20).

6. The method according to claim 4, characterized in that: The solid phase extraction column C (30) is filled with a solid adsorbent with a derivatization functional group, wherein the derivatization functional group is phenylboronic acid, which is used to derive the neutral plant hormone brassinosteroid. The compound containing the phenylboronic acid group is modified on the surface of the solid adsorbent through electrostatic interaction, and during elution, the electrostatic interaction is destroyed so that the derivatized brassinosteroid falls off the surface of the solid adsorbent.

7. The method according to claim 5, characterized in that: The activation solution is either acetonitrile or methanol; the loading solution is a pure water solution; the eluent is methanol-water or acetonitrile-water solution, wherein the volume fraction of methanol or acetonitrile is not greater than 10%; the eluent volume is 20-200 μL.

8. The method according to claim 5, characterized in that: The flow rate of the activation solution is 0.5-1 mL / min; the flow rates of the loading solution and the eluent are 0.1-0.5 mL / min; and the flow rate of the mobile phase during elution is 0.2-1 mL / min.

9. The method according to claim 4, characterized in that: The full range of plant hormones includes auxin, abscisic acid, gibberellin, cytokinin, jasmonic acid, salicylic acid, ethylene precursors, brassinosteroids, strigolactones and polypeptide hormones.

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