Flexible row extraction column structure suitable for high-throughput operation and separation device

Abstract

The utility model provides a flexible row extraction column structure suitable for high-throughput operation and a separation device. The extraction column structure and a liquid storage pipe are sleeved for use; the extraction column structure is inserted into the liquid storage pipe and is pressed down for filtration, to-be-filtered liquid stored in the liquid storage pipe enters from an opening in the bottom of the filter pipe, sequentially penetrates through the sieve plate assembly (6) and the filter membrane assembly (3) and then enters an inner cavity of the pipe body, and extraction liquid is obtained after pressing down for filtration; through design optimization of the sieve plate assembly (6) and the filter membrane assembly (3), the extraction column structure can be used for liquid-liquid separation or solid-liquid separation, and when the extraction column structure is used, the extraction column structure is inserted into a liquid storage pipe and pressed down to obtain a separated product. The extraction column structure has important application value in realization of high throughput and automatic detection.

Description

Technical Field

[0001] The utility model belongs to the technical field of extraction, and in particular relates to a flexible linked extraction column structure and a separation device suitable for high-throughput operation. Background Art

[0002] Solid-liquid separation (such as protein precipitation, etc.) and liquid-liquid extraction are very commonly used pretreatment methods for organic compound testing (including clinical applications). These two pretreatment methods can cover about 90% of clinical testing needs.

[0003] Protein precipitation is a separation method that destroys the hydration of protein molecules or weakens the factors of intermolecular repulsion, so that the solubility of protein in water is reduced and precipitated to convert into solid. Common methods include: salting out, heavy metal salt precipitation of protein, alkaloid reagent and certain acid precipitation of protein, isoelectric precipitation, etc. At present, when performing protein precipitation pretreatment, it is necessary to use centrifugation and other means to separate the liquid phase and the solid phase clearly, and then carefully absorb the supernatant. In the above process, it is necessary to avoid inhaling protein to avoid affecting the subsequent analysis steps. The 96-well protein precipitation plate is an existing product that can achieve high-throughput operation, but the use of protein precipitation plates has many limitations. Therefore, the development of a pretreatment device that can achieve high-throughput operation and is easy to use for solid-liquid separation has important application value in the detection of samples such as proteins.

[0004] Liquid-liquid extraction is a method of transferring solutes from one solvent to another by utilizing the difference in solubility or distribution coefficient of a substance in two immiscible (or slightly soluble) solvents. It is widely used in industries such as chemistry, metallurgy, and food, and is commonly used in the petroleum refining industry. Liquid-liquid extraction is a commonly used pretreatment method for testing organic compounds. For compounds with high sensitivity requirements, especially fat-soluble compounds, liquid-liquid extraction is a pretreatment method with a relatively pure treatment liquid and low cost. It has a wide range of application value that is difficult to replace in the field of clinical mass spectrometry.

[0005] Due to the small volume required for clinical mass spectrometry, liquid-liquid extraction is usually performed using a 1.5-2 mL centrifuge tube. The organic phase (such as ethyl acetate, methyl tert-butyl ether, n-hexane, etc. or mixed solvents in different proportions) that is immiscible with the aqueous phase is fully mixed with the aqueous matrix such as serum and plasma by means of vortexing, and the fat-soluble compounds are extracted into the organic phase by using the different distribution coefficients of the two phases. The two phases are then separated by means of centrifugation, and the upper organic phase is carefully aspirated, and then nitrogen is blown dry, concentrated and re-dissolved. In the above process, it is necessary to avoid inhaling the lower aqueous phase, otherwise it may cause subsequent analysis failure. The existing SLE plates on the market can use the principle of diatomaceous earth adsorption to achieve high throughput of liquid extraction, but in actual applications, due to the addition of fillers, it is often necessary to change the original extraction liquid ratio and increase the amount of extraction liquid to ensure the effect similar to the original liquid-liquid extraction. Therefore, the development of a pretreatment device that can achieve high-throughput operation and is easy to use for liquid-liquid separation has important application value in clinical testing.

[0006] CN1938585A discloses a solid phase extraction tube, which has an inflow side sintered plate, a solid phase filler and an outflow side sintered plate in a tubular extraction tube body opened at the upper and lower ends, starting from the upper side, and a blocking portion protruding inwardly and continuously or intermittently blocking the outer periphery of the outflow side sintered plate is provided on the lower inner surface of the extraction tube body; or a holding portion constituting a conical head with a smaller diameter toward the lower side and used to hold the outflow side sintered plate is formed on the lower inner surface of the extraction tube body, and the extraction tube has a fitting portion for inserting the lower end of the other extraction tube body from the upper end to a prescribed position and maintaining its inserted state, the fitting portion is composed of an inner conical surface formed with a larger diameter toward the upper side at least within a prescribed range on the inner surface of the extraction tube body on the inserted side and an outer conical surface formed with a larger diameter toward the upper side at least within a prescribed range on the outer surface of the extraction tube body on the inserting side, and the inner conical surface and the outer conical surface are formed to have substantially the same inclination angle. However, the device has a complex structure and is not suitable for high-throughput sample processing.

[0007] CN210187192U discloses a test tube for liquid-liquid microextraction, comprising an upper tube body and a lower tube body, wherein the upper end of the upper tube body is provided with an opening, the lower end of the upper tube body is connected to the upper end of the lower tube body, the lower end of the lower tube body is a closed bottom, and the inner diameter of the lower tube body is greater than the inner diameter of the upper tube body. However, the above test tube cannot meet the requirements of high-throughput operation, nor can it be used for experiments requiring large extraction doses.

[0008] Therefore, it is necessary to design a flexible clinical mass spectrometry extraction column structure suitable for high-throughput operation in response to the above problems, which has important application value in realizing high-throughput and automated detection. Utility Model Content

[0009] In view of the shortcomings of the prior art, the utility model aims to provide a flexible parallel extraction column structure and a separation device suitable for high-throughput operation. The extraction column structure can be used for liquid-liquid separation or solid-liquid separation. When in use, the extraction column structure is inserted into a liquid storage tube and pressed down to obtain a separated product.

[0010] In order to achieve the purpose of this utility model, the utility model adopts the following technical solutions:

[0011] In the first aspect, the utility model provides a flexible row extraction column structure suitable for high-throughput operation, wherein the extraction column structure is used in conjunction with a liquid storage tube; the extraction column structure is inserted into the liquid storage tube and pressed down for filtration, and the liquid to be filtered stored in the liquid storage tube enters from the bottom opening of the filter tube and sequentially passes through the sieve plate assembly 6 and the filter membrane assembly 3 before entering the tube body cavity, and the extraction liquid is obtained after pressing down for filtration;

[0012] The row extraction column structure is obtained by connecting at least two centrifuge-free filter tubes;

[0013] The centrifuge-free filter tube comprises a tube body with an opening at the bottom, and the tube body consists of a first liquid storage chamber 1 and a second liquid storage chamber 7;

[0014] The outer wall diameter of the tube body corresponding to the first liquid storage cavity 1 is smaller than the outer wall diameter of the tube body corresponding to the second liquid storage cavity 7;

[0015] The bottom opening of the inner cavity of the tube body of the first liquid storage cavity 1 is provided with a sieve plate assembly 6 and a filter membrane assembly 3 in sequence from bottom to top;

[0016] The second liquid storage chamber 7 is used to expand the volume of the centrifugal filter tube;

[0017] The sieve plate assembly 6 is assembled at the bottom of the inner cavity of the tube body through the limiting kit 4;

[0018] The limiting kit 4 and the filter membrane assembly 3 are fixedly connected by a sealing ring 5; the limiting kit has three functions: one is to support the filter membrane assembly 3 to prevent it from falling, the second is to clamp the sieve plate assembly 6 and act as an adapter, and the third is to fix / limit the sealing ring (the sealing ring can prevent the lower layer of liquid from flowing out of the tube wall and force all liquids to be filtered from the sieve plate assembly 6); assembling the sieve plate assembly 6 at the bottom of the inner cavity of the tube body through the limiting kit can provide a certain degree of protection for the sieve plate assembly 6 and avoid damaging the structure of the sieve plate assembly 6. At the same time, providing a sealing ring can prevent leakage.

[0019] Preferably, the pore size of the sieve plate assembly 6 is 5-50 μm, for example, it can be 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm or 50 μm.

[0020] Preferably, the pore size of the filter membrane assembly 3 is smaller than or equal to the pore size of the sieve plate assembly 6 .

[0021] Preferably, the centrifuge-free filter tubes are connected to each other via a connecting structure, and the connecting point of the connecting structure is arranged on the tube body of the centrifuge-free filter tube.

[0022] Preferably, the extraction column structure is used for liquid-liquid separation, and the filter membrane assembly 3 comprises a water absorbent layer 301 and a water blocking membrane layer 302 which are stacked in sequence from bottom to top.

[0023] Preferably, the sieve plate assembly 6 comprises sieve plates and / or baffles stacked in sequence from bottom to top.

[0024] Preferably, the upper portion of the sieve plate is a cylindrical structure, and the cylindrical structure is nested in the limiting kit 4 .

[0025] Preferably, the baffle is disposed at the lower part of the water absorbent layer 301 to prevent particles in the water absorbent layer 301 from falling.

[0026] In the utility model, the centrifugal filter tube is pressed into the liquid storage tube, the solution is first roughly filtered through the sieve plate assembly 6, and then filtered through the filter membrane assembly, and the clarified filtrate enters the inner cavity of the tube body. The baffle can be used for filtering on the one hand, and can be used to prevent particles in the water absorbent layer from falling downward on the other hand.

[0027] Preferably, the water absorbent layer 301 is selected from any one of a magnesium sulfate layer, a sodium sulfate layer, a silica gel layer or a molecular sieve layer, or a combination of two or more thereof.

[0028] Preferably, the mass of the water absorbent layer 301 is 10-100 mg, for example, it can be 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg or 100 mg, etc.

[0029] Preferably, the particle / pore size of the water absorbent layer 301 is 20-1000 μm.

[0030] Preferably, the water-blocking film layer 302 is selected from any one of a polyacrylamide copolymer layer, a polyethylene resin layer, a vinyl epoxy resin layer, an acrylonitrile layer, an acrylate polymer layer or a polyethylene glycol layer, or a combination of two or more thereof.

[0031] Preferably, the thickness of the water-blocking film layer 302 is 1 mm-1 cm, for example, it may be 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm or 1 cm.

[0032] Preferably, the pore size of the water-blocking film layer 302 is 5-50 μm, for example, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm or 50 μm.

[0033] It should be noted that the water-blocking film layer in the present invention may include only one layer of water-blocking film or may include two or more layers. Depending on the detection items, a combination of filter membranes of the same or different types of materials may be used.

[0034] Preferably, a clamping ring 2 is further provided on the upper portion of the filter membrane assembly 3 in the inner cavity of the tube body of the first liquid storage cavity 1 .

[0035] Preferably, the clamp ring 2 is a hollow cylindrical structure without filtering function, and the filtrate is stored in the tube body inside the clamp ring and on the upper part of the clamp ring. The clamp ring 2 is used to limit the movement of the filter membrane assembly 3.

[0036] Preferably, the extraction column structure is used for solid-liquid separation, and the filter membrane assembly 3 is composed of at least one filter membrane layer.

[0037] Preferably, the filter membrane layer is selected from any one of a polytetrafluoroethylene layer (PTFE), a hydrophilic PTFE layer, a nylon layer, a polyethersulfone layer, a polyethylene layer (PE) or a polypropylene layer (PP).

[0038] In the present invention, according to different detection items, a combination of filter membranes of the same or different materials can be used, including at least one layer of filter membrane.

[0039] In the present invention, the pore size of the filter membrane layer is 0.22-1 μm, for example, it can be 0.22 μm, 0.25 μm, 0.45 μm, 0.5 μm, 0.6 μm, 0.75 μm, 0.8 μm or 1 μm.

[0040] Preferably, the sieve plate assembly 6 is a sieve plate, and the lower part of the sieve plate is adapted to the bottom of the liquid storage tube.

[0041] Preferably, a clamp ring 2 is further provided on the upper part of the filter membrane assembly 3 in the inner cavity of the tube body of the first liquid storage cavity 1;

[0042] Preferably, the clamp ring 2 is a hollow cylindrical structure without filtering function, and the filtrate is stored in the tube body inside the clamp ring and on the upper part of the clamp ring. The clamp ring 2 is used to limit the movement of the filter membrane assembly 3.

[0043] In a second aspect, the utility model provides a flexible separation device suitable for high-throughput operation, the device comprising a liquid storage tube and the flexible parallel extraction column structure suitable for high-throughput operation described in the first aspect;

[0044] The liquid storage tube and the extraction column structure are used in combination, the liquid to be filtered is stored in the liquid storage tube, the extraction column structure is pressed into the liquid storage tube, and the liquid to be filtered passes through the sieve plate assembly 6 and the filter membrane assembly 3 from the bottom of the centrifuge-free filter tube and enters the inner cavity of the tube body of the centrifuge-free filter tube.

[0045] The flexible row-and-row extraction column structure suitable for high-throughput operation provided in the utility model can be well applied to liquid-liquid extraction separation. When performing liquid-liquid extraction separation, the following operation can be adopted: add sample, aqueous phase and organic phase into the liquid storage tube, let stand after mixing, and after the aqueous phase and the organic phase are initially separated, press the flexible row-and-row extraction column structure suitable for high-throughput operation described in the first aspect into the liquid storage tube, and the organic phase passes through the sieve plate assembly 6 and the filter membrane assembly 3 from the bottom of the extraction column structure into the tube body cavity of the extraction column structure, and the aqueous phase is isolated by the water-blocking membrane in the filter assembly 3.

[0046] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0047] Compared with the prior art, the utility model has the following beneficial effects:

[0048] The extraction column structure for liquid-liquid separation described in the utility model can match the commercially available collection plate (i.e., liquid storage tube) when processing samples. It is relatively large in size and can be covered with a small column or a standard silicone cover to ensure a large pre-treatment liquid volume and oscillation intensity; no centrifugation is required during operation, and only a simple downward pressure operation is required to complete the separation, which can save about half the time. While simplifying the operation, it is compatible with a multi-channel pipette for high-throughput operation, and also takes into account flexibility (can be broken apart for use), reduces the pipetting steps, and reduces the errors that may occur during the pipetting steps. The current automation and high throughput of liquid-liquid extraction pretreatment are mainly achieved through solid-phase supported liquid-liquid extraction (SLE), and the established liquid-liquid extraction method needs to be optimized when transferred to SLE, such as changing the type, proportion and volume of the extraction solvent; and when using the liquid-liquid extraction column structure of the utility model, the original method can be applied without optimization, eliminating the disadvantage that the method is not universal during automation. During the operation of SLE, it is usually necessary to wait for 5-10 minutes for the sample to be adsorbed, and then elute the substance to be tested by adding an extractant as an eluent. Depending on the brand, gravity elution or positive / negative pressure elution is used, and the elution time is long, which reduces the speed of pre-treatment and even requires additional equipment. The use of the structure of the utility model not only does not reduce the speed of pre-treatment, but also eliminates the centrifugation step and is suitable for multi-channel pipettes, which can improve the efficiency of pre-treatment and has a significant effect.

[0049] The extraction column structure for solid-liquid separation described in the utility model is used together with the liquid storage tube when processing samples. The extraction column structure is inserted into the liquid storage tube and filtered by pressing down. The liquid to be filtered stored in the liquid storage tube enters from the bottom opening of the filter tube and passes through the sieve plate assembly 6 and the filter membrane assembly 3 in sequence before entering the inner cavity of the tube body. The extraction liquid is obtained after filtering by pressing down. The extraction column structure is simple in structure, easy to operate, has low requirements for operators and high separation efficiency. When using, there is no need to carefully absorb the supernatant, so that the purpose of easily removing the supernatant without centrifugation is achieved, which saves separation time and is not easy to cause contamination to the sample. It avoids the shortcomings of traditional solid-liquid separation that requires human attention and intervention, and can achieve high throughput and automation. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 It is a schematic diagram of a single centrifuge-free filter tube in a flexible parallel extraction column structure suitable for high-throughput operation, wherein 1-first liquid storage chamber, 2-clamp ring, 3-filter membrane assembly, 4-limiting kit, 5-sealing ring, 6-sieve plate assembly, 7-second liquid storage chamber.

[0051] Figure 2 It is a schematic diagram of the structure of the filter membrane assembly, wherein 301 is a water absorbent layer, and 302 is a water blocking membrane layer. DETAILED DESCRIPTION

[0052] It should be understood that, in the description of the present utility model, the terms "upper", "lower", "top", "bottom", "inside", "outside", etc. indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the present utility model and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present utility model. In addition, the terms "first", "second", etc. are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, features defined as "first", "second", etc. may explicitly or implicitly include one or more of the features. In the description of the present utility model, unless otherwise specified, "multiple" means two or more.

[0053] It should be noted that, in the description of the present utility model, unless otherwise clearly specified and limited, the terms "set", "connected" and "connected" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection. For ordinary technicians in this field, the specific meanings of the above terms in the present utility model can be understood through specific circumstances.

[0054] The technical solution of the utility model is further explained below with reference to the accompanying drawings and through specific implementation methods.

[0055] In a specific embodiment, the utility model provides a flexible row extraction column structure suitable for high-throughput operation, the extraction column structure is obtained by connecting at least two centrifuge-free filter tubes, the centrifuge-free filter tube includes a tube body with a bottom opening, the tube body is composed of a first liquid storage chamber 1 and a second liquid storage chamber 7; the outer wall diameter of the tube body corresponding to the first liquid storage chamber 1 is smaller than the outer wall diameter of the tube body corresponding to the second liquid storage chamber 7; the bottom opening of the inner cavity of the tube body of the first liquid storage chamber 1 is sequentially arranged with a sieve plate assembly 6 and a filter membrane assembly 3 from bottom to top; the second liquid storage chamber 7 is used to expand the volume of the centrifuge-free filter tube, which is suitable for large-volume liquid-liquid extraction; the sieve plate assembly 6 is assembled at the bottom of the inner cavity of the tube body through a limiting kit 4; the limiting kit 4 and the filter membrane assembly 3 are fixedly connected by a sealing ring 5; the pore size of the sieve plate assembly 6 is 5-50μm; the pore size of the filter membrane assembly 3 is less than or equal to the pore size of the sieve plate assembly 6; the centrifuge-free filter tubes are connected by a connecting structure, and the connection point of the connecting structure is arranged on the tube body of the centrifuge-free filter tube.

[0056] The flexible row extraction column structure suitable for high-throughput operation provided by the utility model can be used for liquid-liquid extraction and separation. The extraction column structure is used in conjunction with a liquid storage tube. The extraction column structure is inserted into the liquid storage tube and filtered by pressing down. The liquid to be filtered stored in the liquid storage tube enters from the bottom opening of the filter tube and passes through the sieve plate assembly 6 and the filter membrane assembly 3 in sequence before entering the tube body cavity. After filtering by pressing down, the extract is obtained. The structural schematic diagram of the filter membrane assembly 3 is shown in FIG. Figure 2 shown.

[0057] The extraction column provided by the utility model has a simple structure, is easy to operate, has low requirements on operators and has high separation efficiency. There is no need to carefully aspirate the supernatant during use, and the purpose of easily removing organic matter without centrifugation is achieved. The extraction and separation time is saved and it is not easy to cause contamination to the sample. The shortcomings of traditional liquid-liquid extraction methods that require human attention and intervention are avoided, and high throughput and automation can be achieved.

[0058] Furthermore, the filter membrane assembly 3 is located at the bottom of the inner cavity of the tube body of the first liquid storage chamber 1. After the filtrate passes through the sieve plate assembly 6, it continues to pass through the filter membrane assembly 3 and enters the upper part of the inner cavity of the tube body of the first liquid storage chamber 1 or enters the inner cavity of the tube body of the second liquid storage chamber 7. The filter membrane assembly 3 includes a water absorbent layer 301 and a water blocking film layer 302 stacked in sequence from bottom to top.

[0059] The water absorbing agent layer 301 is selected from any one of a magnesium sulfate layer, a sodium sulfate layer, a silica gel layer or a molecular sieve layer, or a combination of two or more thereof. The mass of the water absorbing agent layer 301 is 10-100 mg. The particle / pore size of the water absorbing agent layer 301 is 20-1000 μm.

[0060] The water-blocking film layer 302 is selected from any one of a polyacrylamide copolymer layer, a polyethylene resin layer, a vinyl epoxy resin layer, an acrylonitrile layer, an acrylate polymer layer or a polyethylene glycol layer, or a combination of two or more thereof. The thickness of the water-blocking film layer 302 is 1 mm-1 cm. The pore size of the film layer in the water-blocking film layer 302 is 5-50 μm.

[0061] Furthermore, the sieve plate assembly 6 includes sieve plates and / or baffles stacked in sequence from bottom to top; the upper part of the sieve plate is a cylindrical structure, and the cylindrical structure is nested in the limiting kit 4; the baffle is arranged at the lower part of the water-absorbing agent layer 301 to prevent particles in the water-absorbing agent layer 301 from falling.

[0062] In another specific embodiment, the utility model provides a liquid-liquid extraction separation method, which includes: adding a sample, an aqueous phase and an organic phase into a liquid storage tube, mixing and letting stand, and after the aqueous phase and the organic phase are initially separated, pressing the flexible parallel extraction column structure suitable for high-throughput operation into the liquid storage tube, and the organic phase passes through the sieve plate assembly 6 and the filter membrane assembly 3 from the bottom of the extraction column structure into the tube body cavity of the extraction column structure, and the aqueous phase is isolated by the water-blocking membrane in the filter assembly 3 to achieve liquid-liquid extraction separation.

[0063] In one embodiment, the utility model provides a flexible parallel extraction column structure suitable for high-throughput operation, such as Figure 1 As shown, the extraction column structure is obtained by connecting at least two centrifuge-free filter tubes, and the centrifuge-free filter tube includes a tube body with an opening at the bottom, and the tube body is composed of a first liquid storage chamber 1 and a second liquid storage chamber 7; the outer wall diameter of the tube body corresponding to the first liquid storage chamber 1 is smaller than the outer wall diameter of the tube body corresponding to the second liquid storage chamber 7; the bottom opening of the inner cavity of the tube body of the first liquid storage chamber 1 is sequentially arranged with a sieve plate assembly 6, a filter membrane assembly 3 and a clamping ring 2 from bottom to top; the second liquid storage chamber 7 is used to expand the volume of the centrifuge-free filter tube, which is suitable for large-volume liquid-liquid extraction; the sieve plate assembly 6 is assembled at the bottom of the inner cavity of the tube body through a limiting kit 4; the limiting kit 4 and the filter membrane assembly 3 are fixedly connected by a sealing ring 5; the clamping ring 2 is a hollow cylindrical structure without filtering effect, and the filtrate is stored in the tube body inside and above the clamping ring, and the clamping ring 2 is used to limit the movement of the filter membrane assembly 3. The pore size of the sieve plate assembly 6 is 5-50 μm; the pore size of the filter membrane assembly 3 is less than or equal to the pore size of the sieve plate assembly 6; the centrifuge-free filter tubes are connected by a connecting structure, and the connecting point of the connecting structure is set on the tube body of the centrifuge-free filter tube.

[0064] The utility model provides a flexible row of extraction columns suitable for high-throughput operation and can be used for solid-liquid separation. The extraction column structure is used in conjunction with a liquid storage tube. The extraction column structure is inserted into the liquid storage tube and filtered by pressing down. The liquid to be filtered stored in the liquid storage tube enters from the bottom opening of the filter tube and passes through the sieve plate assembly 6, the filter membrane assembly 3 and the retaining ring 2 in sequence before entering the inner cavity of the tube body. The extraction liquid is obtained after filtering by pressing down.

[0065] The extraction column provided by the utility model has a simple structure, is easy to operate, has low requirements on operators and has high separation efficiency. There is no need to carefully aspirate the supernatant during use, and the purpose of easily removing the supernatant without centrifugation is achieved, which saves separation time and is not easy to contaminate the sample. The shortcomings of traditional solid-liquid separation that requires human attention and intervention are avoided, and high throughput and automation can be achieved.

[0066] Further, the filter membrane assembly 3 is located at the bottom of the inner cavity of the tube body of the first liquid storage chamber 1. After the filtrate passes through the sieve plate assembly 6, it continues to pass through the filter membrane assembly 3 and enters the upper part of the inner cavity of the tube body of the first liquid storage chamber 1 or enters the inner cavity of the tube body of the second liquid storage chamber 7. The filter membrane assembly 3 is composed of at least one filter membrane layer. The materials of the filter membrane layers are independently selected from polytetrafluoroethylene (PTFE), hydrophilic PTFE, nylon, polyethersulfone, polyethylene (PE) or polypropylene (PP) and the like.

[0067] Furthermore, the sieve plate assembly 6 is a sieve plate, and the lower part of the sieve plate is a round bottom structure or a pointed bottom structure; the sieve plate is adapted to the bottom of the liquid storage tube.

[0068] The shape of the lower part of the sieve plate depends on the bottom structure of the liquid storage tube, which can be a round bottom structure or a pointed bottom structure, which is respectively adapted to the bottom shape and size of the round bottom collection plate or the pointed bottom liquid storage tube; the structure of the lower part of the sieve plate is larger than the structure of the upper part of the sieve plate, which can increase the surface area. After the filter tube is pushed into the liquid storage tube, the contact area between the structure of the sieve plate and the liquid to be filtered increases, thereby enhancing the separation effect of the filter tube on the filtrate. In addition, the pore size of the appropriate sieve plate allows the filtrate to pass through the sieve plate and enter the column tube cavity while blocking the lower layer protein precipitation or other impurities from entering the filtrate; the filtrate is first coarsely filtered through the sieve plate, and then filtered through the filter membrane assembly, and the clarified filtrate enters the inner cavity of the tube body through the clamp ring.

[0069] The sieve plate is assembled at the bottom of the inner cavity of the tube body through the stopper kit 4; the stopper kit 4 and the filter membrane assembly 3 are fixedly connected through the sealing ring 5. At the same time, the outer wall of the tube body of the centrifuge-free filter tube and the liquid storage tube are sealed by the sealing ring 5 to form a closed space, and the atmospheric pressure is increased so that the liquid enters the inner cavity of the tube body from the sieve plate assembly 6, the filter membrane assembly 3 and the clamp ring 2.

[0070] The technical solution of the present invention is further described below by specific implementation methods. Those skilled in the art should understand that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0071] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0072] Example 1

[0073] This embodiment provides a flexible parallel extraction column structure suitable for high-throughput operation, wherein the extraction column structure is used for liquid-liquid separation.

[0074] The extraction column structure is used in conjunction with a liquid storage tube; the extraction column structure is inserted into the liquid storage tube and pressed down for filtration, the liquid to be filtered stored in the liquid storage tube enters from the bottom opening of the filter tube and passes through the sieve plate assembly 6 and the filter membrane assembly 3 in sequence before entering the tube body cavity, and the extraction liquid is obtained after pressing down for filtration; the extraction column structure is obtained by connecting 8 centrifuge-free filter tubes. The centrifugal filter tube comprises a tube body with a bottom opening, and the tube body consists of a first liquid storage chamber 1 and a second liquid storage chamber 7; the outer wall diameter of the tube body corresponding to the first liquid storage chamber 1 is smaller than the outer wall diameter of the tube body corresponding to the second liquid storage chamber 7; the bottom opening of the inner cavity of the tube body of the first liquid storage chamber 1 is sequentially provided with a sieve plate assembly 6 and a filter membrane assembly 3 from bottom to top; the second liquid storage chamber 7 is used to expand the volume of the centrifugal filter tube; the sieve plate assembly 6 is assembled at the bottom of the inner cavity of the tube body through a limiting kit 4; the limiting kit 4 and the filter membrane assembly 3 are fixedly connected through a sealing ring 5; the limiting kit has three functions, one is to support the filter membrane assembly 3 to prevent it from falling, the second is to clamp the sieve plate and act as an adapter, and the third is to fix / limit the sealing ring (the sealing ring can prevent the lower layer of liquid from flowing out of the tube wall and force all liquids to be filtered from the sieve plate); assembling the sieve plate at the bottom of the inner cavity of the tube body through the limiting kit can play a certain protective role on the sieve plate and avoid damaging the sieve plate structure. At the same time, the sealing ring is provided to prevent leakage.

[0075] The pore size of the sieve plate assembly 6 is 20 μm.

[0076] The sieve plate assembly 6 comprises a sieve plate and a baffle plate stacked in sequence from bottom to top; the filter membrane assembly 3 comprises a water absorbent layer 301 and a water blocking membrane layer 302 stacked in sequence from bottom to top.

[0077] The water-absorbing agent layer 301 is a magnesium sulfate layer; the mass of the water-absorbing agent layer 301 is 60 mg; and the particle size of the water-absorbing agent layer 301 is 100-800 μm.

[0078] The water-blocking film layer 302 is a polyethylene resin layer; the thickness of the water-blocking film layer 302 is 5 mm; the pore size of the film layer in the water-blocking film layer 302 is 10 μm.

[0079] The upper part of the sieve plate is a cylindrical structure, and the cylindrical structure is nested in the limiting kit 4; the baffle is arranged at the lower part of the water-absorbing agent layer 301 to prevent the particles in the water-absorbing agent layer 301 from falling.

[0080] Example 2

[0081] The present embodiment provides a flexible row extraction column structure suitable for high-throughput operation, and the extraction column structure is used for liquid-liquid separation. The difference between the extraction column structure and Example 1 is that the extraction column structure is obtained by connecting two centrifuge-free filter tubes; the pore size of the sieve plate assembly 6 is 30μm. The water-absorbing agent layer 301 is a silica gel layer; the mass of the water-absorbing agent layer 301 is 80mg; the particle size of the water-absorbing agent layer 301 is 500-1000μm. The water-blocking film layer 302 is a polyacrylamide copolymer layer; the thickness of the water-blocking film layer 302 is 9mm; the pore size of the film layer in the water-blocking film layer 302 is 20μm.

[0082] Example 3

[0083] The present embodiment provides a flexible row extraction column structure suitable for high-throughput operation, and the extraction column structure is used for liquid-liquid separation. The difference between the extraction column structure and Example 1 is that the extraction column structure is obtained by connecting 12 centrifuge-free filter tubes; the pore size of the sieve plate assembly 6 is 10μm. The water-absorbing agent layer 301 is a molecular sieve layer; the mass of the water-absorbing agent layer 301 is 20mg; the pore size of the water-absorbing agent layer 301 is 20-500μm. The water-blocking film layer 302 is a vinyl epoxy resin layer; the thickness of the water-blocking film layer 302 is 2mm; the pore size of the film layer in the water-blocking film layer 302 is 10μm.

[0084] Example 4

[0085] This embodiment provides a flexible parallel extraction column structure suitable for high-throughput operation, and the extraction column structure is used for liquid-liquid separation. The difference between the extraction column structure and the embodiment 1 is that the pore size of the sieve plate assembly 6 is 2 μm.

[0086] Example 5

[0087] This embodiment provides a flexible parallel extraction column structure suitable for high-throughput operation, and the extraction column structure is used for liquid-liquid separation. The difference between the extraction column structure and the embodiment 1 is that the pore size of the sieve plate assembly 6 is 60 μm.

[0088] Example 6

[0089] This embodiment provides a flexible parallel extraction column structure suitable for high-throughput operation, and the extraction column structure is used for liquid-liquid separation. The difference between the extraction column structure and the embodiment 1 is that the filter membrane assembly 3 does not contain a water absorbent layer 301.

[0090] Example 7

[0091] This embodiment provides a flexible parallel extraction column structure suitable for high-throughput operation, and the extraction column structure is used for liquid-liquid separation. The difference between the extraction column structure and the embodiment 1 is that the filter membrane assembly 3 does not contain a water-blocking membrane layer 302 .

[0092] Example 8

[0093] This embodiment provides a flexible parallel extraction column structure suitable for high-throughput operation, wherein the extraction column structure is used for solid-liquid separation.

[0094] The extraction column structure is used in conjunction with a liquid storage tube; the extraction column structure is inserted into the liquid storage tube and pressed down for filtration, and the liquid to be filtered stored in the liquid storage tube enters from the bottom opening of the filter tube and passes through the sieve plate assembly 6, the filter membrane assembly 3 and the retaining ring 2 in sequence before entering the inner cavity of the tube body, and the extract is obtained after pressing down for filtration; the extraction column structure is obtained by connecting 12 centrifuge-free filter tubes. The centrifuge-free filter tube includes a tube body with an opening at the bottom, and the tube body is composed of a first liquid storage cavity 1 and a second liquid storage cavity 7; the outer wall diameter of the tube body corresponding to the first liquid storage cavity 1 is smaller than the outer wall diameter of the tube body corresponding to the second liquid storage cavity 7; the sieve plate assembly 6, the filter membrane assembly 3 and the retaining ring 2 are sequentially arranged at the bottom opening of the inner cavity of the tube body of the first liquid storage cavity 1 from bottom to top; the second liquid storage cavity 7 is used to expand the volume of the centrifuge-free filter tube; the sieve plate assembly 6 is assembled at the bottom of the inner cavity of the tube body through a limiting kit 4; the limiting kit 4 and the filter membrane assembly 3 are sealed by a sealing ring 5; the limiting The kit has three functions: one is to support the filter membrane assembly 3 to prevent it from falling, the second is to clamp the sieve plate and act as an adapter, and the third is to fix / limit the sealing ring (the sealing ring can prevent the lower layer of liquid from flowing out of the tube wall, forcing all liquid to be filtered from the sieve plate); assembling the sieve plate at the bottom of the inner cavity of the tube body through the limiting kit can provide a certain degree of protection for the sieve plate and avoid damaging the sieve plate structure. At the same time, providing a sealing ring can prevent leakage; the clamping ring 2 is a hollow cylindrical structure without filtering function, and the filtrate is stored in the tube body inside the clamping ring and on the upper part of the clamping ring. The clamping ring 2 is used to limit the movement of the filter membrane assembly 3.

[0095] The sieve plate assembly 6 is a sieve plate, which is matched with the bottom of the liquid storage tube, and the pore size of the sieve plate is 10 μm.

[0096] The filter membrane assembly 3 contains a filter membrane layer, which is a polytetrafluoroethylene (PTFE) layer, and the pore size of the filter membrane layer is 0.22 μm.

[0097] Example 9

[0098] This embodiment provides a flexible parallel extraction column structure suitable for high-throughput operation, and the extraction column structure is used for solid-liquid separation. The difference between the extraction column structure and the embodiment 8 is that the pore size of the sieve plate is 20 μm.

[0099] Example 10

[0100] This embodiment provides a flexible parallel extraction column structure suitable for high-throughput operation, and the extraction column structure is used for solid-liquid separation. The difference between the extraction column structure and the embodiment 8 is that the filter membrane assembly 3 contains two filter membrane layers, one of which is a hydrophilic PTFE layer with a pore size of 0.22 μm, and the other is a nylon layer with a pore size of 0.45 μm.

[0101] Embodiment 11

[0102] This embodiment provides a flexible parallel extraction column structure suitable for high-throughput operation, and the extraction column structure is used for solid-liquid separation. The difference between the extraction column structure and the embodiment 8 is that the filter membrane assembly 3 contains three filter membrane layers, which are nylon layers from bottom to top, with pore sizes of 1 μm and 0.45 μm, and one polypropylene layer (PP) with a pore size of 0.22 μm.

[0103] Example 12

[0104] This embodiment provides a flexible parallel extraction column structure suitable for high-throughput operation, and the extraction column structure is used for solid-liquid separation. The difference between the extraction column structure and the embodiment 8 is that the filter membrane assembly 3 has only one two-layer filter membrane layer made of nylon, with pore sizes of 0.8 μm and 0.22 μm respectively.

[0105] Embodiment 13

[0106] This embodiment provides a flexible parallel extraction column structure suitable for high-throughput operation, and the extraction column structure is used for solid-liquid separation. The difference between the extraction column structure and the embodiment 8 is that the filter membrane assembly 3 has only one filter membrane layer of PP with a pore size of 0.22 μm.

[0107] Test Example 1

[0108] The extraction column structure in Example 1-7 was used to perform liquid-liquid separation and verify the separation effect. The experimental steps are as follows:

[0109] Dihydrotestosterone (DHT) is detected by liquid chromatography, and the detection process specifically includes the following steps:

[0110] 1. Testing conditions

[0111] The experimental conditions for liquid chromatography detection are shown in Table 1, Table 2 and Table 3:

[0112] Table 1

[0113]

[0114]

[0115] Table 2

[0116]

[0117] Table 3

[0118]

[0119] 2. Sample pretreatment:

[0120] (1) Using LLE method to process samples:

[0121] Add 200 μL of standard curve sample or plasma spiked sample, 25 μL of IS internal standard solution and 1 mL of extractant to the 96-deep well collection plate in sequence, mix thoroughly and push into the row extraction column structure until the row extraction separation column encounters the water layer and cannot be pressed. Take all the clear liquid filtered from the row extraction separation column, blow dry with nitrogen at 45°C, re-dissolve with 100 μL of derivatization reagent, derivatize at 60°C for 15 minutes, mix well, and load the sample. The results show that the upper layer solution of Example 5 is mixed with aqueous phase liquid leakage, and the aqueous phase in Example 7 enters the extraction column cavity completely, and the upper clear liquid is not suitable for machine detection. However, the force required to press the row column in Example 4 into the deep well collection plate is very large, so the sieve plate aperture in this example is too small and is not suitable for the row liquid-liquid separation column.

[0122] The accuracy of the linear curve samples of Examples 1 and 6 were 89.1%-103.5% and 86.3%-113.2%, respectively. However, the recoveries of three spiked concentrations of dihydrotestosterone of 150pg / mL, 500pg / mL and 8ng / mL in real plasma samples were 86.8%, 98.3% and 92.3% in Example 1, and 78.5%, 90.6% and 80.3% in Example 6. The accuracy of the linear curve samples of Example 2 was 93.4-125.8%, which could not meet the requirements of quantitative analysis for the accuracy of the standard curve. The accuracy of the linear curve samples of Example 3 was 87.9-112.3%, which met the requirements of the analysis for the accuracy of the standard curve, but its internal standard response was 30-50% of that of Example 1, which was not the optimal solution for projects with relatively high sensitivity requirements.

[0123] In summary, under the conditions of appropriate sieve plate pore size, water-blocking membrane material, water-blocking membrane thickness and type of water absorbent, the parallel liquid-liquid extraction separation column can ensure the accuracy of method development under the premise of no centrifugation, easy operation, efficient pretreatment and high throughput.

[0124] Test Example 2

[0125] The extraction column structure in Examples 8-13 was used to perform solid-liquid separation and verify the separation effect. The experimental steps are as follows:

[0126] The working curve and the concentrations of low, medium and high quality control solutions are prepared as shown in Table 4 (in μg / mL):

[0127] Table 4

[0128] / S-6 S-5 S-4 S-3 S-2 S-1 QC-L QC-M QC-H Amikacin 100 50 10 5 2 1 5 10 80 Posaconazole 20 10 2 1 0.4 0.2 1 2 16

[0129] The concentrations of amikacin-d5 and posaconazole-d4 in the internal standard working solution (IS) prepared in 50% methanol were 200 μg / mL and 10 μg / mL, respectively, for the standard curve.

[0130] The standard curve and quality control were prepared with 4% BSA, and the quality control concentration standard was added to the serum sample for detection. The detection conditions are shown in Table 5:

[0131] Table 5

[0132]

[0133]

[0134] Sample treatment: In a 96-well deep-well plate, add 50 μL of serum sample to be tested, 5 μL of internal standard working solution and 200 μL of 5% formic acid-acetonitrile solution as a precipitant, vortex for 30 seconds to mix, or use an oscillating incubator to mix at 1300 rpm for 2 minutes at room temperature, let stand for about 2 minutes to allow the solution to separate, press into a filter cartridge, take 10 μL of the filtrate, dilute with 90 μL of 0.1% ammonia aqueous solution, mix, and inject 5 μL.

[0135] The calibration curve was drawn with the ratio of the compound peak area to the internal standard peak area as the ordinate and the ratio of the compound concentration to the internal standard concentration as the abscissa. Except for the amikacin in Examples 8 and 9, which was adsorbed and could not obtain a linear equation and recovery rate, the linear R values ​​of posaconazole in Examples 8-9 and the two compounds in Examples 10-13 were all greater than 0.99, and the back-calculated values ​​of each point were within 85-115%, which met the requirements. The specific results are shown in Table 6.

[0136] Table 6

[0137]

[0138]

[0139] From the data in the table, it can be seen that for some compounds (such as amikacin), the filter membrane material is more important. The PTFE filter membrane has a strong adsorption effect on the compound, resulting in a very small peak area of ​​the compound when using the PTFE filter membrane, thus affecting the quantification; and the hydrophilic PTFE will reduce the response of posaconazole, thereby affecting the recovery rate. In general, the filter membrane material, pore size and number of layers of the row separation column have little effect on compound detection. From the perspective of user experience, when the number of filter membrane layers is 2-3, due to the effect of layer-by-layer filtration, the pressure during the downward pressure process is small, it is easier to handle, and the user experience is better.

[0140] In summary, the utility model provides a flexible parallel extraction column structure suitable for high-throughput operation, and the extraction column structure can be used for liquid-liquid extraction and solid-liquid extraction. The use of the structure of the utility model can eliminate the centrifugation step, and is applicable to methods such as multi-channel pipettes, which can improve the efficiency of pretreatment and has significant effects.

[0141] The applicant declares that the above is only a specific implementation method of the present utility model, but the protection scope of the present utility model is not limited thereto. The technicians in the relevant technical field should understand that any changes or substitutions that can be easily thought of by the technicians in the relevant technical field within the technical scope disclosed in the present utility model fall within the protection scope and disclosure scope of the present utility model.

Claims

1. A flexible parallel extraction column structure suitable for high-throughput operation, characterized in that: The extraction column structure is used in conjunction with a liquid storage tube; the extraction column structure is inserted into the liquid storage tube and filtered by pressing downward, and the liquid to be filtered stored in the liquid storage tube enters from the bottom opening of the filter tube and passes through the sieve plate assembly (6) and the filter membrane assembly (3) in sequence before entering the inner cavity of the tube body, and the extraction liquid is obtained after filtering by pressing downward; The row extraction column structure is obtained by connecting at least two centrifuge-free filter tubes; The centrifuge-free filter tube comprises a tube body with an opening at the bottom, wherein the tube body consists of a first liquid storage chamber (1) and a second liquid storage chamber (7); The diameter of the outer wall of the tube body corresponding to the first liquid storage cavity (1) is smaller than the diameter of the outer wall of the tube body corresponding to the second liquid storage cavity (7); The bottom opening of the inner cavity of the tube body of the first liquid storage cavity (1) is provided with a sieve plate assembly (6) and a filter membrane assembly (3) in sequence from bottom to top; The second liquid storage chamber (7) is used to expand the volume of the centrifuge-free filter tube; The sieve plate assembly (6) is assembled at the bottom of the inner cavity of the tube body through a limiting kit (4); The limiting kit (4) and the filter membrane assembly (3) are fixedly connected via a sealing ring (5).

2. The flexible row extraction column structure suitable for high-throughput operation according to claim 1, characterized in that: The pore size of the sieve plate assembly (6) is 5-50 μm; The pore size of the filter membrane assembly (3) is smaller than or equal to the pore size of the sieve plate assembly (6); The centrifuge-free filter tubes are connected to each other via a connecting structure, and a connecting point of the connecting structure is arranged on the tube body of the centrifuge-free filter tube.

3. The flexible parallel extraction column structure suitable for high-throughput operation according to claim 2, characterized in that: The extraction column structure is used for liquid-liquid separation, and the filter membrane assembly (3) comprises a water absorbent layer (301) and a water-blocking membrane layer (302) which are stacked in sequence from bottom to top.

4. The flexible parallel extraction column structure suitable for high-throughput operation according to claim 3, characterized in that: The sieve plate assembly (6) comprises sieve plates and / or baffles stacked in sequence from bottom to top.

5. The flexible parallel extraction column structure suitable for high-throughput operation according to claim 4, characterized in that: The upper part of the sieve plate is a cylindrical structure, and the cylindrical structure is nested in the limiting kit (4); The baffle is arranged at the lower part of the water absorbent layer (301) and is used to prevent particles in the water absorbent layer (301) from falling.

6. The flexible parallel extraction column structure suitable for high-throughput operation according to claim 3, characterized in that: The water absorbent layer (301) is selected from any one of a magnesium sulfate layer, a sodium sulfate layer, a silica gel layer or a molecular sieve layer, or a combination of two or more thereof; The mass of the water absorbing agent layer (301) is 10-100 mg; The particle / pore size of the water-absorbing agent layer (301) is 20-1000 μm; The water-blocking film layer (302) is selected from any one or a combination of two or more of a polyacrylamide copolymer layer, a polyethylene resin layer, a vinyl epoxy resin layer, an acrylonitrile layer, an acrylate polymer layer or a polyethylene glycol layer; The thickness of the water-blocking film layer (302) is 1 mm-1 cm; The pore size of the membrane layer in the water-blocking membrane layer (302) is 5-50 μm.

7. The flexible parallel extraction column structure suitable for high-throughput operation according to claim 3, characterized in that: A clamping ring (2) is also provided on the upper part of the filter membrane assembly (3) in the inner cavity of the tube body of the first liquid storage cavity (1); The clamp ring (2) is a hollow cylindrical structure without filtering function. The filtrate is stored in the tube inside the clamp ring and on the upper part of the clamp ring. The clamp ring (2) is used to limit the movement of the filter membrane assembly (3).

8. The flexible parallel extraction column structure suitable for high-throughput operation according to claim 2, characterized in that: The extraction column structure is used for solid-liquid separation, and the filter membrane component (3) is composed of at least one filter membrane layer; The filter membrane layer is selected from any one of a polytetrafluoroethylene layer, a hydrophilic polytetrafluoroethylene layer, a nylon layer, a polyethersulfone layer, a polyethylene layer or a polypropylene layer; The sieve plate assembly (6) is a sieve plate, and the lower part of the sieve plate is adapted to the bottom of the liquid storage tube.

9. The flexible parallel extraction column structure suitable for high-throughput operation according to claim 8, characterized in that: A clamping ring (2) is also provided on the upper part of the filter membrane assembly (3) in the inner cavity of the tube body of the first liquid storage cavity (1); The clamp ring (2) is a hollow cylindrical structure without filtering function. The filtrate is stored in the tube inside the clamp ring and on the upper part of the clamp ring. The clamp ring (2) is used to limit the movement of the filter membrane assembly (3).

10. A flexible separation device suitable for high throughput operation, characterized in that The device comprises a liquid storage tube and a flexible parallel extraction column structure suitable for high-throughput operation according to any one of claims 1 to 9; The liquid storage tube and the extraction column structure are used in combination, the liquid to be filtered is stored in the liquid storage tube, the extraction column structure is pressed into the liquid storage tube, and the liquid to be filtered passes through the sieve plate assembly (6) and the filter membrane assembly (3) from the bottom of the centrifuge-free filter tube and enters the inner cavity of the tube body of the centrifuge-free filter tube.

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