Liquid chromatography monolithic column and preparation method thereof

Abstract

The invention provides a liquid chromatography monolithic column and a preparation method thereof, and the preparation method comprises the following steps: S1, preparing a blocky porous monolithic material, and processing the blocky porous monolithic material into a strip-shaped column core; s2, coating the outer surface of the column core with a structural adhesive, plugging the structural adhesive into a liquid chromatography hollow column tube, and enabling two ends of the column core to extend out of two ends of the liquid chromatography hollow column tube; s3, curing and forming the structural adhesive, and trimming the column core and the end of the liquid chromatogram hollow column tube; and S4, assembling a sieve plate and a joint at two ends of the liquid chromatography hollow column tube to obtain the liquid chromatography monolithic column. According to the brand new preparation method of the liquid chromatography monolithic column, the porous blocky monolithic material is directly processed into the column core, the column core and the liquid chromatography hollow column tube are assembled into the liquid chromatography column, the preparation method is easy to operate and low in preparation cost, in-situ reaction is not needed, the material selection range of the chromatography monolithic column is widened, and the preparation success rate is high.

Description

Technical Field

[0001] The invention relates to the technical field of liquid chromatography separation materials, and in particular to a liquid chromatography monolithic column and a preparation method thereof. Background Art

[0002] A liquid chromatography monolithic column is a column structure in liquid chromatography, which is different from a traditional packed column. A monolithic column is composed of a single piece of porous material, rather than being filled with a packing material (such as small particles). Monolithic columns are usually made of polymer or gel materials and have many continuous micropores and channels, which constitute the internal structure of the monolithic column. Compared with traditional packed columns, liquid chromatography monolithic columns have excellent separation efficiency, lower pressure drop, wider pH range, and better mechanical stability. They have been widely used in liquid chromatography separations and have begun to replace traditional packed columns in some fields.

[0003] However, in the prior art, the liquid chromatography monolithic column is prepared in situ in a hollow column tube, such as the preparation methods disclosed in patent documents CN101975835A, CN113209669A, and CN103869013A. This preparation method has the following problems: (1) Low success rate: When the solution state changes to the solid state, there will be a significant volume shrinkage during the phase transition from liquid to solid. It is easy to produce void defects in the overall chromatographic column, which ultimately leads to preparation failure.

[0004] (2) High preparation cost: polymerization, phase transformation, pore making, flushing, sintering, and modification all need to be completed in the column tube, which involves many steps. In addition, in order to minimize the volume shrinkage during phase transformation, the preparation process is relatively complicated and the preparation cost is high.

[0005] (3) Limited selection of column materials: There are only a limited number of material systems that are suitable for in-situ preparation in the column tube and for transformation from liquid to solid porous structure, which restricts the application and promotion of liquid chromatography monolithic columns. Summary of the invention

[0006] In view of the deficiencies of the prior art, the purpose of the present invention is to develop a method for preparing a liquid chromatography monolithic column to solve the problems of low preparation success rate, high cost and narrow material selection range.

[0007] To achieve the above object, the present invention provides a method for preparing a liquid chromatography monolithic column, comprising the following steps: S1, preparing a block-shaped porous monolithic material, and processing it into a long strip-shaped column core; S2. Apply the structural glue to the outer surface of the column core and insert it into the hollow column tube of liquid chromatography, with the two ends of the column core extending from the two ends of the column tube; S3, the structural adhesive is cured and formed, and the ends of the column core and the column tube are trimmed; S4. Assemble sieve plates and joints at both ends of the liquid chromatography hollow column tube to obtain a liquid chromatography monolithic column.

[0008] The present invention provides a new method for preparing a liquid chromatography monolithic column, in which a porous block monolithic material is directly processed into a column core, and assembled with a liquid chromatography hollow column tube into a liquid chromatography column. The preparation method has simple operation, low preparation cost, and does not require in-situ reaction, thus expanding the material selection range of the chromatographic monolithic column and having a high preparation success rate.

[0009] In a preferred or optional embodiment, the porous monolithic material is selected from one of porous oxides, porous sintered ceramics, foamed metals, metal organic frameworks, and covalent organic frameworks.

[0010] In a preferred or optional embodiment, the porous oxide is selected from one of porous silicon dioxide, porous zirconium dioxide and porous aluminum oxide. The preparation method of the present invention has a wide range of material selection and can be applied to a variety of material systems; and can prepare composite material monolithic columns.

[0011] In a preferred or optional embodiment, the porous monolithic material has pores of various sizes, wherein the pore size of the largest pore is no greater than 20 micrometers, and the pore size of the smallest pore is no less than 0.3 nanometers. The pore size range of the block-shaped porous monolithic material is limited to ensure separation efficiency while reducing the penetration of the structural glue into the column core.

[0012] In a preferred or optional embodiment, the structural adhesive is selected from one of epoxy adhesive, polyimide adhesive, polyurethane adhesive, acrylic adhesive, and organic silica gel. By coating with the structural adhesive, the gap between the column core and the column tube surface can be avoided, which is beneficial to improving the chromatographic separation efficiency.

[0013] In a preferred or optional embodiment, the method further comprises: step S5, connecting the liquid chromatography monolithic column to the outside, cleaning the column core, and modifying it with a stationary phase to obtain a liquid chromatography monolithic column modified with a stationary phase chemical reagent. The cleaning and modification of the monolithic column core are similar to the traditional liquid chromatography preparation method, and different types of stationary phases can be bonded to separate different media.

[0014] In a preferred or optional embodiment, in step S1, the cross-sectional shape of the processed column core is selected from one of circular, square and diamond.

[0015] In a preferred or optional embodiment, in step S1, the length of the processed column core is greater than the length of the empty liquid chromatography column tube, and the diameter of the column core or the length of the longest diagonal is smaller than the inner diameter of the empty liquid chromatography column tube.

[0016] The column core is installed in an empty column tube of a liquid chromatography and reacts in the liquid phase of the container. The volume shrinkage during the phase change process is not likely to cause internal defects, so the preparation success rate is high.

[0017] In a preferred or optional embodiment, in step S3, the curing temperature is lower than the denaturation temperature of the porous monolithic material and the structural adhesive. Heating can accelerate curing and control the temperature to avoid excessive temperature affecting material properties.

[0018] The present invention also provides a liquid chromatography monolithic column, which is prepared by the above preparation method. The liquid chromatography monolithic column is fully compatible with existing liquid chromatography instruments, and the connection methods of components such as sieve plates and end heads are completely consistent, which is convenient for promotion and application.

[0019] In summary, compared with the prior art, the present invention has the following beneficial effects: (1) A wide range of material selection: The porous block material is directly processed into a column core. In addition to the traditional porous silica and porous polystyrene systems, this preparation method can be applied to porous oxides, porous sintered ceramics, foam metals, metal-organic frameworks (MOFs), covalent organic frameworks (COFs) and other systems.

[0020] (2) Suitable for preparing composite monolithic columns: It is difficult to prepare composite monolithic columns by the in-situ preparation method of the conventional column tube. However, by adopting the preparation method of the present invention, composite monolithic columns can be prepared by simply mixing the composite materials and sintering them into a monolithic material.

[0021] (3) High preparation success rate: The column core is installed in the empty column tube of the liquid chromatography and reacts in the liquid phase of the container. The volume shrinkage during the phase change process is not easy to cause internal defects, so the preparation success rate is high.

[0022] (4) The separation efficiency of the liquid chromatography monolithic column is higher: the structural glue is coated on the surface of the column core, which can completely fill the gap between the column core and the surface of the column tube, which is beneficial to improving the chromatographic separation efficiency.

[0023] (5) Suitable for popularization and application: The preparation method of the present invention is simple, the preparation cost is low, the connection method of the sieve plate, the end head and other components is consistent, and it is compatible with existing liquid chromatography instruments, which is convenient for popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a structural diagram of the porous ceramic monolithic column core in Example 1.

[0025] Figure 2 This is a chromatogram of the porous ceramic liquid chromatography monolithic column separating a sugar mixture in Example 5.

[0026] Figure 3This is a chromatogram of the separation of alkylbenzenes using a silica reverse phase liquid chromatography monolithic column in Example 6. DETAILED DESCRIPTION

[0027] In order to make the above-mentioned purposes, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. It should be noted that the following embodiments are only used to illustrate the implementation method and typical parameters of the present invention, and are not used to limit the parameter range described in the present invention. Reasonable changes derived therefrom are still within the scope of protection of the claims of the present invention.

[0028] It should be noted that the endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

[0029] The specific embodiment of the present invention provides a liquid chromatography monolithic column and a preparation method thereof, the preparation method comprising the following steps: S1. Prepare a block-shaped porous monolithic material and process it into a long strip-shaped column core.

[0030] In a specific embodiment, the porous monolithic material may be porous oxides, porous sintered ceramics, foamed metals, framework compounds (MOFs), covalent organic frameworks (COFs), etc.; porous oxides and porous ceramics are preferred; porous oxides may be porous silicon dioxide, porous zirconium dioxide, porous aluminum oxide, etc. The preparation method has a wide range of material selection and can be applied to a variety of material systems; and is suitable for preparing composite monolithic columns, which only requires mixing the composite materials and sintering them into monolithic materials, and the operation is simple.

[0031] In a specific embodiment, the porous monolithic material has pores of various sizes, which can achieve liquid phase separation; wherein the pore size of the largest pore is no greater than 20 microns, and the pore size of the smallest pore is no less than 0.3 nanometers.

[0032] In a specific embodiment, the cross-section of the processed long strip column core can be circular, square, diamond, etc., preferably circular. The diameter of the column core or the length of the longest diagonal line is smaller than the inner diameter of the empty column tube of the liquid chromatography, which is convenient for overall packing and assembly; and the length of the column core is greater than the length of the empty column tube of the liquid chromatography.

[0033] S2. Apply the structural glue to the outer surface of the column core and insert it into the hollow column tube of liquid chromatography, with the two ends of the column core extending from both ends of the column tube.

[0034] In a specific embodiment, the structural adhesive may be epoxy adhesive, polyimide adhesive, polyurethane adhesive, acrylic adhesive, organic silica gel, etc. By coating with the structural adhesive, the gap between the column core and the surface of the column tube can be avoided; and because the maximum pore size of the column core is less than 20 microns, the penetration depth of the structural adhesive into the interior of the column core is very small, which is conducive to improving the chromatographic separation efficiency.

[0035] S3. The structural adhesive is cured and formed, and the ends of the column core and the column tube are trimmed.

[0036] In a specific embodiment, the curing time of the structural adhesive can be shortened by increasing the temperature, but the temperature should be lower than the denaturation temperature of the porous monolithic material and the structural adhesive.

[0037] S4. Assemble sieve plates and joints at both ends of the liquid chromatography hollow column tube to obtain a liquid chromatography monolithic column.

[0038] In a specific embodiment, the connection method between the hollow column tube of liquid chromatography and the sieve plate and the end is similar to that of a traditional liquid chromatography column: the sieve plate is put on the end of the column tube, and the external thread at the end of the column tube and the internal thread at the end are used to tighten them together.

[0039] S5. Connect the liquid chromatography hollow column tube to the outside, clean the column core, and modify it with a stationary phase to obtain a liquid chromatography monolithic column modified with a stationary phase chemical reagent.

[0040] In a specific embodiment, the cleaning and modification of the monolithic column core are similar to those of conventional liquid chromatography, and a reverse phase stationary phase, a hydrophilic stationary phase, a chiral stationary phase, etc. can be bonded.

[0041] The above method directly processes the porous block-shaped monolithic material into a column core, and assembles it with a liquid chromatography hollow column tube into a liquid chromatography column. The preparation method is simple to operate, low in preparation cost, does not require in-situ reaction, expands the material selection range of the chromatographic monolithic column, is suitable for preparing composite material monolithic columns, has a high preparation success rate, and the liquid chromatography monolithic column has a higher separation efficiency.

[0042] The technical solutions and effects of the present invention are described below through specific embodiments.

[0043] Example 1

[0044] The porous ceramic liquid chromatography monolithic column was prepared as follows: (1) Take a commercially available porous ceramic rod with a nominal diameter of 200 nm, turn it into a porous ceramic monolithic column core with a diameter of 4 mm and a length of 6 cm, activate it with 6M hydrochloric acid, wash it, and vacuum dry it. The porous ceramic monolithic column core prepared in this step is as follows: Figure 1 shown.

[0045] (2) Take commercially available AB epoxy glue, mix well, and coat the porous ceramic monolithic column core; take a 4.6 mm inner diameter, 5 cm long liquid chromatography stainless steel empty column tube, and fill the empty column tube with glue; slowly push the column core coated with glue into the empty column tube, put it in an oven, and cure it overnight at 60°C in vacuum.

[0046] (3) Trim the excess column core at both ends of the column tube to the end of the empty liquid chromatography column tube, and ultrasonically clean and dry it in ethanol.

[0047] (4) Take the sieve plate and end cap that match the column tube, put the sieve plate on the end of the column tube, tighten the end cap, and obtain a porous ceramic liquid chromatography monolithic column.

[0048] The porous ceramic liquid chromatography monolithic column prepared in Example 1 was connected to a liquid chromatograph and flushed with 100% methanol for 30 minutes. The methanol flow rate was adjusted to 0.5, 1, 2, and 3 mL / min, and the measured back pressures were 1.2, 2.1, 4.2, and 6.3 MPa, respectively.

[0049] Example 2

[0050] The porous ceramic reversed phase liquid chromatography monolithic column was prepared as follows: (1) The porous ceramic liquid chromatography monolithic column obtained in Example 1 was connected to a high-pressure chromatography pump, rinsed with 6M hydrochloric acid, pure water, and ethanol for 2 hours, and dried in a vacuum oven at 50° C. overnight.

[0051] (2) Pour a 1% toluene solution of octadecyldimethylsilyl chloride into the column tube, cap both ends of the column tube tightly, place it in an oven, and react at 120°C for 12 hours.

[0052] (3) Repeat step (2) twice to obtain a porous ceramic reversed-phase liquid chromatography monolithic column.

[0053] Example 3

[0054] The silica monolithic column was prepared as follows: (1) In a 20 mL polytetrachloroethylene-lined reactor, add 1 g PEG-2000 and 10 mL 0.5 M acetic acid aqueous solution and stir until PEG is completely dissolved; then add 8 mL methyl orthosilicate (TMOS) and stir vigorously for 20 min in an ice bath to obtain a uniform transparent solution; add 5 mL 1 M urea and stir evenly, then seal the vessel immediately and age it in an oven at 120 °C for 24 h to obtain a silica monolithic column gel block.

[0055] (2) After the gel block is taken out as a whole, it is soaked and rinsed with water and anhydrous ethanol, air-dried at 60 °C for 12 h, vacuum-dried at 60 °C for 12 h, and finally calcined at 600 °C for 5 h in a muffle furnace to obtain a silica monolith.

[0056] (3) The silica monolithic column block was turned into a silica monolithic column core with a diameter of 4 mm and a length of 6 cm, which was then activated with 6M hydrochloric acid, cleaned, and vacuum dried.

[0057] (4) Take commercially available AB epoxy glue, mix well, and coat the entire silica column core; take a 4.6 mm inner diameter, 5 cm long empty stainless steel column tube for liquid chromatography, and fill the empty column tube with glue; slowly push the column core coated with glue into the empty column tube, place it in an oven, and cure it overnight at 60°C in vacuum.

[0058] (5) Trim the excess column core at both ends of the column tube to the end of the empty liquid chromatography column tube, and ultrasonically clean and dry it in ethanol.

[0059] (6) Take the sieve plate and end cap that match the column tube, put the sieve plate on the end of the column tube, tighten the end cap, and obtain a silica monolithic column.

[0060] The silica monolithic column prepared in this example was connected to a liquid chromatograph and flushed with 100% methanol for 30 minutes. The methanol flow rate was adjusted to 0.5, 1, 2, and 3 mL / min, and the measured back pressures were 0.6, 1.1, 2.2, and 3.3 MPa, respectively.

[0061] Example 4

[0062] The process of preparing a silica reversed phase liquid chromatography monolithic column is as follows: (1) The silica monolithic column obtained in Example 3 was connected to a high-pressure chromatography pump, rinsed with 6M hydrochloric acid, pure water, and ethanol for 2 hours, and dried in a vacuum oven at 50°C overnight.

[0063] (2) Pour a 1% toluene solution of octadecyldimethylsilyl chloride into the column tube, cap both ends of the column tube tightly, place it in an oven, and react at 120°C for 12 hours.

[0064] (3) Pour a 1% hexamethyldisilazane toluene solution into the column tube, cap both ends of the column tube tightly, place it in an oven, and react at 80°C for 12 hours.

[0065] (4) Repeat steps (2) and (3) twice to obtain a silica reverse phase liquid chromatography monolithic column.

[0066] Example 5

[0067] The porous ceramic liquid chromatography monolithic column prepared in Example 1 was used to separate monosaccharides, disaccharides and trisaccharides, with 82 / 18 (v / v) acetonitrile / 200 mM ammonium acetate buffer (pH = 5) as the mobile phase, a flow rate of 2 mL / min, a column temperature of 22°C, a drift tube temperature of the evaporative light scattering detector of 130°C, and a N2 nebulizer gas flow rate of 4 L / min.

[0068] The separation results are as follows Figure 2 As shown in the figure, the six compounds are ribose (a), xylose (b), glucose (c), sucrose (d), trehalose (e), and raffinose (f). The chromatographic dead time is about 1.5 minutes, and these small molecule sugars are effectively separated on the porous ceramic liquid chromatography monolithic column.

[0069] Example 6

[0070] The five alkylbenzenes were separated using the silica reverse phase liquid chromatography monolithic column prepared in Example 4, with separation conditions of acetonitrile / water (65:35), a flow rate of 1.0 mL / min, and a detection wavelength of 210 nm.

[0071] The separation results are as follows Figure 3 As shown in the figure, the five compounds are toluene (a), ethylbenzene (b), propylbenzene (c), butylbenzene (d), and pentylbenzene (e). The chromatographic dead time is about 2.5 minutes, and these alkylbenzene small molecules are effectively separated on the silica reversed phase liquid chromatography monolithic column.

[0072] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

Claims

1. A method for preparing a liquid chromatography monolithic column, characterized in that: The following steps are involved: S1. Prepare block-shaped porous monolithic material and process it into a long strip of column core; S2. Apply the structural glue all over the outer surface of the column core and stuff it into the liquid chromatographic hollow column tube. Both ends of the column core extend out from both ends of the liquid chromatographic hollow column tube; S3. Structural glue is cured and molded, and the column core and the ends of the liquid chromatographic hollow column tube are trimmed; S4. Assemble the screen plate and connector at both ends of the hollow column tube of the liquid chromatography to obtain a liquid chromatography integral column.

2. The method for preparing a liquid chromatography monolithic column according to claim 1, characterized in that: The porous monolithic material is selected from at least one of a porous oxide, a porous sintered ceramic, a foam metal, a metal organic frame, and a covalent organic frame.

3. The method for preparing a liquid chromatography monolithic column according to claim 2, characterized in that: The porous oxide is selected from at least one of porous silica, porous zirconium dioxide, and porous aluminum trioxide.

4. The method for preparing a liquid chromatography monolithic column according to claim 2, characterized in that: The porous monolithic material has holes of various sizes, the pore size of the largest hole is not greater than 20 microns and the pore size of the smallest hole is not less than 0.3 nanometers.

5. The method for preparing a liquid chromatography monolithic column according to claim 1, characterized in that: The structural glue is selected from one of epoxy glue, polyimide glue, polyurethane glue, acrylic glue, and organic silicone glue.

6. The method for preparing a liquid chromatography monolithic column according to claim 1, characterized in that: It also includes: step S5, connecting the liquid chromatography whole column with the outside world, cleaning the column core, modifying it with stationary chemical reagent, and obtaining the stationary phase modified liquid chromatography whole column.

7. The method for preparing a liquid chromatography monolithic column according to any one of claims 1 to 6, characterized in that: In the step S1, the cross-sectional shape of the obtained column core is selected from one of a circle, a square, and a diamond.

8. The method for preparing a liquid chromatography monolithic column according to claim 7, characterized in that: In the step S1, the length of the column core is processed and the length of the empty column tube of the liquid chromatography is larger than the length of the empty column tube of the liquid chromatography is smaller than the diameter of the inner cavity of the empty column tube of the liquid chromatography.

9. The method for preparing a liquid chromatography monolithic column according to any one of claims 1 to 6, characterized in that: In the step S3, the curing forming temperature is lower than the denaturation temperature of the porous whole material and the structural glue.

10. A liquid chromatography monolithic column, characterized in that:

1. The preparation method according to any one of claims 1-9.

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