CN114939401A - Method for preparing hydrophilic interaction chromatography functional group fixing phase - Google Patents

Method for preparing hydrophilic interaction chromatography functional group fixing phase Download PDF

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Publication number
CN114939401A
CN114939401A CN202210227484.8A CN202210227484A CN114939401A CN 114939401 A CN114939401 A CN 114939401A CN 202210227484 A CN202210227484 A CN 202210227484A CN 114939401 A CN114939401 A CN 114939401A
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preparing
emulsifier
hydrophilic interaction
seed
interaction chromatography
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任连兵
仲玉
丁士忠
贺森
彭顺飞
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Nanjing Genshan Biotechnology Co ltd
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/264—Synthetic macromolecular compounds derived from different types of monomers, e.g. linear or branched copolymers, block copolymers, graft copolymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/22—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the construction of the column
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/265—Adsorption chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
    • B01J20/28011—Other properties, e.g. density, crush strength
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28016—Particle form
    • B01J20/28021—Hollow particles, e.g. hollow spheres, microspheres or cenospheres

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Abstract

The invention discloses a preparation method of a fixed phase containing hydrophilic interaction chromatography functional groups, which is characterized by comprising the following steps: based on the swellable seed polymer microsphere, a polymerizable nitrile group monomer, a specific monomer, a pore-forming agent, a cross-linking agent and an initiator are swelled in the seed microsphere, and after polymerization reaction, the nitrile group hydrolysis reaction is carried out to obtain the polystyrene polymer microsphere which is monodisperse in particle size, specific in pore size and contains a large number of amide groups on the surface. The method has the advantages of simple preparation, mild reaction conditions, good controllability, low production cost and the like.

Description

Method for preparing hydrophilic interaction chromatography functional group-containing fixed phase
Technical Field
The invention relates to a method for preparing polymer microspheres for a chromatographic stationary phase, in particular to a method for preparing polymer microspheres for a chromatographic stationary phase, which comprises the following steps: a method for preparing polymer microspheres by introducing hydrophilic interaction chromatography functional groups into reversed phase chromatography filler stationary phase.
Background
Reverse phase liquid chromatography is the most widely used mode of chromatography in current chromatographic separation and analysis, and can realize high-efficiency separation of most of weak-polarity and medium-polarity compounds by depending on the strength of interaction between a hydrophobic fixed phase and a solute.
With the development of research fields such as biomedical technology, food safety, environmental monitoring and the like, the strongly polar and hydrophilic small molecular substances rapidly become important research objects in the fields of analytical chemistry and biochemistry, but generally, the compounds are weakly retained on a reversed-phase liquid chromatography filler, so that the separation is difficult.
The amide group has good stability and hydrophilicity, the influence of ion exchange effect on the amide group modified filler when separating an ionic compound is small, the retention of a sample on an amide bonding phase is less influenced by pH, irreversible adsorption is not easy to generate, and the stability is better when the filler is used for a long time; amide columns do not generally require ionic mobile phases and are therefore well suited for use in conjunction with mass spectrometry, primarily for the separation of polypeptides, and also for the separation of oligosaccharides and various sugar-containing compounds.
The polystyrene microsphere has good mechanical strength, stable chemical property and strong surface hydrophobicity; can realize the advantages of use at high flow rate and the like, meets the requirements of rapid and efficient separation and purification, and is an excellent reversed phase chromatographic separation medium. The amide functional group is introduced into the polystyrene microsphere, so that the retention of the strongly polar and hydrophilic micromolecular substances can be better realized under the reversed phase chromatography retention condition, and the chromatographic separation is realized.
Disclosure of Invention
The invention aims to solve the problem that a hydrophilic chromatographic functional group is introduced into the structure of the reversed-phase chromatographic packing to enhance the retention capacity of the packing on strong-polarity and hydrophilic micromolecular substances; the introduced hydrophilic chromatographic functional group is an amide group and is realized by controlling and hydrolyzing a nitrile functional group.
The invention adopts a 'seed swelling method' to prepare a microsphere containing hydrophilic chromatographic functional groups, and the principle is that uniform polymer microspheres with smaller grain sizes are prepared by using methods such as dispersion polymerization or seed swelling polymerization, and the like, then the uniform polymer microspheres are used as seed microspheres, emulsion of monomers and initiators enters the original seed microspheres through one-step or multi-step swelling, the seed microspheres are uniformly enlarged, polymerization is initiated after the swelling balance is achieved, thus porous polymer microspheres can be prepared, the original grain size uniformity of the seed microspheres is kept, and amide groups are obtained by controlling hydrolysis nitrile functional groups.
The technical scheme of the invention is as follows: a method for preparing a fixed phase containing hydrophilic interaction chromatography functional groups comprises the following steps:
(1) preparing an oil phase: weighing a proper amount of oil phase monomer M1, oil phase monomer M2, pore-forming agent P4, cross-linking agent C2, emulsifier S3 and ultrapure water, mixing according to the mass ratio of 2:1-5:1-3:1-3:0.02:10-20, and carrying out ultrasonic emulsification for 2-15min to obtain an oil phase for later use;
(2) preparation of an aqueous phase: weighing a proper amount of stabilizer, adding deionized water, heating to dissolve, and cooling to room temperature after the stabilizer is completely dissolved to obtain a water phase;
(3) swelling the oil phase: adding the oil-phase emulsion in the step (1) into the seed polymer microspheres, uniformly stirring, and keeping the system temperature at 10-70 ℃ for 30-200 min;
(4) swelling of an initiator: appropriate amount of initiator I4, emulsifier S3 and water are added according to the mass ratio of 0.05-0.15: 0.05-0.1:1-5, ultrasonic emulsifying for 2-15min, and adding into (3)
(5) Polymerization: adding the (4) into the (3), uniformly stirring, heating to 60-90 ℃, and preserving heat for 6-24 hours; after the reaction is finished, cooling to room temperature for cleaning;
(6) cleaning: pumping the polymer microspheres obtained in the step (5), cleaning with ethanol or methanol or acetone, and drying for later use;
(7) hydrolysis: dispersing the microspheres obtained in the step (6) in dilute acid, heating to 60-80 ℃, and preserving heat for 3-6 h; and after the reaction is finished, cooling to room temperature, washing with deionized water, and cleaning to obtain the polystyrene polymer microspheres with amide groups on the surfaces.
Wherein the monomer M1 is acrylonitrile, or 5-hexenenitrile, or 2-butenenitrile, or methacrylonitrile; the monomer M2 is styrene, or methyl styrene, or halogenated styrene.
Wherein the pore-foaming agent P4 is toluene, or n-hexanol, or heptane, or dodecanol, or cyclohexanol, or isobutanol, or any mixture thereof.
Wherein the crosslinking agent C2 is any one or a mixture of divinylbenzene, diallyl phthalate or triallyl benzenetricarboxylate;
wherein the emulsifier S3 is an ionic emulsifier, a nonionic emulsifier or an ionic and nonionic compound emulsifier; wherein the ionic emulsifier is: alkyl benzene sulfonates, or alkyl succinate sulfonates, or alkyl diphenyl ether sulfonates, such as SDS, SDBS; wherein the non-ionic emulsifier is alkylphenol polyoxyethylene, or benzyl phenol polyoxyethylene, or phenethyl phenol polyoxyethylene, or fatty alcohol polyoxyethylene, or fatty amine polyoxyethylene, such as Tween 80, Tween 40, Tween 20, Tween 60, span 65, span 85, span 80, Tritro X-401, Tritro X-405, or Tritro X-100;
the initiator I4 is any one or a mixture of more of benzoyl peroxide, diisobutyronitrile peroxide, dimethyl azobisisobutyrate and azobisisobutylamidine;
wherein the stabilizer is polyvinyl alcohol, or polyethylene glycol, or polyvinylpyrrolidone, or hydroxymethyl cellulose, or hydroxypropyl cellulose, or hydroxyethyl cellulose, or carboxymethyl cellulose, or beta-cyclodextrin, or beta-methyl cyclodextrin, or hydroxyapatite, or a mixture of any two or more of them;
wherein (7) the dilute acid obtained by hydrolysis is sulfuric acid, hydrochloric acid, nitric acid or a mixture of the three;
wherein the particle size of the microsphere is 3-50 um; the pore diameter of the microspheres is
Figure BDA0003536686250000021
The invention has the characteristics that:
the invention relates to a hydrophilic interaction chromatography functional group fixing phase which adopts a seed swelling method, wherein a nitrile group polymerizable monomer is swelled into seed microspheres, and then synthesized into spheres through free radicals, and then hydrolyzed to obtain polystyrene polymer microspheres with a large amount of amide groups on the surfaces; overcomes the defects that the acrylamide monomer has too strong water solubility and is difficult to swell into the seed microspheres. The invention can realize the range of 3-50 microns to obtain the polymer microsphere product with uniform particle size, and the CV value is less than 3 percent. In addition, the method has the following advantages: the synthetic method has the advantages of simplicity, easy control, convenient amplification production and the like, and has potential application value in the fields of bioseparation, biomedical detection and the like.
Drawings
FIG. 1 is a flow chart of a preparation process of poly (acrylamide-styrene-divinylbenzene) polymer microspheres according to the present invention;
FIG. 2 is a schematic diagram of a chemical reaction principle of poly (acrylamide-styrene-divinylbenzene) polymer microspheres according to the present invention;
FIG. 3 is an optical microscope photograph of fixed phase microspheres containing hydrophilic interaction chromatography functional groups according to a fourth embodiment of the present invention;
Detailed Description
The preparation method of the fixed phase containing hydrophilic interaction chromatography functional group according to the invention is further described in the following by combining the attached drawings and specific examples.
The seed polymer microspheres are polystyrene microspheres, are prepared by a dispersion polymerization method or a swelling method, the preparation method is determined according to the particle size of the needed seed polymer microspheres, and when the particle size of the seed polymer microspheres is less than 5 mu m, dispersion polymerization is adopted; the swelling method is adopted when the particle size of the seed polymer microsphere is larger than 5 mu m.
Dispersion polymerization process
160mL of ethanol and 20mL of ultrapure water are weighed and added into a 500mL round-bottom flask, a reflux condenser tube, mechanical stirring, a nitrogen ventilating conduit and a thermometer are arranged on a reaction bottle, 2.0g of dispersing agent PVP and 1.0g of auxiliary dispersing agent dodecanol are added, stirring and mixing are carried out uniformly, nitrogen is introduced into the flask, and the system is heated to 60 ℃; weighing 1.0g of dodecanethiol and 0.14g of azobisisobutyronitrile, dissolving in 20g of styrene, adding into the reaction bottle, keeping the temperature for reaction for 20 hours to obtain a polystyrene seed solution with uniform particles, and cleaning to obtain the polystyrene seed solution with the particle size of 2.4 microns.
Swelling seed preparation
Weighing 1g of polystyrene seed microspheres with the particle size of 2.4 mu m prepared by the method in a 250mL round-bottom flask; weighing 0.11g of azobisisobutyronitrile, 38g of styrene, 0.4g of dodecanethiol, 0.1g of SDS and 20mL of ultrapure water into a 100mL beaker, emulsifying to obtain an emulsion with the particle size of about 10 micrometers, adding the emulsion into the flask, heating to 60 ℃, reacting for 3 hours under heat preservation, adding 0.5g of PEG4000/40g of ultrapure water solution, reacting for 20 hours at 80 ℃ to obtain a homogeneous polystyrene seed solution, and cleaning to obtain a homogeneous polystyrene seed solution with the particle size of 8.0 micrometers.
Example one
Preparation of poly (acrylonitrile-styrene-divinylbenzene) polymer microspheres
1. 10L of aqueous solution containing 10g/L of polyvinyl alcohol (PVA) and 2.5g/L of Sodium Dodecyl Sulfate (SDS) is prepared to be used as aqueous phase solution for preparing a microsphere reaction system with uniform particle size.
2. 1g of the swellable seed microspheres having a monodispersed particle size of 2.4 μm were suspended and dispersed in 200mL of the above aqueous solution containing 10g/L of PVA and 2.5g/L of SDS to obtain a seed microsphere suspension dispersion solution.
3. 20g of acrylonitrile, 45g of styrene, 20g of 80% divinylbenzene and 20g of n-hexanol were mixed and added to 200mL of an aqueous solution containing 10g/L of PVA and 2.5g/L of SDS to obtain a mixed system in which oil and water phases were separated. The oil-water two-phase layered mixed system is prepared into emulsion through ultrasonic action, the used ultrasonic power is 500W, the ultrasonic time is 9s, the interval time is 3s, the repetition times are 90 times, and the diameter of the dispersed oily small droplets in the final emulsion is observed to be less than 1 mu m under an optical microscope.
4. And adding the 3 emulsion into 200mL of seed microsphere suspension solution, adding 10g of 10% sodium nitrite solution, placing the swelling mixture system in an oil bath at 60 ℃, and swelling for 2 hours under the mechanical stirring action of 150 rpm.
5. 1g of benzoyl peroxide is made into emulsion by 10g/L of PVA and 2.5g/L of SDS solution; adding into the above 4 solution, and swelling for 20 min.
6. After the swelling is finished, the mechanical stirring speed is increased to 280 rpm; heating to 80 deg.C, polymerizing for 16 hr to obtain poly (acrylonitrile-styrene-divinylbenzene) polymer microsphere with particle diameter of 10.8 μm, CV value of 2.5%, and crosslinking degree of 16%.
Example two
Preparation of porous poly (acrylonitrile-styrene-divinylbenzene) polymer microspheres with uniform particle size
1. 10L of water solution containing 5g/L of cellulose and 2.5g/L of Sodium Dodecyl Benzene Sulfonate (SDBS) is prepared to be used as aqueous phase solution for preparing the microsphere reaction system with uniform particle size.
2. 1g of seed microspheres with uniform monodisperse particle size of 8 mu m are suspended and dispersed in 300mL of the aqueous solution containing 5g/L of cellulose and 2.5g/L of SDBS to obtain a seed microsphere suspension dispersion solution.
3. 15g of acrylonitrile, 35g of styrene, 20g of divinylbenzene and 30g of toluene were mixed and added to 200mL of an aqueous solution containing 5g/L of cellulose and 2.5g/L of SDBS to obtain a mixed system in which two phases of oil and water were separated. The oil-water two-phase layered mixed system is prepared into emulsion through ultrasonic action, the used ultrasonic power is 500W, the ultrasonic time is 9s, the interval time is 3s, the repetition times are 90 times, and the diameter of the dispersed oily small droplets in the final emulsion is observed to be less than 1 mu m under an optical microscope.
4. And adding the 3 emulsion into 300mL of seed microsphere suspension solution, adding 10g of 10% sodium nitrite solution, placing the swelling mixture system in an oil bath at 60 ℃, and swelling for 2 hours under the mechanical stirring action of 150 rpm.
5. Preparing 1g of azobisisobutyronitrile into emulsion by using 5g/L of cellulose and 2.5g/L of SDBS solution; adding into the solution 4, and swelling for 20 min.
6. After the swelling is finished, the mechanical stirring speed is increased to 280 rpm; heating to 80 ℃ and polymerizing for 16 hours to obtain the porous poly (acrylonitrile-styrene-divinylbenzene) polymer microspheres with the particle size of 32.8 microns, the CV value of 2.8 percent and the crosslinking degree of 23 percent and uniform particle size.
EXAMPLE III
Preparation of porous poly (acrylonitrile-styrene-diallyl phthalate) polymer microspheres with uniform particle size
1. 10L of aqueous solution containing 10g/L of polyvinylpyrrolidone and 2.5g/L of Tritro X-405 is prepared to be used as aqueous phase solution for preparing the microsphere reaction system with uniform particle size.
2. 1g of seed microspheres with uniform particle size of 8.0 μm and monodisperse particles was suspended and dispersed in 300mL of the above aqueous solution containing 10g/L of polyvinylpyrrolidone and 2.5g/L of Tritro X-405 to obtain a seed microsphere suspension dispersion solution.
3. 15g of methacrylonitrile, 40g of styrene, 20g of diallyl phthalate and 25g of cyclohexanol were mixed and added to 200mL of an aqueous solution containing 10g/L of polyvinylpyrrolidone and 2.5g/L of Tritro X-405 to obtain a mixed system in which two phases of oil and water were separated. The oil-water two-phase layered mixed system is prepared into emulsion through ultrasonic action, the used ultrasonic power is 500W, the ultrasonic time is 9s, the interval time is 3s, the repetition times are 90 times, and the diameter of the dispersed oily small droplets in the final emulsion is observed to be less than 1 mu m under an optical microscope.
4. And adding the 3 emulsion into 300mL of seed microsphere suspension solution, adding 10g of 10% sodium nitrite solution, placing the swelling mixture system in an oil bath at 60 ℃, and swelling for 2 hours under the mechanical stirring action of 150 rpm.
5. Preparing 1g of dimethyl azodiisobutyrate into emulsion by using 10g/L of polyvinylpyrrolidone and 2.5g/L of Tritro X-405 solution; adding into the above 4 solution, and swelling for 20 min.
6. After the swelling is finished, the mechanical stirring speed is increased to 280 rpm; heating to 80 deg.C, polymerizing for 16 hr to obtain porous poly (methacrylonitrile-styrene-diallyl phthalate) polymer microsphere with particle diameter of 33.1 μm, CV value of 2.8%, and 23% crosslinking degree.
Example four
Preparation of poly (acrylamide-styrene-divinylbenzene) polymer microspheres
Dispersing 10g of the poly (acrylonitrile-styrene-divinylbenzene) polymer microspheres in 100mL of 1mol/L sulfuric acid solution, mechanically stirring, and heating to 80 ℃ for reaction for 3 hours; after the reaction is finished, carrying out suction filtration and washing to obtain the poly (acrylamide-styrene-divinylbenzene) polymer microspheres.
EXAMPLE five
Preparation of porous poly (acrylamide-styrene-divinylbenzene) polymer microspheres
Cleaning the porous poly (acrylonitrile-styrene-divinylbenzene) polymer microspheres obtained in the second embodiment with ethanol to remove the pore-forming agent, and then washing the residual ethanol with deionized water; dispersing 30mL of porous poly (acrylonitrile-styrene-divinylbenzene) polymer microspheres in 100mL of 1mol/L sulfuric acid solution, mechanically stirring, and heating to 80 ℃ for reaction for 3 hours; after the reaction is finished, carrying out suction filtration and washing to obtain the poly (acrylamide-styrene-divinylbenzene) polymer microspheres.
It should be noted that the description of the present invention and the accompanying drawings illustrate preferred embodiments of the present invention, but the present invention may be embodied in many different forms and is not limited to the embodiments described in the present specification, which are provided as additional limitations to the present invention and to provide a more thorough understanding of the present disclosure. Moreover, the above technical features are combined with each other to form various embodiments which are not listed above, and all the embodiments are regarded as the scope of the present invention described in the specification; further, modifications and variations will occur to those skilled in the art in light of the foregoing description, and it is intended to cover all such modifications and variations as fall within the true spirit and scope of the invention as defined by the appended claims.

Claims (9)

1. A method for preparing a fixed phase containing hydrophilic interaction chromatography functional groups is characterized in that: a swellable polymer 'seed' microsphere swells polymerizable nitrile monomers, specific monomers, a pore-forming agent, a cross-linking agent and an initiator to be swelled into the seed microsphere, and nitrile functional groups are hydrolyzed after polymerization reaction to obtain the polystyrene high-molecular microsphere which is monodisperse in particle size, specific in pore diameter and contains a large number of amide groups on the surface.
2. A method of preparing a fixed phase comprising hydrophilic interaction chromatography functionality as claimed in claim 1, comprising the steps of:
(1) preparing an oil phase: weighing appropriate amount of oil phase monomer M1, oil phase monomer M2, pore-forming agent P4, cross-linking agent C2, emulsifier S3 and ultrapure water, mixing according to the mass ratio of 2:1-5:1-3:1-3:0.02:10-20, carrying out ultrasonic emulsification for 2-15min,
obtaining an oil phase for later use;
(2) preparation of an aqueous phase: weighing a proper amount of stabilizer, adding deionized water, heating to dissolve, after the stabilizer is completely dissolved,
cooling to room temperature to obtain water phase;
(3) swelling the oil phase: adding the oil-phase emulsion in the step (1) into the seed polymer microspheres, uniformly stirring, and keeping the system temperature at 10-70 ℃ for 30-200 min;
(4) swelling of an initiator: appropriate amount of initiator I4, emulsifier S3 and water are added according to the mass ratio of 0.05-0.15: 0.05-0.1:1-5, ultrasonic emulsifying for 2-15min, and adding into (3)
(5) Polymerization: adding the (4) into the (3), uniformly stirring, heating to 60-90 ℃, and preserving heat for 6-24 h; after the reaction is finished, cooling to room temperature for cleaning;
(6) cleaning: pumping the polymer microspheres obtained in the step (5), cleaning with ethanol or methanol or acetone, and drying for later use;
(7) hydrolysis: dispersing the microspheres obtained in the step (6) in dilute acid, heating to 60-80 ℃, and preserving heat for 3-6 h; and after the reaction is finished, cooling to room temperature, washing with deionized water, and cleaning to obtain the polystyrene polymer microspheres with amide groups on the surfaces.
3. The method for preparing a fixed phase containing hydrophilic interaction chromatography functional groups as claimed in claim 2, wherein the monomer M1 is acrylonitrile, or 5-hexenenitrile, or 2-butenenitrile, or methacrylonitrile; the monomer M2 is styrene, methyl styrene or halogenated styrene.
4. The method according to claim 2, wherein the porogen P4 is toluene, n-hexanol, heptane, dodecanol, cyclohexanol, isobutanol, or any mixture thereof.
5. The method for preparing the fixed phase containing the hydrophilic interaction chromatography functional group as claimed in claim 2, wherein the cross-linking agent C2 is any one or a mixture of divinyl benzene, diallyl phthalate or triallyl benzenetricarboxylate.
6. The method for preparing the fixed phase containing the hydrophilic interaction chromatographic functional group according to claim 2, wherein the emulsifier S3 is an ionic emulsifier, a nonionic emulsifier or a compound emulsifier of an ionic emulsifier and a nonionic emulsifier; wherein the ionic emulsifier is: alkyl benzene sulfonates, alkyl succinate sulfonates, alkyl diphenyl ether sulfonates, such as SDS, SDBS; wherein the non-ionic emulsifier is alkylphenol polyoxyethylene, or benzyl phenol polyoxyethylene, or phenethyl phenol polyoxyethylene, or fatty alcohol polyoxyethylene, or fatty amine polyoxyethylene, such as Tween 80, Tween 40, Tween 20, Tween 60, span 65, span 85, span 80, Tritro X-401, Tritro X-405, or Tritro X-100.
7. The method for preparing the fixed phase containing the hydrophilic interaction chromatographic functional group as claimed in claim 2, wherein the initiator I4 is any one or a mixture of benzoyl peroxide, diisobutyronitrile peroxide, dimethyl azobisisobutyrate or azobisisobutylamidine.
8. The method according to claim 2, wherein the stabilizer is polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, beta-cyclodextrin, beta-methyl cyclodextrin, or hydroxyapatite, or a mixture of any two or more thereof.
9. The method for preparing a fixed phase containing hydrophilic interaction chromatography functional groups according to claim 2, wherein the particle size of the microspheres is 3-50 um; the pore diameter of the microspheres is
Figure RE-RE-FDA0003765895570000011
CN202210227484.8A 2022-03-08 2022-03-08 Method for preparing hydrophilic interaction chromatography functional group fixing phase Pending CN114939401A (en)

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