Monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase and preparation method and application thereof
阅读说明：本技术 一种单分散β-环糊精功能化聚合物微球手性固定相及其制备方法和应用 (Monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase and preparation method and application thereof ) 是由 龚波林 王肖肖 于 2021-03-03 设计创作，主要内容包括：本发明提供了一种单分散β-环糊精功能化聚合物微球手性固定相及其制备方法和应用,涉及色谱固定相技术领域。以单分散聚甲基丙烯酸环氧丙酯微球为基质,在其表面引入3-氯丙基,与二乙基二硫代氨基甲酸钠反应,加入β-环糊精功能单体进行可逆加成断裂链转移聚合反应,形成单分散β-环糊精功能化聚合物微球手性固定相。该单分散β-环糊精功能化聚合物微球手性固定相制备过程简单、反应条件温和、反应可控,可用于高效液相色谱,高效快速地完成对映体的分离。(The invention provides a monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase and a preparation method and application thereof, and relates to the technical field of chromatographic stationary phases. Taking monodisperse poly (glycidyl methacrylate) microspheres as a matrix, introducing 3-chloropropyl on the surface of the monodisperse poly (glycidyl methacrylate) microspheres, reacting with sodium diethyldithiocarbamate, adding a beta-cyclodextrin functional monomer to perform reversible addition fragmentation chain transfer polymerization reaction, and forming a monodisperse beta-cyclodextrin functional polymer microsphere chiral stationary phase. The monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase has the advantages of simple preparation process, mild reaction conditions and controllable reaction, can be used for high performance liquid chromatography, and can efficiently and quickly complete the separation of enantiomers.)
1. A monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase is characterized in that monodisperse poly (glycidyl methacrylate) microspheres are used as a matrix, 3-chloropropyl is introduced to the surface of the monodisperse poly (glycidyl methacrylate) microspheres, the monodisperse poly (glycidyl methacrylate) microspheres react with sodium diethyldithiocarbamate, and beta-cyclodextrin functional monomers are added to perform reversible addition fragmentation chain transfer polymerization reaction to form the monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase;
the beta-cyclodextrin functional monomer is (methacryloyl) holophenylcarbamoyl beta-cyclodextrin.
2. The chiral stationary phase of claim 1, wherein the particle size of the chiral stationary phase is 6.5 μm, the pore diameter is 18nm, and the specific surface area is 180.6m2/g。
3. A preparation method of the monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase as claimed in claim 1, is characterized by comprising the following steps:
(1) preparation of seed liquid of monodisperse poly (glycidyl methacrylate): ultrasonically dispersing 5-10mL of glycidyl methacrylate, 0.1-0.2g of azobisisobutyronitrile and 1.0-2.0g of polyvinylpyrrolidone in 60-90mL of absolute ethyl alcohol, heating to 50-70 ℃ under the protection of nitrogen for polymerization reaction for 16-36h, fully washing and centrifuging a product by using the absolute ethyl alcohol, adding 30-60mL of sodium dodecyl sulfate solution, and standing to obtain a seed solution of monodisperse glycidyl methacrylate;
(2) preparing monodisperse poly (glycidyl methacrylate) microspheres: respectively taking 2-6mL of glycidyl methacrylate, 2-6mL of ethylene glycol dimethacrylate, 2-6mL of toluene, 2-6mL of cyclohexanol and 0.2-0.36g of azobisisobutyronitrile, ultrasonically dispersing uniformly, adding 20-35mL of 5% (w/v) polyvinyl alcohol, 45-75mL of 0.2% (w/v) sodium dodecyl sulfate and 25-45mL of distilled water, mixing uniformly, placing in a cell crusher for 30-60min until the solution is in an emulsion state, adding 5-9mL of seed liquid of the poly (glycidyl methacrylate) obtained in the step (1), swelling and reacting for 16-48h at the temperature of 25-35 ℃, heating to 50-70 ℃ under the protection of nitrogen, reacting for 18-36h, washing with absolute ethyl alcohol and distilled water after the reaction is finished, performing Soxhlet extraction on the obtained product in tetrahydrofuran for 48-60h, washing with absolute ethyl alcohol, and drying in vacuum to obtain monodisperse poly (glycidyl methacrylate) microspheres;
(3) surface modification of disulfide bond of monodisperse poly (glycidyl methacrylate) microsphere: taking 2.0-6.0g of the monodisperse poly (glycidyl methacrylate) microspheres obtained in the step (2) to disperse in 100-300 mL0.1mol.L-1H of (A) to (B)2SO4Reacting in the solution at 40-80 ℃ for 6-20h to obtain hydrolyzed poly (glycidyl methacrylate) microspheres, dispersing 2.0-5.0g of the hydrolyzed poly (glycidyl methacrylate) microspheres in 80-150mL of anhydrous toluene under ultrasonic, dropwise adding 4-10mL of 3-chloropropyltrimethoxysilane, refluxing in 120 deg.C oil bath for 24-48h under nitrogen protection, washing the product with anhydrous ethanol, vacuum drying to obtain chloropropyl functional poly (glycidyl methacrylate) microsphere, ultrasonically dispersing 1.0-3.0g sodium diethyldithiocarbamate in 50-120mL anhydrous tetrahydrofuran, adding 1.0-4.0g of chloropropyl functionalized poly (glycidyl methacrylate) microspheres, reacting for 12-48h at 40-80 ℃ under the protection of nitrogen, and washing products with tetrahydrofuran and methanol in sequence;
(4) preparing a beta-cyclodextrin functional monomer: dissolving 1.0-3.0g of dried beta-cyclodextrin in 50-100mL of dried pyridine, dropwise adding 3-9mL of dried tetrahydrofuran, stirring, adding 3-15mL of phenyl isocyanate, reacting in an oil bath at 80-120 ℃ for 10-24h, removing pyridine and unreacted phenyl isocyanate in a reaction solution under a reduced pressure condition after the reaction is finished, dissolving the residue with ethanol, pouring the dissolved residue into 250mL of water, separating out a solid, filtering, and recrystallizing a filter cake with ethanol to obtain an earthy yellow beta-cyclodextrin functional monomer;
(5) preparing a monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase: taking 1.0-3.0g of disulfide bond modified poly (glycidyl methacrylate) microspheres obtained in the step (3), adding 40-120mL of dry tetrahydrofuran, performing ultrasonic dispersion, adding 0.2-2.5g of beta-cyclodextrin functional monomer obtained in the step (4) and 10-30mg of azobisisobutyronitrile, performing ultrasonic dispersion, performing oil bath reaction at 50-70 ℃ for 12-36h under the protection of nitrogen, washing with tetrahydrofuran and methanol in sequence, and performing vacuum drying to obtain a monodisperse beta-cyclodextrin functional polymer microsphere chiral stationary phase;
the dry tetrahydrofuran in the step (4) contains 0.170-0.85mL of methacryloyl chloride.
4. The method for preparing the monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase according to claim 3, wherein the standing time in the step (1) is 10-24 h; the vacuum drying condition in the step (2) is that the drying is carried out in a vacuum drying oven at the temperature of 30-60 ℃ for 12-24 h; the vacuum drying condition in the step (3) is that the drying is carried out in a vacuum drying oven at the temperature of 40-80 ℃ for 6-24 h; the stirring in the step (4) is carried out for 10-20h at the temperature of 20-60 ℃; the vacuum drying condition in the step (5) is drying in a vacuum drying oven at 50 ℃ for 24 hours.
5. The method for preparing the monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase according to claim 3, wherein the ultrasonic dispersion time in the step (5) is 10-40 min.
6. The use of the chiral stationary phase of monodisperse β -cyclodextrin functionalized polymer microspheres of claim 1, wherein the chiral stationary phase of monodisperse β -cyclodextrin functionalized polymer microspheres can be used for chiral separation of enantiomers.
Technical Field
The invention relates to the technical field of chromatographic stationary phases, in particular to a monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase and a preparation method and application thereof.
Background
The separation of enantiomers has become an important and challenging task due to certain differences in the biological activity, metabolic mechanisms, toxicity, etc. of enantiomeric drugs. Chiral Stationary Phase (CSPs) methods are an effective method for separating enantiomers by immobilizing a chiral selector in a chromatographic stationary phase and separating enantiomers by utilizing the difference in the interaction between the enantiomers in a mobile phase and the chiral selector in the stationary phase. The cyclodextrin-based chiral stationary phase is a widely used chiral stationary phase because the unique hollow structure of cyclodextrin can form an inclusion compound with an enantiomer guest molecule, and the difference of the strength of the formed inclusion compound enables the cyclodextrin-functionalized stationary phase to be used for separating enantiomers.
At present, the preparation of cyclodextrin chiral stationary phase is mainly carried out by physical coating and chemical bonding. The physical coating method is to fix the cyclodextrin derivative on the surface of the matrix by utilizing the adsorption performance of the matrix, and the cyclodextrin derivative generally has better water solubility and is easy to run off under the condition of reverse phase chromatography, so that the chiral fixation of the cyclodextrin prepared by the physical coating method is more suitable for separating enantiomers under the condition of normal phase chromatography. The chemical bonding method is to bond the cyclodextrin derivative to the surface of the substrate via a spacer by chemical bonding. The stationary phase obtained by the method is relatively stable, and the application range of the mobile phase is wide, so that the research is relatively wide. The earliest was the introduction of nitrogen-containing linker groups such as amines, amides and carbamates as spacer arms to bond cyclodextrin derivatives to the silica microsphere surface. However, the spacer arm is unstable and easy to hydrolyze due to the existence of nitrogen atom in the chiral stationary phase, and the complex synthetic route and low loading of cyclodextrin make the cyclodextrin chiral stationary phase containing nitrogen linker have strong limitation in aqueous mobile phase. In order to improve the stability of the stationary phase, researchers have used ether linkage groups that are not easily hydrolyzed to prepare chiral stationary phases. Currently, most commercial cyclodextrin stationary phases are also prepared via these ether linkage groups. However, such chiral columns are only suitable for the resolution of enantiomers in reversed phase mode and thus have some limitations in application range.
In recent years, radical polymerization has been widely used because of its mild reaction conditions, and because of its low necessity of strictly purifying monomers and solvents, it is possible to obtain polymers having a large molecular weight. Researchers have prepared a series of cyclodextrin chiral stationary phases by grafting cyclodextrin derivatives onto functionalized silica microspheres by a free radical polymerization method. These immobilizates have a very good resolution effect on drugs, herbicides and carboxylic acid-containing compounds in the forward mode, and are more stable in many solvents than carbamate-based immobilizates. However, in the process of synthesizing these cyclodextrin chiral stationary phases by classical radical polymerization, the inherent slow initiation and easy termination elementary reactions determine the relative molecular mass of the obtained stationary phases and the distribution, composition structure and the like of the stationary phases to be uncontrollable. Therefore, in order to improve the reaction activity and obtain the chiral stationary phase with a specific structure, the controllable/active free radical polymerization technology is introduced into the preparation of the chiral stationary phase. Wang et al first polymerized glycidyl methacrylate on silica microspheres via Atom Transfer Radical (ATRP) polymerization to obtain a "comb" cyclodextrin chiral stationary phase. The ATRP method improves the reaction activity, but some vinyl monomers containing carboxyl, hydroxyl, amido or halogen are difficult to be directly polymerized by the ATRP method, the preparation process is complicated by adopting a method of protecting groups generally, and in addition, the ATRP method generally uses a transition metal compound as a catalyst and is difficult to remove in polymers, so that the application of the ATRP method in chiral stationary phase synthesis is limited. Therefore, there is still a need to explore an effective synthesis method for synthesizing a beta-cyclodextrin chiral stationary phase.
Recently, a reversible addition fragmentation chain transfer polymerization (RAFT) method has attracted attention because of its controllable activity, wide monomer application range and mild reaction conditions. RAFT polymerization avoids the defect that transition metal catalysts, organic ligands and the like are difficult to remove from polymerization products in an atom transfer radical polymerization method, and solves the polymerization limitation of monomers with alkene functional groups. The chiral stationary phase prepared by the RAFT method can introduce high-density functional groups and proper spacer arms, and can ensure that the polymer hybrid material is in a monodisperse state. Therefore, the preparation of the cyclodextrin chiral stationary phase on the surface of the monodisperse poly (glycidyl methacrylate) microspheres by a reversible addition-fragmentation chain transfer (RAFT) polymerization method by utilizing the characteristic of high specific surface area of the polymer microspheres can become an effective method for synthesizing the chiral stationary phase.
The invention content is as follows:
the invention aims to provide a monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase, a preparation method and application thereof, which can be used as a stationary phase of high performance liquid chromatography to efficiently and quickly separate enantiomers.
The invention provides a monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase, which is prepared by introducing 3-chloropropyl to the surface of a monodisperse poly (glycidyl methacrylate) microsphere serving as a substrate, reacting with sodium diethyldithiocarbamate, and adding a beta-cyclodextrin functional monomer to perform reversible addition fragmentation chain transfer polymerization to form the monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase (beta-CD-CSP), wherein the structural formula of the monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase is shown in figure 1;
the beta-cyclodextrin functional monomer is (methacryloyl) holophenylcarbamoyl beta-cyclodextrin.
Preferably, the particle size of the monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase is 6.5 mu m, the pore diameter is 18nm, and the specific surface area is 180.6m2/g。
The invention provides a preparation method of the monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase, which comprises the following steps (the preparation method is schematically shown in figure 2):
(1) preparation of seed liquid of monodisperse poly (glycidyl methacrylate): ultrasonically dispersing 5-10mL of glycidyl methacrylate, 0.1-0.2g of azobisisobutyronitrile and 1.0-2.0g of polyvinylpyrrolidone in 60-90mL of absolute ethyl alcohol, heating to 50-70 ℃ under the protection of nitrogen for polymerization reaction for 16-36h, fully washing and centrifuging a product by using the absolute ethyl alcohol, adding 30-60mL of sodium dodecyl sulfate solution, and standing to obtain a seed solution of monodisperse glycidyl methacrylate;
(2) monodisperse poly (epoxypropyl methacrylate)Preparing microspheres: respectively taking 2-6mL of glycidyl methacrylate, 2-6mL of ethylene glycol dimethacrylate, 2-6mL of toluene, 2-6mL of cyclohexanol and 0.2-0.36g of azobisisobutyronitrile, ultrasonically dispersing uniformly, adding 20-35mL of 5% (w/v) polyvinyl alcohol, 45-75mL of 0.2% (w/v) sodium dodecyl sulfate and 25-45mL of distilled water, mixing uniformly, placing in a cell crusher for 30-60min until the solution is in an emulsion state, adding 5-9mL of seed liquid of the poly (glycidyl methacrylate) obtained in the step (1), swelling and reacting for 16-48h at the temperature of 25-35 ℃, heating to 50-70 ℃ under the protection of nitrogen, reacting for 18-36h, washing with absolute ethyl alcohol and distilled water after the reaction is finished, performing Soxhlet extraction on the obtained product in tetrahydrofuran for 48-60h, washing with anhydrous ethanol, and vacuum drying to obtain monodisperse poly (glycidyl methacrylate) microsphere (P)GMA/EDMA)；
(3) Surface modification of disulfide bond of monodisperse poly (glycidyl methacrylate) microsphere: taking 2.0-6.0g of the monodisperse poly (glycidyl methacrylate) microspheres obtained in the step (2) to disperse in 100-300 mL0.1mol.L-1H of (A) to (B)2SO4Reacting in the solution at 40-80 ℃ for 6-20h to obtain hydrolyzed poly (glycidyl methacrylate) microspheres, dispersing 2.0-5.0g of the hydrolyzed poly (glycidyl methacrylate) microspheres in 80-150mL of anhydrous toluene under ultrasonic, dropwise adding 4-10mL of 3-chloropropyltrimethoxysilane, refluxing in 120 deg.C oil bath for 24-48h under nitrogen protection, washing the product with anhydrous ethanol, vacuum drying to obtain chloropropyl functional poly (glycidyl methacrylate) microsphere, ultrasonically dispersing 1.0-3.0g sodium diethyldithiocarbamate in 50-120mL anhydrous tetrahydrofuran, adding 1.0-4.0g of chloropropyl functionalized poly (glycidyl methacrylate) microspheres, reacting for 12-48h at 40-80 ℃ under the protection of nitrogen, and washing products with tetrahydrofuran and methanol in sequence;
(4) preparing a beta-cyclodextrin functional monomer: dissolving 1.0-3.0g of dried beta-cyclodextrin in 50-100mL of dried pyridine, dropwise adding 3-9mL of dried tetrahydrofuran, stirring, adding 3-15mL of phenyl isocyanate, reacting in an oil bath at 80-120 ℃ for 10-24h, removing pyridine and unreacted phenyl isocyanate in a reaction solution under a reduced pressure condition after the reaction is finished, dissolving the residue with ethanol, pouring the dissolved residue into 250mL of water, separating out a solid, filtering, and recrystallizing a filter cake with ethanol to obtain an earthy yellow beta-cyclodextrin functional monomer;
(5) preparing a monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase: taking 1.0-3.0g of disulfide bond modified poly (glycidyl methacrylate) microspheres obtained in the step (3), adding 40-120mL of dry tetrahydrofuran, performing ultrasonic dispersion, adding 0.2-2.5g of beta-cyclodextrin functional monomer obtained in the step (4) and 10-30mg of azobisisobutyronitrile, performing ultrasonic dispersion, performing oil bath reaction at 50-70 ℃ for 12-36h under the protection of nitrogen, washing with tetrahydrofuran and methanol in sequence, and performing vacuum drying to obtain a monodisperse beta-cyclodextrin functional polymer microsphere chiral stationary phase;
the dry tetrahydrofuran in the step (4) contains 0.170-0.85mL of methacryloyl chloride.
Preferably, the standing time in the step (1) is 10-24 h; the vacuum drying condition in the step (2) is that the drying is carried out in a vacuum drying oven at the temperature of 30-60 ℃ for 12-24 h; the vacuum drying condition in the step (3) is that the drying is carried out in a vacuum drying oven at the temperature of 40-80 ℃ for 6-24 h; the stirring in the step (4) is carried out for 10-20h at the temperature of 20-60 ℃; the vacuum drying condition in the step (5) is drying in a vacuum drying oven at 50 ℃ for 24 hours.
Preferably, the time for ultrasonic dispersion in the step (5) is 10-40 min.
The preparation method of the monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase comprises the following specific steps:
(1) preparation of seed liquid of monodisperse poly (glycidyl methacrylate): ultrasonically dispersing 5-10mL of glycidyl methacrylate, 0.1-0.2g of azobisisobutyronitrile and 1.0-2.0g of polyvinylpyrrolidone in 60-90mL of absolute ethyl alcohol, placing on a rotary evaporator, heating to 50-70 ℃ under the protection of nitrogen for polymerization reaction for 16-36h, fully washing and centrifuging a product by using the absolute ethyl alcohol, adding 30-60mL of lauryl sodium sulfate solution, placing in a refrigerator, standing and precipitating for 10-24h to obtain seed liquid of monodisperse glycidyl methacrylate;
(2) preparing monodisperse poly (glycidyl methacrylate) microspheres: respectively taking 2-6mL of glycidyl methacrylate, 2-6mL of ethylene glycol dimethacrylate, 2-6mL of toluene, 2-6mL of cyclohexanol and 0.2-0.36g of azobisisobutyronitrile, ultrasonically dispersing uniformly, adding 20-35mL of 5% (w/v) polyvinyl alcohol, 45-75mL of 0.2% (w/v) sodium dodecyl sulfate and 25-45mL of distilled water, mixing uniformly, putting into a cell crusher, working for 30-60min until the solution is in an emulsion state, adding 5-9mL of seed solution of polypropylene methacrylate, swelling and reacting for 16-48h at 25-35 ℃, heating to 50-70 ℃ under the protection of nitrogen, reacting for 18-36h, washing with absolute ethyl alcohol and distilled water after the reaction is finished, soxhlet extracting the obtained product in tetrahydrofuran for 48-60h, washing with absolute ethyl alcohol, and drying in a vacuum drying oven at 30-60 ℃ for 12-24h to obtain monodisperse poly (glycidyl methacrylate) microspheres;
(3) surface modification of disulfide bond of monodisperse poly (glycidyl methacrylate) microsphere: taking 2.0-6.0g of the monodisperse poly (glycidyl methacrylate) microspheres obtained in the step (2) to disperse in 100-300 mL0.1mol.L-1H of (A) to (B)2SO4Reacting in the solution at 40-80 ℃ for 6-20h to obtain hydrolyzed poly (glycidyl methacrylate) microspheres, ultrasonically dispersing 2.0-5.0g of the hydrolyzed poly (glycidyl methacrylate) microspheres in 80-150mL of anhydrous toluene, dropwise adding 4-10mL of 3-chloropropyltrimethoxysilane (NQ-54), refluxing in an oil bath at 120 ℃ for 24-48h under the protection of nitrogen, washing the product with anhydrous ethanol, drying in a vacuum drying oven at 40-80 ℃ for 6-24h to obtain chloropropyl functionalized poly (glycidyl methacrylate) microspheres, ultrasonically dispersing 1.0-3.0g of sodium diethyldithiocarbamate in 50-120mL of anhydrous tetrahydrofuran, adding 1.0-4.0g of chloropropyl functionalized poly (glycidyl methacrylate) microspheres, reacting at 40-80 ℃ for 12-48h under the protection of nitrogen, washing the product with tetrahydrofuran and methanol in turn;
(4) preparing a beta-cyclodextrin functional monomer: dissolving 1.0-3.0g of dried beta-cyclodextrin in 50-100mL of dried pyridine, dropwise adding 3-9mL of dried tetrahydrofuran (containing 0.170-0.85mL of methacryloyl chloride), stirring at 20-60 ℃ for 10-20h, adding 3-15mL of phenyl isocyanate, reacting in an oil bath at 80-120 ℃ for 10-24h, removing pyridine and unreacted phenyl isocyanate in the reaction solution under the condition of reduced pressure after the reaction is finished, dissolving the residue with ethanol, pouring the solution into a beaker filled with 250mL of water, separating out a large amount of solids, filtering, and recrystallizing a filter cake with ethanol to obtain an earthy yellow beta-cyclodextrin functional monomer;
(5) preparing a monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase: and (3) taking 1.0-3.0g of disulfide bond modified poly (glycidyl methacrylate) microspheres obtained in the step (3), adding 40-120mL of dry tetrahydrofuran, performing ultrasonic dispersion for 10-40min, then adding 0.2-2.5g of beta-cyclodextrin functional monomers obtained in the step (4) and 10-30mg of azodiisobutyronitrile, performing ultrasonic dispersion for 10-40min, performing oil bath reaction for 12-36h at 50-70 ℃ under the protection of nitrogen, washing with tetrahydrofuran and methanol in sequence, and drying in a vacuum drying oven at 50 ℃ for 24h to obtain the monodisperse beta-cyclodextrin functional polymer microsphere chiral stationary phase.
The invention also provides an application of the monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase, and the monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase can be used for chiral separation of enantiomers.
Specifically, the filling method of the monodisperse β -cyclodextrin functionalized polymer microsphere chiral stationary phase in a high performance liquid chromatography column is as follows:
taking 2-4g of monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase, adding 20-40mL of methanol into the monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase, ultrasonically dispersing for 20-60min, placing the obtained chiral stationary phase homogenate into a homogenate tank, and pressing the homogenate into a high performance liquid chromatography column tube through a pneumatic pump under the condition that the methanol is used as a displacement liquid, wherein the specification of the chromatography column is 150 multiplied by 4.6mm (I.D.), and the filling pressure is 15-30 MPa.
The invention has the beneficial effects that:
(1) the invention provides a monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase and a preparation method thereof.
(2) The monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase provided by the invention can be used as a stationary phase of high performance liquid chromatography, and can efficiently and quickly complete the separation of enantiomers. The alkyl chain in the methacrylated beta-cyclodextrin provides proper spacer arm length, so that the density of the functional monomer grafted on the surface of the poly (glycidyl methacrylate) microsphere is high, and enantiomer molecules can easily enter the functionalized cyclodextrin molecules, thereby being beneficial to realizing chiral resolution. In addition, the phenyl carbamate on the cyclodextrin rim expands the cavity of the cyclodextrin stationary phase, which facilitates the formation of inclusion complexes with various types of enantiomer compounds in the reversed phase mode and improves pi-pi interactions, dipole-dipole interactions, hydrogen bonds and hydrophobic interactions with the enantiomers in the normal phase mode.
Drawings
FIG. 1 is a schematic diagram of the structure of a beta-CD-CSP.
Fig. 2 is a schematic diagram of the preparation of a monodisperse magnetic chiral stationary phase.
FIG. 3 is an infrared spectrum of a material: (A) hydrolysis of PGMA/EDMA(B) chloropropyl-functionalized PGMA/EDMA(C) disulfide bond modified PGMA/EDMA，(D)β-CD-CSP。
FIG. 4 is a scanning electron micrograph, in which (A) is PGMA/EDMAAnd (B) is beta-CD-CSP.
FIG. 5 shows the structural formulae of enantiomeric compound (A) (+/-) -1-phenyl-1-propanol, (B) (+/-) -2-phenylpropionic acid, (C) (+/-) -styrene oxide and (D) ibuprofen.
FIG. 6 is a chromatogram of the separation of (A) (+/-) -1-phenyl-1-propanol, (B) (+/-) -2-phenylpropionic acid, (C) (+/-) -styrene oxide and (D) ibuprofen under reverse phase chromatographic conditions.
Detailed Description
In order to make the technical means, the creation features, the achievement objects and the effects of the invention easy to understand, the invention is further explained with the specific embodiments, but the embodiments are only the preferred embodiments of the invention, and not all. Based on the embodiments in the implementation, other embodiments obtained by those skilled in the art without any creative efforts belong to the protection scope of the present invention.
The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
The preparation method of the monodisperse magnetic chiral stationary phase comprises the following specific steps:
the preparation method of the monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase comprises the following specific steps:
(1) preparation of seed liquid of monodisperse poly (glycidyl methacrylate): ultrasonically dispersing 5mL of glycidyl methacrylate, 0.1g of azodiisobutyronitrile and 1.0g of polyvinylpyrrolidone in 60mL of absolute ethyl alcohol, placing on a rotary evaporator, heating to 50 ℃ under the protection of nitrogen for polymerization reaction for 16h, fully washing and centrifuging a product by using the absolute ethyl alcohol, adding 30mL of a sodium dodecyl sulfate solution, placing in a refrigerator, standing and precipitating for 10h to obtain a seed solution of monodisperse poly (glycidyl methacrylate);
(2) preparing monodisperse poly (glycidyl methacrylate) microspheres: respectively taking 2mL of glycidyl methacrylate, 2mL of ethylene glycol dimethacrylate, 2mL of toluene, 2mL of cyclohexanol and 0.2g of azobisisobutyronitrile, ultrasonically dispersing uniformly, adding 20mL of 5% (w/v) polyvinyl alcohol, 45mL of 0.2% (w/v) sodium dodecyl sulfate and 25mL of distilled water, uniformly mixing, putting into a cell disruptor, working for 30min until the solution is in an emulsion state, adding 5mL of seed solution of polypropylene methacrylate, carrying out swelling reaction for 16h at 25 ℃, heating to 70 ℃ under the protection of nitrogen, reacting for 18h, washing with absolute ethyl alcohol and distilled water after the reaction is finished, carrying out Soxhlet extraction on the obtained product in tetrahydrofuran for 48h, washing with absolute ethyl alcohol, and drying in a vacuum drying oven at 30 ℃ for 24h to obtain monodisperse polypropylene methacrylate microspheres;
(3) surface modification of disulfide bond of monodisperse poly (glycidyl methacrylate) microsphere: taking 2.0g of the monodisperse poly (glycidyl methacrylate) microspheres obtained in the step (2) to disperse in 100 mL0.1mol.L-1H of (A) to (B)2SO4Reacting in the solution at 40 deg.C for 6h to obtain hydrolyzed poly (glycidyl methacrylate) microspheres, and dispersing 2.0g hydrolyzed poly (glycidyl methacrylate) microspheres in 80m under ultrasonicDropwise adding 4mL of 3-chloropropyl trimethoxyl silane into L anhydrous toluene, refluxing in an oil bath at 120 ℃ for 24 hours under the protection of nitrogen, washing a product with absolute ethyl alcohol, drying in a vacuum drying oven at 40 ℃ for 48 hours to obtain chloropropyl functionalized polypropylene oxide methacrylate microspheres, ultrasonically dispersing 1.0g of sodium diethyldithiocarbamate into 50mL of anhydrous tetrahydrofuran, adding 1.0g of chloropropyl functionalized polypropylene oxide methacrylate microspheres, reacting at 40 ℃ for 12 hours under the protection of nitrogen, and sequentially washing the product with tetrahydrofuran and methanol;
(4) preparing a beta-cyclodextrin functional monomer: dissolving 1.0g of dried beta-cyclodextrin in 50mL of dried pyridine, dropwise adding 3mL of dried tetrahydrofuran (containing 0.170mL of methacryloyl chloride), stirring at 20 ℃ for 10h, adding 3mL of phenyl isocyanate, reacting in an oil bath at 80 ℃ for 10h, removing pyridine and unreacted phenyl isocyanate in the reaction solution under the condition of reduced pressure after the reaction is finished, dissolving the residue with ethanol, pouring the solution into a beaker filled with 250mL of water, separating out a large amount of solids, filtering, and recrystallizing a filter cake with ethanol to obtain earthy yellow (methacryloyl) holophenylcarbamoyl beta-cyclodextrin, namely a beta-cyclodextrin functional monomer;
(5) preparing a monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase: and (3) taking 1.0g of disulfide bond modified poly (glycidyl methacrylate) microspheres obtained in the step (3), adding 40mL of dry tetrahydrofuran, performing ultrasonic dispersion, adding 0.2g of the beta-cyclodextrin functional monomer obtained in the step (4) and 10mg of azodiisobutyronitrile, performing ultrasonic dispersion uniformly, performing oil bath reaction at 50 ℃ for 12h under the protection of nitrogen, washing with tetrahydrofuran and methanol in sequence, and drying in a vacuum drying oven at 50 ℃ for 24h to obtain the monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase (beta-CD-CSP).
The filling method of the monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase in the high performance liquid chromatography column comprises the following steps:
taking 2g of beta-CD-CSP, adding 20mL of methanol into the beta-CD-CSP, performing ultrasonic dispersion for 20min, placing the obtained chiral stationary phase homogenate into a homogenate tank, and pressing the homogenate into a high performance liquid chromatography column tube by a pneumatic pump under the condition that the methanol is used as a displacement liquid, wherein the specification of the chromatography column is 150 multiplied by 4.6mm (I.D.), and the filling pressure is 15 MPa.
Example 2
The preparation method of the monodisperse magnetic chiral stationary phase comprises the following specific steps:
the preparation method of the monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase comprises the following specific steps:
(1) preparation of seed liquid of monodisperse poly (glycidyl methacrylate): ultrasonically dispersing 8mL of glycidyl methacrylate, 0.15g of azodiisobutyronitrile and 1.5g of polyvinylpyrrolidone in 75mL of absolute ethyl alcohol, placing on a rotary evaporator, heating to 60 ℃ under the protection of nitrogen, carrying out polymerization reaction for 24 hours, fully washing and centrifuging a product by using the absolute ethyl alcohol, adding 45mL of a sodium dodecyl sulfate solution, placing in a refrigerator, standing, and precipitating for 18 hours to obtain a seed solution of monodisperse poly (glycidyl methacrylate);
(2) preparing monodisperse poly (glycidyl methacrylate) microspheres: respectively taking 4mL of glycidyl methacrylate, 4mL of ethylene glycol dimethacrylate, 4mL of toluene, 4mL of cyclohexanol and 0.28g of azobisisobutyronitrile, ultrasonically dispersing uniformly, adding 30mL of 5% (w/v) polyvinyl alcohol, 60mL of 0.2% (w/v) sodium dodecyl sulfate and 35mL of distilled water, uniformly mixing, putting into a cell disruptor, working for 45min until the solution is in an emulsion state, adding 7.5mL of seed solution of glycidyl methacrylate, swelling and reacting for 24h at 30 ℃, heating to 60 ℃ under the protection of nitrogen, reacting for 24h, washing with absolute ethyl alcohol and distilled water after the reaction is finished, Soxhlet extracting the obtained product in tetrahydrofuran for 55h, washing with absolute ethyl alcohol, and drying in a vacuum drying oven at 50 ℃ for 20h to obtain monodisperse glycidyl methacrylate microspheres;
(3) surface modification of disulfide bond of monodisperse poly (glycidyl methacrylate) microsphere: 4.0g of monodisperse poly (glycidyl methacrylate) microspheres obtained in the step (2) are dispersed in 200 mL0.1mol.L-1H of (A) to (B)2SO4Reacting in the solution at 60 ℃ for 12h to obtain hydrolyzed poly (glycidyl methacrylate) microspheres, dispersing 3.5g of the hydrolyzed poly (glycidyl methacrylate) microspheres in 120mL of anhydrous toluene under ultrasonic treatment, and dropwise adding 7mL of 3-chloropropyltrimethoxysilaneRefluxing in an oil bath at 120 ℃ for 45 hours under the protection of nitrogen, washing the product with absolute ethyl alcohol, drying in a vacuum drying oven at 60 ℃ for 12 hours to obtain chloropropyl functional poly (glycidyl methacrylate) microspheres, ultrasonically dispersing 2.0g of sodium diethyldithiocarbamate in 90mL of anhydrous tetrahydrofuran, adding 2.5g of chloropropyl functional poly (glycidyl methacrylate) microspheres, reacting at 60 ℃ for 24 hours under the protection of nitrogen, and sequentially washing the product with tetrahydrofuran and methanol;
(4) preparing a beta-cyclodextrin functional monomer: dissolving 2.0g of dried beta-cyclodextrin in 80mL of dried pyridine, dropwise adding 6mL of dried tetrahydrofuran (containing 0.520mL of methacryloyl chloride), stirring at 40 ℃ for 15h, adding 9mL of phenyl isocyanate, reacting in a 100 ℃ oil bath for 18h, removing pyridine and unreacted phenyl isocyanate in a reaction solution under a reduced pressure condition after the reaction is finished, dissolving the residue with ethanol, pouring the residue into a beaker containing 250mL of water, separating out a large amount of solids, filtering, and recrystallizing a filter cake with ethanol to obtain earthy yellow (methacryloyl) holophenylcarbamoyl beta-cyclodextrin, namely a beta-cyclodextrin functional monomer;
(5) preparing a monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase: and (3) taking 2.0g of disulfide bond modified poly (glycidyl methacrylate) microspheres obtained in the step (3), adding 80mL of dry tetrahydrofuran, performing ultrasonic dispersion for 20min, adding 1.5g of the beta-cyclodextrin functional monomer obtained in the step (4) and 20mg of azodiisobutyronitrile, performing ultrasonic dispersion for 20min, uniformly dispersing, performing oil bath reaction for 24h at 60 ℃ under the protection of nitrogen, washing with tetrahydrofuran and methanol in sequence, and drying in a vacuum drying oven at 50 ℃ for 24h to obtain the monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase (beta-CD-CSP).
The filling method of the monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase in the high performance liquid chromatography column comprises the following steps:
taking 2.5g of beta-CD-CSP, adding 30mL of methanol into the beta-CD-CSP, performing ultrasonic dispersion for 40min, loading the obtained chiral stationary phase homogenate into a homogenate tank, and pressing the homogenate into a high performance liquid chromatography column tube by a pneumatic pump under the condition that the methanol is used as a displacement liquid, wherein the specification of the chromatography column is 150 multiplied by 4.6mm (I.D.), and the filling pressure is 25 MPa.
Example 3
(1) Preparation of seed liquid of monodisperse poly (glycidyl methacrylate): ultrasonically dispersing 10mL of glycidyl methacrylate, 0.2g of azodiisobutyronitrile and 2.0g of polyvinylpyrrolidone in 90mL of absolute ethyl alcohol, placing on a rotary evaporator, heating to 70 ℃ under the protection of nitrogen, carrying out polymerization reaction for 36h, fully washing and centrifuging a product by using the absolute ethyl alcohol, adding a sodium dodecyl sulfate solution, placing in a refrigerator, standing, and precipitating for 24h to obtain a seed solution of monodisperse poly (glycidyl methacrylate);
(2) preparing monodisperse poly (glycidyl methacrylate) microspheres: respectively taking 6mL of glycidyl methacrylate, 6mL of ethylene glycol dimethacrylate, 6mL of toluene, 6mL of cyclohexanol and 0.36g of azobisisobutyronitrile, ultrasonically dispersing uniformly, adding 35mL of 5% (w/v) polyvinyl alcohol, 75mL of 0.2% (w/v) sodium dodecyl sulfate and 45mL of distilled water, uniformly mixing, putting into a cell disrupter, working for 60min until the solution is in an emulsion state, adding 9mL of seed solution of glycidyl methacrylate, swelling and reacting for 48h at 35 ℃, heating to 50 ℃ under the protection of nitrogen, reacting for 36h, washing with absolute ethyl alcohol and distilled water after the reaction is finished, Soxhlet extracting the obtained product in tetrahydrofuran for 60h, washing with absolute ethyl alcohol, and drying in a vacuum drying oven at 60 ℃ for 12h to obtain monodisperse glycidyl methacrylate microspheres;
(3) surface modification of disulfide bond of monodisperse poly (glycidyl methacrylate) microsphere: taking 6.0g of the monodisperse poly (glycidyl methacrylate) microspheres obtained in the step (2) to disperse in 300 mL0.1mol.L-1H of (A) to (B)2SO4Reacting in a solution at 80 ℃ for 20 hours to obtain hydrolyzed poly (glycidyl methacrylate) microspheres, ultrasonically dispersing 5.0g of the hydrolyzed poly (glycidyl methacrylate) microspheres in 150mL of anhydrous toluene, dropwise adding 10mL of 3-chloropropyltrimethoxysilane, refluxing in an oil bath at 120 ℃ for 48 hours under the protection of nitrogen, washing the product with anhydrous ethanol, drying in a vacuum drying oven at 80 ℃ for 24 hours to obtain chloropropyl functionalized poly (glycidyl methacrylate) microspheres, ultrasonically dispersing 3.0g of sodium diethyldithiocarbamate in 120mL of anhydrous tetrahydrofuran, and adding 4.0g of chloropropylReacting the functionalized poly (glycidyl methacrylate) microspheres for 48 hours at 80 ℃ under the protection of nitrogen, and sequentially washing products by using tetrahydrofuran and methanol;
(4) preparing a beta-cyclodextrin functional monomer: dissolving 3.0g of dried beta-cyclodextrin in 100mL of dried pyridine, dropwise adding 9mL of dried tetrahydrofuran (containing 0.850mL of methacryloyl chloride), stirring at 60 ℃ for 20h, adding 15mL of phenyl isocyanate, reacting in an oil bath at 120 ℃ for 24h, removing pyridine and unreacted phenyl isocyanate in the reaction solution under the condition of reduced pressure after the reaction is finished, dissolving the residue with ethanol, pouring the residue into a beaker containing 250mL of water, separating out a large amount of solids, filtering, and recrystallizing the filter cake with ethanol to obtain earthy yellow (methacryloyl) holophenylcarbamoyl beta-cyclodextrin, namely the beta-cyclodextrin functional monomer;
(5) preparing a monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase: and (3) taking 3.0g of disulfide bond modified poly (glycidyl methacrylate) microspheres obtained in the step (3), adding 120mL of dry tetrahydrofuran, performing ultrasonic dispersion for 40min, adding 2.5g of the beta-cyclodextrin functional monomer obtained in the step (4), 30mg of azobisisobutyronitrile, performing ultrasonic dispersion for 40min, performing oil bath reaction for 36h at 70 ℃ under the protection of nitrogen, washing with tetrahydrofuran and methanol in sequence, and drying in a vacuum drying oven at 50 ℃ for 24h to obtain the monodisperse beta-cyclodextrin functional polymer microsphere chiral stationary phase (beta-CD-CSP).
The filling method of the monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase in the high performance liquid chromatography column comprises the following steps:
taking 4g of beta-CD-CSP, adding 40mL of methanol into the beta-CD-CSP, performing ultrasonic dispersion for 60min, loading the obtained chiral stationary phase homogenate into a homogenate tank, and pressing the homogenate into a high performance liquid chromatography column tube by a pneumatic pump under the condition that the methanol is used as a displacement liquid, wherein the specification of the chromatography column is 150 multiplied by 4.6mm (I.D.), and the filling pressure is 30 MPa.
Example 4
Taking the beta-CD-CSP prepared in example 2 as an example, the detection of the chiral stationary phase of the monodisperse beta-cyclodextrin functionalized polymer microsphere is carried out:
2.1 detection by infrared spectroscopy, the results are shown in FIG. 3: at 1490-1372cm-1Both have-COO-symmetrical and-asymmetrical vibration absorption peaks, which shows that P is hydrolyzedGMA/EDMA(FIG. 3(A)), chloropropyl functionalized PGMA/EDMA (FIG. 3(B)), disulfide modified PGMA/EDMA (FIG. 3(C)) and beta-CD-CSP (FIG. 3(D)) have the same core (P)GMA/EDMA). 3525cm in curve A-1Has a broad absorption peak of PGMA/EDMAStretching vibration peak of-OH after hydrolysis. P functionalized in comparison with chloropropylGMA/EDMAAt 2958cm-1Nearby occurrence of-CH3and-CH2The absorption peak of the internal C-H bond is 1062cm-1A stretching vibration peak of Si-O appears at the position, and the length is 653cm-1The absorption peak at C-Cl bond was enhanced while 3525cm-1the-OH absorption peak is weakened, and these results show that chloropropyl is successfully grafted on PGMA/EDMAOf (2) is provided. Curve C, C-S and C ═ S at 619cm respectively-1And 1060cm-1And a new absorption peak appears, which indicates that the disulfide bond grafting is successful. In the FT-IR spectrum of beta-CD-CSP, the newly appeared spectrum was 1648cm-1Belongs to the vibration absorption peak of C ═ O, and the appearance of the characteristic peak indicates that the functionalized beta-cyclodextrin monomer is successfully grafted to the surface of the polymer microsphere.
2.2 Observation of beta-CD-CSP by transmission electron microscopy, the results are shown in FIG. 4: polyepoxypropyl methacrylate (P)GMA/EDMA) The microspheres (FIG. 4(A)) are monodisperse, porous on the surface and uniformly distributed, and have a particle size of about 5.0. mu.m. The mean diameter of the beta-CD-CSP (FIG. 4(B)) was about 6.5. mu.m. And PGMA/EDMACompared with microspheres, the surface porous structure of the beta-CD-CSP is more uniform, the surface aperture is reduced, the particle size is obviously increased, and the successful grafting of the functionalized beta-cyclodextrin monomer to the P is provedGMA/EDMAThe surface of the microsphere.
2.3 preparation of mobile phase and sample: adjusting the pH of the mobile phase with triethylamine acetate buffer (volume ratio of triethylamine to water is 1:1000), ultrasonically treating acetonitrile and buffer mobile phase with an ultrasonic instrument for 30min before use, wherein the sample is prepared into a solution of 3mg/mL with methanol, and the samples are respectively (A) (+/-) -1-phenyl-1-propanol, (B) (+/-) -2-phenylpropionic acid, (C) (+/-) -styrene oxide and (D) ibuprofen, and the structural formula is shown in FIG. 5.
Reverse phase chromatography conditions: mobile phase: (A) acetonitrile/0.1% acetic acid (35:65, v/v); (B) acetonitrile/0.1% acetic acid (77:23, v/v); (C) acetonitrile/20 mmol ammonium acetate buffer solution (pH 3.119) (95:5, v/v); (D) acetonitrile/20 mmol ammonium acetate buffer solution (pH 3.106) (97:3, v/v); flow rate: 1.0 mL/min; sample introduction amount: 10 mu L of the solution; ultraviolet detection wavelength: 254 nm.
The chromatogram for the enantiomeric separation under reverse phase chromatography conditions is shown in fig. 6, and the separation results are shown in table 1. Wherein the retention factor (k) is represented by the formula k ═ tR–t0)/t0Calculating where t isRRepresents the sample retention time, t0Representing dead time. The separation factor (alpha) is represented by k2/k1Calculated and the degree of separation (Rs) is given by the formula Rs ═ 2 (t)R2–tR1)/(W1+W2) Calculating where t isR2And tR1Respectively represents the retention times of the second optically pure isomer and the first optically pure isomer, W1And W2Respectively, the widths of the bottoms of the chromatographic peaks.
Table 1: separation of enantiomers under reverse phase conditions:
TABLE 1 separation of enantiomers under reversed phase conditions
As can be seen from fig. 6 and the results in table 1, the separation effect of 4 enantiomeric compounds on the monodisperse β -cyclodextrin functionalized polymer microsphere chiral stationary phase prepared in example 2 of the present invention is good, and the speed is high; in addition, 4 kinds of enantiomer compounds are separated by using the monodisperse beta-cyclodextrin functionalized polymer microsphere chiral stationary phase prepared by other embodiments of the invention, and the separation effect is good and the speed is high.
The foregoing is a preferred embodiment of the present invention, and is not intended to limit the invention in any way, so that modifications and equivalents may be made thereto without departing from the spirit and scope of the invention.