A packing for a chromatographic column for separating glycated hemoglobin and a method for preparing the same

Abstract

This application relates to the field of packing technology, and discloses a packing material for a chromatography column used to separate glycated hemoglobin and its preparation method. The invention uses hydrophobic monomers to synthesize 5 μm non-porous polymer-based spheres via dispersion polymerization, followed by hydrophilication treatment, and further hydrophilication to increase the linker length. Simultaneously, ion exchange groups are introduced to obtain a weak cation exchange packing material with an average particle size of 5 μm. This invention, while reducing non-specific adsorption through hydrophilication treatment without affecting the swelling and mechanical strength of the packing material in water, further improves the hydrophilicity of the microspheres by increasing the linker length. This results in improved salt tolerance of the hydrophilicated polymer and a significant reduction in non-specific adsorption. Furthermore, the analytical packing material prepared by this invention has uniform particle size, good separation effect, and long service life; in particular, the separation degree is significantly improved after further hydrophilication.

Description

Technical Field

[0001] This invention belongs to the field of packing technology, and particularly relates to a packing material for a chromatography column for separating glycated hemoglobin and its preparation method. Background Technology

[0002] Glycated hemoglobin (HbA1c) is a type of hemoglobin that reflects the average glucose concentration in the blood over the past 1-2 months. Therefore, it is commonly used in diabetes screening tests and to assess the blood glucose control status of diabetic patients. High-performance liquid chromatography (HPLC) is widely used due to its high accuracy and ability to detect HbA1c in a short time.

[0003] In the high-performance liquid chromatography (HPLC) process for detecting glycated hemoglobin, the packing matrix needs to have a certain degree of hydrophobicity to reduce swelling in water. However, excessive hydrophobicity can lead to poor packing dispersion and non-specific adsorption. A common method to improve the hydrophilicity of the packing is to introduce a large amount of hydrophilic monomers into the synthesis of the substrate spheres. However, this can lead to a decrease in the mechanical strength of the packing and swelling in water, thus reducing accuracy.

[0004] Previous column chromatography packing materials for determining glycated hemoglobin were mostly synthesized using suspension polymerization, which resulted in uneven particle size distribution. To achieve uniform particle size distribution, fractionation methods were typically employed. However, fractionation is complex, leads to significant product loss, and current fractionation techniques have limitations in adjusting particle size distribution width, failing to adequately suppress peak broadening caused by sample diffusion due to uneven particle size distribution. Therefore, there is an urgent need for a new analytical packing material for column chromatography used to separate glycated hemoglobin, along with its preparation method, to overcome these problems. Summary of the Invention

[0005] The present invention aims to provide a packing material for a chromatography column for separating glycated hemoglobin and a method for preparing the same. The analytical packing material has good durability, can efficiently and stably detect glycated hemoglobin, and can achieve the separation of hemoglobin.

[0006] To achieve the above objectives, the present invention provides a method for preparing packing material for a chromatography column used for separating glycated hemoglobin, comprising:

[0007] Step S1: Mix and emulsify the monomer, crosslinking agent, initiator and polyvinyl alcohol aqueous solution to obtain polymer seed balls;

[0008] Step S2: Mix the polymer seed spheres with the activating reagent and react to obtain polymer-based spheres;

[0009] Step S3: Mix the polymer-based spheres, allyl glycidyl ether and sodium sulfate solution, dissolve them in a solvent, stir, add sodium hydroxide solution, and react to obtain allyl functionalized polymer microspheres;

[0010] Step S4: Add allyl functionalized polymer microspheres to a solvent and carry out the first reaction in an aqueous sodium hydroxide solution. After the reaction is completed, add allyl glycidyl ether and carry out the second reaction to obtain modified polymer microspheres.

[0011] Step S5: Mix the modified polymer microspheres, ligand monomers and water, and react them in the presence of a free radical initiator or oxidant to obtain a 5 μm cation exchange packing.

[0012] Preferably, in step S1, the monomer is any one or more of methyl methacrylate monomers, ethyl methacrylate, cyclohexyl methacrylate, vinyl glycidyl ether, glycidyl methacrylate monomers, and styrene; all monomers are hydrophobic monomers, which enable the filler to not swell or hardly swell in an aqueous medium.

[0013] Preferably, in step S1, the crosslinking agent is any one or more of divinylbenzene, ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, triethylene glycol dimethacrylate phthalate, and pentaerythritol tetramethacrylate.

[0014] Preferably, in step S1, the initiator is any one or more of azobisisobutyronitrile, dimethyl azobisisobutyrate, benzoyl peroxide, and diacyl peroxide.

[0015] Preferably, in step S1, the mass ratio of monomer, crosslinking agent, initiator and polyvinyl alcohol aqueous solution is 1:(0.2~0.3):(0.01~0.02):(3~4).

[0016] Preferably, in step S1, the emulsification time is 3-5 min, and the stirring speed during emulsification is 700-1000 rpm.

[0017] Preferably, in step S1, the reaction temperature is 70~90℃ and the reaction time is 10~13 h.

[0018] Preferably, in step S1, the polymer in the polymer seed ball is one of polymethyl methacrylate (PMMA), polyglycerol methacrylate (PGMA), or polystyrene-divinylbenzene copolymer.

[0019] Preferably, in step S2, the activating reagent is any one or more of a strong acid solution, a strong base solution, and a chloromethylating reagent; the strong acid solution includes any one or more of sulfuric acid, hydrochloric acid, and nitric acid; the strong base solution includes any one or more of sodium hydroxide, potassium hydroxide, and lithium hydroxide; and the chloromethylating reagent includes one or more of chloromethyl methyl ether and chloromethyl diethyl ether.

[0020] Preferably, in step S2, the reaction conditions are: reaction temperature 25~70℃, reaction time 20~24 h, and rotation speed 160~180 rpm.

[0021] Preferably, in step S2, the mass ratio of the polymer seed pellets to the activation solvent is 1:(1.5~6).

[0022] The reaction of polymer seed beads with activating reagents introduces active functional groups onto the surface of inert polymer microspheres, providing reaction sites for subsequent grafting. Specifically, when the polymer seed beads are polymethyl methacrylate (PMMA), under alkaline conditions, the ester groups on their surface undergo hydrolysis to generate sodium carboxylate, which is then converted into free carboxyl groups after acidification, achieving surface carboxylation. When the polymer seed beads are polyglycerol methacrylate (PGMA), under acidic conditions, the epoxy groups on their surface undergo ring-opening reactions to generate chlorohydrin intermediates, which are then converted into ortho-dihydroxy structures after alkaline treatment, achieving surface hydroxylation. This activation treatment transforms the surface of the inert microspheres into a functionalized surface rich in carboxyl or hydroxyl groups, thereby providing covalent bonding sites for the subsequent grafting reaction of allyl glycidyl ether.

[0023] When the polymer seed spheres are polystyrene-divinylbenzene copolymers, the chloromethylation reaction of polystyrene-divinylbenzene copolymer microspheres with chloromethyl methyl ether follows a Friedel-Crafts alkylation mechanism. In the reaction system, chloromethyl methyl ether forms an electrophile, namely a chloromethyl cation or a close ion pair, under acidic catalysis (usually hydrogen chloride or trace amounts of formic acid produced from the decomposition of paraformaldehyde). This electrophile attacks the benzene rings on the polystyrene segments on the microsphere surface, resulting in electrophilic substitution and the introduction of chloromethyl groups onto the microsphere surface. Due to the non-porous structure of the microspheres, the reaction sites are limited to the surface, resulting in a high density of surface active sites, and adjacent benzene rings are easily attacked by the electrophile. The reaction is carried out at 50°C for 24 hours to ensure a complete and uniform surface chloromethylation reaction.

[0024] Preferably, in step S3, the stirring speed is 140~160 rpm and the stirring time is 1~2 h.

[0025] Preferably, in step S3, the reaction temperature is 20~30℃ and the reaction time is 14~16 h.

[0026] Preferably, in step S3, the solvent is a 50% dimethyl sulfoxide aqueous solution.

[0027] Preferably, in step S3, the mass ratio of the polymer-based spheres, allyl glycidyl ether, sodium sulfate, solvent, and strong alkaline solution is 1:(0.3~0.5):(0.01~0.02):(8~10):(0.5~5).

[0028] The active functional groups on the surface of activated polymer microspheres undergo ring-opening or nucleophilic substitution reactions with the epoxy groups of allyl glycidyl ether, achieving covalent grafting of allyl groups onto the microsphere surface. Specifically, when the microsphere surface is carboxyl, the carboxyl group and epoxy group undergo ring-opening to form an ester bond; when the microsphere surface is hydroxyl, the hydroxyl group and epoxy group undergo ring-opening to form an ether bond; when the microsphere surface is chloromethyl, the chloromethyl group undergoes a nucleophilic substitution reaction with the epoxy group under alkaline conditions, also forming an ether bond. Through the above reactions, allyl glycidyl ether molecules are chemically fixed to the microsphere surface, thereby introducing allyl double bonds that can participate in subsequent reactions. Simultaneously, due to the intrinsic characteristics of this ring-opening or substitution reaction: on the one hand, a new hydroxyl group will inevitably be generated on the microsphere surface after the epoxy group ring-opens; on the other hand, due to reaction kinetics limitations, some unreacted epoxy groups and a small amount of incompletely converted carboxyl, hydroxyl, or chloromethyl groups may remain on the microsphere surface. Therefore, the allyl functionalized polymer microspheres obtained in step S3 do not contain only allyl double bonds, but are polymer microspheres containing allyl double bonds, hydroxyl groups, and residual epoxy groups.

[0029] Preferably, in step S4, the solvent is any one or more of 2-methylallyl alcohol and 3-buten-1-ol.

[0030] Preferably, in step S4, the reaction conditions for the first and second reactions are a temperature of 30°C, a rotation speed of 160 rpm, and a time of 16 h.

[0031] Preferably, in step S4, the mass ratio of the allyl functionalized polymer microspheres, solvent, sodium hydroxide aqueous solution, and allyl glycidyl ether is 1:0.4:2:0.6.

[0032] The residual epoxy and hydroxyl groups on the microsphere surface are utilized for further etherification reactions, thereby introducing more short chains with double bonds and additional hydroxyl groups to increase the grafting density of allyl groups on the surface and the hydrophilicity of the microspheres. A two-step reaction strategy is employed: first, allyl compounds such as 2-methylallyl alcohol or 3-buten-1-ol are added to react with the residual epoxy or hydroxyl groups on the microsphere surface; subsequently, allyl glycidyl ether is added to further extend the surface grafted chains. Through this stepwise grafting strategy, the density of allyl double bonds on the microsphere surface is significantly improved, and thicker, more flexible allyl side chains are formed, providing more reaction sites for subsequent click reactions.

[0033] When 2-methylallyl alcohol is used, under alkaline conditions, the hydroxyl anion of 2-methylallyl alcohol acts as a nucleophile, attacking the residual epoxy groups on the microsphere surface, resulting in an epoxy ring-opening reaction. This reaction introduces a second allyl double bond and simultaneously generates a new hydroxyl group. Additionally, the hydroxyl groups on the microsphere surface can also undergo a dehydration etherification reaction with 2-methylallyl alcohol under strongly alkaline conditions, similarly fixing the allyl structure to the microsphere surface. The combined effect of these reactions results in a denser distribution of allyl groups on the microsphere surface.

[0034] When 3-buten-1-ol is used, its molecular structure also contains a hydroxyl group that can participate in nucleophilic reactions and a terminal allyl group. The hydroxyl group can also undergo epoxy ring-opening or etherification reactions with residual epoxy groups or hydroxyl groups on the microsphere surface, thus being covalently fixed to the microsphere surface. 3-Buten-1-ol has one more methylene group in its carbon chain than 2-methylallylol. While they differ in the flexibility and steric hindrance of the introduced chain segments, they share a consistent reaction pathway.

[0035] Preferably, in step S5, the free radical initiator is azobisisobutyronitrile, and the mass ratio of the free radical initiator to the modified polymer microspheres is (0.1~0.2):(10~20); the oxidant is oxygen, which is introduced by bubbling at a rate of 1-3 bubbles / s; and the ligand monomer is any one or more of mercaptosuccinic acid, mercaptoglutaric acid, acrylic acid, butenoic acid, butenedioic acid, pentenedioic acid, sodium bisulfite, and sodium sulfate.

[0036] Preferably, in step S5, the reaction temperature is 40~70℃ and the reaction time is 10~12 h.

[0037] Preferably, in step S5, the mass ratio of the ligand monomer, the modified polymer microspheres and water is 1:(10~20):(8~10).

[0038] Functional ligands are grafted onto the surface of microspheres via radical-driven "thiol-ene" click chemistry or nucleophilic addition reactions, ultimately yielding strong cation exchange fillers. The reactants are not simply epoxy systems, but modified polymer microspheres with surface-containing allyl double bonds, benzyl structures, or activated allyl structures. This multifunctional surface provides multiple reaction pathways for ligand grafting. Specifically, sodium bisulfite can introduce sulfonic acid groups through two pathways: firstly, sodium bisulfite undergoes nucleophilic addition reactions with allyl or activated double bonds on the microsphere surface to form stable sulfonate structures; secondly, sodium bisulfite can also undergo nucleophilic substitution reactions with benzyl halogens or similar activated sites, similarly introducing sulfonic acid groups. Because the intermediates are fixed on the microsphere surface during the reaction, intermolecular side reactions are less likely to occur, thus stable transformation can be achieved without the need for additional complexing agents.

[0039] The present invention also provides a packing material for a chromatography column for separating glycated hemoglobin, which is prepared by the above-described preparation method.

[0040] The packing material is an analytical packing material used in high performance liquid chromatography (HPLC) and is applied to the clinical diagnosis, detection, and pathological research of diabetes, as well as the screening of gestational diabetes.

[0041] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0042] (1) In the detection of glycated hemoglobin by high performance liquid chromatography, the packing matrix is ​​required to have a certain degree of hydrophobicity to reduce swelling in water. To address this issue, this invention uses hydrophobic monomers to synthesize hydrophobic crosslinked polymers as base spheres. This invention uses hydrophobic monomers, such as methyl methacrylate, to obtain Polymer seed spheres with a uniform particle size of 5 μm through dispersion polymerization. Polymer-based spheres are obtained by hydrolyzing the ester groups under alkaline conditions. The Polymer-based spheres are then hydrophilized by selecting allyl glycidyl ethers, which have dual active functional groups of allyl and epoxy groups. Through ring-opening of the epoxy groups and reactions with functional groups, the base spheres are hydrophilized to obtain allyl functionalized polymer microspheres.

[0043] (2) This invention increases the linker length by adding 2-methylallyl alcohol or 3-buten-1-ol to react with allyl functionalized polymer microspheres in an alkaline solution, followed by a secondary reaction with allyl glycidyl ether. The treated microspheres and thiol-containing ligand monomers, such as mercaptosuccinic acid or mercaptoglutaric acid, react with the double bonds on the microsphere surface to generate carboxylated modified polymer microspheres, further improving hydrophilicity. Excessive hydrophobicity of the filler leads to poor dispersibility and non-specific adsorption. Strong hydrophilicity allows the surface to easily form hydrogen bonds and other interactions with water molecules, thus forming a hydrated layer on the surface, preventing other substances from directly contacting the surface and reducing non-specific adsorption. This invention, based on the preparation of base spheres using hydrophobic monomers and hydrophilic treatment, increases the linker length and utilizes hydrophilic groups such as thiol and hydroxyl groups on the links to further enhance the hydrophilicity of the microspheres and reduce non-specific adsorption. In addition, there is a certain interaction between the long chain segments of the microsphere upper arm, which can be connected by winding, bonding and other methods to form a barrier, generate greater steric hindrance, further protect the hydrophilic layer and the base sphere, improve the salt resistance of the filler and extend its service life. Attached Figure Description

[0044] Figure 1 This is a SEM image of the polymethyl methacrylate microspheres prepared in Example 1.

[0045] Figure 2 This is a SEM image of the polymethyl methacrylate microspheres prepared in Example 2.

[0046] Figure 3 This is a SEM image of the polyglycerol methacrylate microspheres prepared in Example 3.

[0047] Figure 4 This is a SEM image of the polyglycerol methacrylate microspheres prepared in Example 4.

[0048] Figure 5 SEM image of the polystyrene-divinylbenzene copolymer microspheres prepared in Example 5.

[0049] Figure 6 The image shows a SEM image of the polystyrene-divinylbenzene copolymer microspheres prepared in Example 6.

[0050] Figure 7 HPLC linear elution chromatogram of the packing material prepared in Example 1 injected into a GlyHb-HNmicro 4.6*20 mm metal pre-packed column.

[0051] Figure 8 HPLC linear elution chromatogram of the packing material prepared for Comparative Example 1 injected into a GlyHb-HNmicro 4.6*20 mm metal pre-packed column. Detailed Implementation

[0052] The following embodiments are only used to illustrate the technical solutions of the present invention more clearly, and should not be used to limit the scope of protection of the present invention.

[0053] The compounds used in the examples and comparative examples were all commercially available products and were not subjected to any further purification treatment.

[0054] Example 1

[0055] A packing material for a chromatography column used to separate glycated hemoglobin, the preparation method of which includes:

[0056] Step S1: Mix 10 g of methyl methacrylate, 2 g of divinylbenzene, 0.1 g of azobisisobutyronitrile and 30 g of 5% polyvinyl alcohol aqueous solution, emulsify at 700 rpm for 5 min, and then react at 70℃ for 13 h. The resulting solid is washed repeatedly with ethanol and water 3 times each, and dried at 80℃ for 12 h to obtain PMMA seed pellet powder with a particle size of 5 μm and no pores on the surface.

[0057] Step S2: Take 10 g of PMMA seed pellet powder, add 15 g of 0.8 M sodium hydroxide solution, react at 50 °C and 140 rpm for 24 h, add 30 g of 0.8 M hydrochloric acid solution, wash the obtained solid with ethanol and water 3 times each, and dry at 60 °C for 10 h to obtain carboxylated PMMA-based pellets.

[0058] Step S3: Take 10 g of carboxylated PMMA microspheres, mix with 3 g of allyl glycidyl ether, 0.1 g of sodium sulfate, and 80 g of 50% dimethyl sulfoxide aqueous solution, and react at 140 rpm for 1 h. Add 5 g of 2 M sodium hydroxide solution and react at 20 °C for 16 h. Wash the obtained solid with ethanol and water three times each, and dry at 80 °C for 12 h to obtain allyl functionalized PMMA microspheres.

[0059] Step S4: Take 5 g of allyl functionalized PMMA microspheres, add 2 g of 2-methylallyl alcohol and 10 g of sodium hydroxide solution, and react for 14 h at 160 rpm and 25 °C. After the reaction is completed, add 3 g of allyl glycidyl ether and continue to react for 14 h at 160 rpm and 25 °C to obtain modified PMMA microspheres.

[0060] Step S5: Take 10 g of modified PMMA microspheres, and react them with 1 g of mercaptosuccinic acid, 80 g of water and 0.1 g of azobisisobutyronitrile at 180 rpm and 50 °C for 12 h to obtain carboxylated modified PMMA microspheres. Wash the carboxylated modified PMMA microspheres with ethanol and water three times each, and dry them at 80 °C for 12 h to obtain a strong cation exchange filler with a particle size of 5 μm.

[0061] Example 2

[0062] A packing material for a chromatography column used to separate glycated hemoglobin, the preparation method of which includes:

[0063] Step S1: Mix 10 g of methyl methacrylate, 3 g of divinylbenzene, 0.2 g of azobisisobutyronitrile (AIB), and 40 g of 5% polyvinyl alcohol aqueous solution, emulsify at 1000 rpm for 5 min, and react at 90 °C for 10 h. The resulting solid is washed three times each with ethanol and water, and dried at 80 °C to obtain PMMA seed pellets with a particle size of 5 μm and a non-porous surface.

[0064] Step S2: Take 10 g of PMMA seed pellet powder, add 20 g of 0.8 M sodium hydroxide solution, react at 70 °C and 180 rpm for 24 h, add 40 g of 0.8 M hydrochloric acid solution, wash the obtained solid with ethanol and water 3 times each, and dry at 80 °C to obtain carboxylated PMMA-based pellets.

[0065] Step S3: Take 10 g of carboxylated PMMA microspheres, 5 g of allyl glycidyl ether, 0.2 g of sodium sulfate, and 100 g of 50% dimethyl sulfoxide aqueous solution and mix them. React at 160 rpm for 2 h. Add 10 g of 2 M sodium hydroxide solution and react at 30 °C for 16 h. Wash the obtained solid with ethanol and water three times each, and dry at 80 °C to obtain allyl functionalized PMMA microspheres.

[0066] Step S4: Take 5 g of allyl-functionalized PMMA microspheres, add 2 g of 3-buten-1-ol and 10 g of sodium hydroxide solution, and react for 16 h at 160 rpm and 30 °C. After the reaction is complete, add 3 g of allyl glycidyl ether, and continue to react for 14 h at 160 rpm and 25 °C to obtain modified PMMA microspheres.

[0067] Step S5: Take 20 g of modified PMMA microspheres, add 1 g of sodium bisulfite, 100 g of water, and 2 O2 bubbles / s. React at 180 rpm and 40℃ for 12 h to obtain sulfonic acid functionalized modified PMMA microspheres. Wash with ethanol and water 3 times each, and dry at 90℃ to obtain a strong cation exchange filler with an average particle size of 5 μm.

[0068] Example 3

[0069] A packing material for a chromatography column used to separate glycated hemoglobin, the preparation method of which includes:

[0070] Step S1: Mix 10 g of glycidyl methacrylate, 2 g of divinylbenzene, 0.1 g of azobisisobutyronitrile and 30 g of 5% polyvinyl alcohol aqueous solution, emulsify at 700 rpm for 5 min, and then react at 70℃ for 13 h. The resulting solid is washed three times each with ethanol and water, and dried at 80℃ for 12 h to obtain PGMA seed pellet powder with a particle size of 5 μm and no pores on the surface.

[0071] Step S2: Take 10 g of PGMA seed pellet powder, add 15 g of 0.8 M hydrochloric acid solution, react at 50℃ and 140 rpm for 24 h, add 30 g of 0.8 M sodium hydroxide solution, wash the obtained solid with ethanol and water 3 times each, and dry at 60℃ for 10 h to obtain hydroxylated PGMA-based pellets.

[0072] Step S3: Take 10 g of hydroxylated PGMA microspheres, mix with 5 g of allyl glycidyl ether, 0.1 g of sodium sulfate, and 80 g of 50% dimethyl sulfoxide aqueous solution, and react at 140 rpm for 1 h. Add 15 g of 2 M sodium hydroxide solution and react at 20 °C for 16 h. Wash the obtained solid with ethanol and water three times each, and dry at 80 °C for 12 h to obtain allyl functionalized PGMA microspheres.

[0073] Step S4: Take 5 g of allyl functionalized PGMA microspheres, add 2 g of 2-methylallyl alcohol and 10 g of sodium hydroxide solution, and react at 160 rpm and 25°C for 14 h; after the reaction is completed, add 3 g of allyl glycidyl ether, and continue to react at 160 rpm and 25°C for 14 h to obtain modified PGMA microspheres.

[0074] Step S5: Take 10 g of modified PGMA microspheres and react them with 1 g of mercaptosuccinic acid, 80 g of water, and 0.2 g of azobisisobutyronitrile at 180 rpm and 50 °C for 12 h to obtain carboxylated modified PGMA microspheres. Wash the carboxylated modified PGMA microspheres three times each with ethanol and water, and dry them at 80 °C for 12 h to obtain a strong cation exchange filler with a particle size of 5 μm.

[0075] Example 4

[0076] A packing material for a chromatography column used to separate glycated hemoglobin, the preparation method of which includes:

[0077] Step S1: Mix 10 g glycidyl methacrylate, 3 g divinylbenzene, 0.2 g azobisisobutyronitrile (AIB), and 40 g of 5% polyvinyl alcohol aqueous solution, emulsify at 1000 rpm for 5 min, and react at 90℃ for 10 h. The resulting solid is washed three times each with ethanol and water, and dried at 80℃ to obtain PGMA seed pellet powder with a particle size of 5 μm and a non-porous surface.

[0078] Step S2: Take 10 g of PGMA seed pellet powder, add 20 g of 0.8 M hydrochloric acid solution, react at 70℃ and 180 rpm for 24 h, add 40 g of 0.8 M hydrochloric acid, wash the obtained solid with ethanol and water 3 times, and dry at 80℃ to obtain hydroxylated PGMA-based pellets.

[0079] Step S3: Take 10 g of hydroxylated PGMA microspheres, 5 g of allyl glycidyl ether, 0.2 g of sodium sulfate, and 100 g of 50% dimethyl sulfoxide aqueous solution and mix them. React at 160 rpm for 2 h. Add 25 g of 2 M sodium hydroxide solution and react at 30 °C for 16 h. Wash the obtained solid with ethanol and water three times each, and dry at 80 °C to obtain allyl functionalized PGMA microspheres.

[0080] Step S4: Take 5 g of allyl-functionalized PGMA microspheres, add 2 g of 3-buten-1-ol and 10 g of sodium hydroxide solution, and react for 16 h at 160 rpm and 30 °C. After the reaction is complete, add 3 g of allyl glycidyl ether, and continue to react for 14 h at 160 rpm and 25 °C to obtain modified PGMA microspheres.

[0081] Step S5: Take 20 g of modified PGMA microspheres, add 1 g of sodium bisulfite, 100 g of water, and 2 O2 bubbles / s. React at 180 rpm and 40℃ for 12 h to obtain sulfonic acid functionalized modified PGMA microspheres. Wash with ethanol and water 3 times each, and dry at 90℃ to obtain a strong cation exchange filler with an average particle size of 5 μm.

[0082] Example 5

[0083] A packing material for a chromatography column used to separate glycated hemoglobin, the preparation method of which includes:

[0084] Step S1: Mix 10 g of styrene, 2 g of divinylbenzene, 0.1 g of azobisisobutyronitrile and 30 g of 5% polyvinyl alcohol aqueous solution, emulsify at 700 rpm for 5 min, and then react at 70℃ for 13 h. The resulting solid is washed repeatedly with ethanol and water 3 times each, and dried at 80℃ for 12 h to obtain polystyrene-divinylbenzene copolymer seed pellet powder with a particle size of 5 μm and a non-porous surface.

[0085] Step S2: Take 10 g of polystyrene-divinylbenzene copolymer seed pellet powder, add 15 g of chloromethyl methyl ether, and react at 50℃ and 140 rpm for 24 h. The resulting solid is washed three times each with ethanol and water, and dried at 60℃ for 10 h to obtain chloromethyl methyl ether. change Polystyrene-divinylbenzene copolymer-based spheres.

[0086] Step S3, take chloromethyl change10 g of polystyrene-divinylbenzene copolymer-based spheres were mixed with 5 g of allyl glycidyl ether, 0.1 g of sodium sulfate, and 80 g of 50% dimethyl sulfoxide aqueous solution. The mixture was reacted at 140 rpm for 1 h. Then, 40 g of 2 M sodium hydroxide solution was added, and the mixture was reacted at 20 °C for 16 h. The resulting solid was washed three times each with ethanol and water, and dried at 80 °C for 12 h to obtain allyl functionalized polystyrene-divinylbenzene copolymer microspheres.

[0087] Step S4: Take 5 g of allyl functionalized polystyrene-divinylbenzene copolymer microspheres, add 2 g of 2-methylallyl alcohol and 10 g of sodium hydroxide solution, and react for 14 h at 160 rpm and 25°C. After the reaction is completed, add 3 g of allyl glycidyl ether and continue to react for 14 h at 160 rpm and 25°C to obtain modified polystyrene-divinylbenzene copolymer microspheres.

[0088] Step S5: Take 10 g of modified polystyrene-divinylbenzene copolymer microspheres, add 1 g of sodium bisulfite, 80 g of water, and bubble with O2 at 2 bubbles / s. React at 180 rpm and 40℃ for 12 h to obtain sulfonic acid functionalized modified polystyrene-divinylbenzene copolymer microspheres. Wash them repeatedly with ethanol and water 3 times each, and dry them at 90℃ to obtain a strong cation exchange filler with an average particle size of 5 μm.

[0089] Example 6

[0090] A packing material for a chromatography column used to separate glycated hemoglobin, the preparation method of which includes:

[0091] Step S1: Mix 10 g of styrene, 3 g of divinylbenzene, 0.2 g of azobisisobutyronitrile (AIB), and 40 g of 5% polyvinyl alcohol aqueous solution. Emulsify at 1000 rpm for 5 min and react at 90 °C for 10 h. The resulting solid is washed three times each with ethanol and water, and dried at 80 °C to obtain polystyrene-divinylbenzene copolymer seed pellets with a particle size of 5 μm and a non-porous surface.

[0092] Step S2: Take 10 g of polystyrene-divinylbenzene copolymer seed pellet powder, add 20 g of chloromethyl methyl ether, react at 70℃ and 180 rpm for 24 h, wash the obtained solid three times each with ethanol and water, and dry at 80℃ to obtain chloromethyl methyl ether. change Polystyrene-divinylbenzene copolymer-based spheres.

[0093] Step S3, take chloromethyl change10 g of polystyrene-divinylbenzene copolymer microspheres, 5 g of allyl glycidyl ether, 0.2 g of sodium sulfate, and 100 g of 50% dimethyl sulfoxide aqueous solution were mixed and reacted at 160 rpm for 2 h. Then, 50 g of 2 M sodium hydroxide solution was added and reacted at 30 °C for 16 h. The resulting solid was washed three times each with ethanol and water and dried at 80 °C to obtain allyl functionalized polystyrene-divinylbenzene copolymer microspheres.

[0094] Step S4: Take 5 g of allyl-functionalized polystyrene-divinylbenzene copolymer microspheres, add 2 g of 3-buten-1-ol and 10 g of sodium hydroxide solution, and react for 16 h at 160 rpm and 30 °C. After the reaction is complete, add 3 g of allyl glycidyl ether, and continue to react for 14 h at 160 rpm and 25 °C to obtain modified polystyrene-divinylbenzene copolymer microspheres.

[0095] Step S5: Take 20 g of modified polystyrene-divinylbenzene copolymer microspheres, add 1 g of sodium bisulfite, 100 g of water, and 2 O2 bubbles / s. React at 180 rpm and 40℃ for 12 h to obtain sulfonic acid functionalized modified polystyrene-divinylbenzene copolymer microspheres. Wash them repeatedly with ethanol and water 3 times each, and dry them at 90℃ to obtain a strong cation exchange filler with an average particle size of 5 μm.

[0096] Comparative Example 1

[0097] A packing material for a chromatography column for separating glycated hemoglobin is prepared in a manner different from that in Example 1, in that the allyl functionalized polymer microspheres prepared in step S3 are not modified in step S4.

[0098] SEM testing: The surface microstructure of the strong cation exchange packing materials with a particle size of 5 μm prepared in Examples 1-6 was shown by scanning electron microscopy.

[0099] Linear elution test: The packing materials prepared in Example 1 and Comparative Example 1 were injected into the injectors of the HPLC system, and the system was started to begin analysis.

[0100] Test solution: whole blood

[0101] Solution A: A phosphate buffer solution with a pH of 5.0;

[0102] Solution B: a phosphate buffer solution with a pH of 7.2;

[0103] Solution C: EDTA hemolytic agent.

[0104] like Figure 7 and Figure 8As shown, the horizontal axis of an HPLC chromatogram typically represents elution time, and the vertical axis represents signal intensity. The packing material prepared in Example 1 exhibited clear and complete peaks at retention times of 0.232, 0.279, 0.373, 0.486, 0.802, and 2.155 minutes. The peak at a retention time of 0.802 minutes was the glycated hemoglobin peak (HbA1 peak), with a peak area of ​​5.47%, indicating a blood glucose level of 5.47 in the blood sample.

[0105] Figure 7 The HbF peak appearing at 0.373 minutes is an unstable peak. The clearer and more complete the peak shape of the unstable peak, the higher the separation degree of the packing material. Figure 8 In the sample, an HbF peak appeared at 0.732 minutes, but its peak shape was unclear and incomplete. Therefore, the packing material prepared in Example 1 has a better separation effect than the packing material prepared in Comparative Example 1.

[0106] The packing materials prepared in Example 1 and Comparative Example 1 were tested on the same blood samples, and the results were compared. Figure 7 and Figure 8 The total peak area in the spectrum shows that... Figure 7 The total area of ​​the middle peak is greater than Figure 8 The total peak area shows that the packing material prepared in Example 1 has lower non-specific adsorption than the packing material prepared in Comparative Example 1 when calibrating glycated hemoglobin.

[0107] By comparing the peak areas of the first 5 peaks in the spectrum. Figure 7 The sum of the peak areas of the HbA1 and HbF peaks is less than Figure 8 The peak area of ​​A0 is greater than that of A0. Figure 8 This demonstrates that the packing material prepared in Example 1 has better salt resistance than the packing material prepared in Comparative Example 1, resulting in a lower total area of ​​the first 5 peaks and a higher peak area at A0 during the actual elution process.

[0108] Experiments have shown that by using hydrophobic monomers to prepare the base spheres and then performing hydrophilic treatment, increasing the length of the connector arm can further improve the separation degree and salt tolerance of the packing material for glycated hemoglobin, making the blood glucose measurement results of the packing material more accurate and extending its service life.

[0109] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing packing material for a chromatography column used to separate glycated hemoglobin, characterized in that, Includes the following steps: S1. Mix and emulsify the monomer, crosslinking agent, initiator and polyvinyl alcohol aqueous solution to obtain polymer seed balls; Step S2: Mix the polymer seed pellets with the activating agent and react to obtain polymer-based pellets; S3. Mix the polymer-based spheres, allyl glycidyl ether and sodium sulfate, dissolve them in a solvent, stir, add sodium hydroxide solution, and react to obtain allyl functionalized polymer microspheres. S4. Allyl functionalized polymer microspheres are added to a solvent and a first reaction is carried out in an aqueous sodium hydroxide solution. After the reaction is completed, allyl glycidyl ether is added to carry out a second reaction to obtain modified polymer microspheres. S5. The modified polymer microspheres, ligand monomers and water are mixed and reacted in the presence of a free radical initiator or oxidant to obtain a 5 μm cation exchange packing.

2. The method for preparing packing material for a chromatography column for separating glycated hemoglobin according to claim 1, characterized in that, In S1, the monomer is any one or more of methyl methacrylate, glycidyl methacrylate, and styrene.

3. The method for preparing packing material for a chromatography column for separating glycated hemoglobin according to claim 1, characterized in that, In S1, the crosslinking agent is divinylbenzene; the initiator is azobisisobutyronitrile.

4. The method for preparing packing material for a chromatography column for separating glycated hemoglobin according to claim 1, characterized in that, In step S1, the mass ratio of monomer, crosslinking agent, initiator and polyvinyl alcohol aqueous solution is 1:(0.2~0.3):(0.01~0.02):(3~4); the emulsification time is 3~5 min, the stirring speed during emulsification is 700~1000 rpm; the reaction temperature is 70~90℃, and the reaction time is 10~13 h.

5. The method for preparing packing material for a chromatography column for separating glycated hemoglobin according to claim 1, characterized in that, In step S2, the activating agent is any one or more of a strong acid solution, a strong alkali solution, and a chloromethylating agent; the strong acid solution is hydrochloric acid; the strong alkali solution is sodium hydroxide; and the chloromethylating agent is chloromethyl ether.

6. The method for preparing packing material for a chromatography column for separating glycated hemoglobin according to claim 1, characterized in that, In step S2, the reaction conditions are: reaction temperature 25~70℃, reaction time 20~24 h, and rotation speed 160~180 rpm; the mass ratio of polymer seed balls to activating reagent is 1:(1.5~6).

7. The method for preparing packing material for a chromatography column for separating glycated hemoglobin according to claim 1, characterized in that, In step S3, the stirring speed is 140-160 rpm and the stirring time is 1-2 h; the reaction temperature is 20-30℃ and the reaction time is 14-16 h; the solvent is 50% dimethyl sulfoxide aqueous solution; the mass ratio of the polymer-based spheres, allyl glycidyl ether, sodium sulfate, solvent, and strong alkali solution is 1:(0.3-0.5):(0.01-0.02):(8-10):(0.5-5).

8. The method for preparing packing material for a chromatography column for separating glycated hemoglobin according to claim 1, characterized in that, In step S4, the solvent is any one or more of 2-methylallyl alcohol and 3-buten-1-ol; the reaction conditions for the first and second reactions are a temperature of 30°C, a rotation speed of 160 rpm, and a time of 16 h; the mass ratio of the allyl functionalized polymer microspheres, solvent, sodium hydroxide aqueous solution, and allyl glycidyl ether is 1:0.4:2:0.

6.

9. The method for preparing packing material for a chromatography column for separating glycated hemoglobin according to claim 1, characterized in that, In step S5, the free radical initiator is azobisisobutyronitrile (AIBN), and the mass ratio of the free radical initiator to the modified polymer microspheres is (0.1~0.2):(10~20); the oxidant is oxygen, which is introduced by bubbling at a rate of 1-3 bubbles / s; the ligand monomer is any one or more of mercaptosuccinic acid and sodium bisulfite; the reaction temperature is 40~70℃, and the reaction time is 10~12 h; the mass ratio of the ligand monomer, modified polymer microspheres, and water is 1:(10~20):(8~10).

10. A packing material for a chromatography column used to separate glycated hemoglobin, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 9.

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