CN113549183A - Preparation method of filler for glycosylated hemoglobin chromatographic column - Google Patents
Preparation method of filler for glycosylated hemoglobin chromatographic column Download PDFInfo
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- CN113549183A CN113549183A CN202110869427.5A CN202110869427A CN113549183A CN 113549183 A CN113549183 A CN 113549183A CN 202110869427 A CN202110869427 A CN 202110869427A CN 113549183 A CN113549183 A CN 113549183A
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- 102000017011 Glycated Hemoglobin A Human genes 0.000 title claims abstract description 31
- 239000000945 filler Substances 0.000 title claims abstract description 27
- 108010014663 Glycated Hemoglobin A Proteins 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 239000000178 monomer Substances 0.000 claims abstract description 47
- 239000004005 microsphere Substances 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 24
- 238000006243 chemical reaction Methods 0.000 claims abstract description 22
- 125000000524 functional group Chemical group 0.000 claims abstract description 19
- 238000012856 packing Methods 0.000 claims abstract description 19
- 239000002245 particle Substances 0.000 claims abstract description 12
- 238000006116 polymerization reaction Methods 0.000 claims abstract description 10
- 230000000379 polymerizing effect Effects 0.000 claims abstract description 7
- 238000005406 washing Methods 0.000 claims abstract description 7
- 239000002131 composite material Substances 0.000 claims abstract description 6
- 238000012799 strong cation exchange Methods 0.000 claims abstract description 6
- 239000002270 dispersing agent Substances 0.000 claims abstract description 5
- 239000002904 solvent Substances 0.000 claims abstract description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 108091005995 glycated hemoglobin Proteins 0.000 claims description 14
- 239000003999 initiator Substances 0.000 claims description 12
- 239000000463 material Substances 0.000 claims description 10
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- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical compound N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 9
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- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 claims description 5
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 5
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- DBCAQXHNJOFNGC-UHFFFAOYSA-N 4-bromo-1,1,1-trifluorobutane Chemical compound FC(F)(F)CCCBr DBCAQXHNJOFNGC-UHFFFAOYSA-N 0.000 claims description 4
- STVZJERGLQHEKB-UHFFFAOYSA-N ethylene glycol dimethacrylate Substances CC(=C)C(=O)OCCOC(=O)C(C)=C STVZJERGLQHEKB-UHFFFAOYSA-N 0.000 claims description 4
- 239000008213 purified water Substances 0.000 claims description 4
- JHUFGBSGINLPOW-UHFFFAOYSA-N 3-chloro-4-(trifluoromethoxy)benzoyl cyanide Chemical compound FC(F)(F)OC1=CC=C(C(=O)C#N)C=C1Cl JHUFGBSGINLPOW-UHFFFAOYSA-N 0.000 claims description 3
- JZINNAKNHHQBOS-AATRIKPKSA-N 3-methylcinnamic acid Chemical compound CC1=CC=CC(\C=C\C(O)=O)=C1 JZINNAKNHHQBOS-AATRIKPKSA-N 0.000 claims description 3
- 239000004342 Benzoyl peroxide Substances 0.000 claims description 3
- OMPJBNCRMGITSC-UHFFFAOYSA-N Benzoylperoxide Chemical compound C=1C=CC=CC=1C(=O)OOC(=O)C1=CC=CC=C1 OMPJBNCRMGITSC-UHFFFAOYSA-N 0.000 claims description 3
- 229920000663 Hydroxyethyl cellulose Polymers 0.000 claims description 3
- 239000004354 Hydroxyethyl cellulose Substances 0.000 claims description 3
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 claims description 3
- 229920003171 Poly (ethylene oxide) Polymers 0.000 claims description 3
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- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- XUDOZULIAWNMIU-UHFFFAOYSA-N delta-hexenoic acid Chemical compound OC(=O)CCCC=C XUDOZULIAWNMIU-UHFFFAOYSA-N 0.000 claims description 3
- 239000012933 diacyl peroxide Substances 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- VOZRXNHHFUQHIL-UHFFFAOYSA-N glycidyl methacrylate Chemical compound CC(=C)C(=O)OCC1CO1 VOZRXNHHFUQHIL-UHFFFAOYSA-N 0.000 claims description 3
- 235000019447 hydroxyethyl cellulose Nutrition 0.000 claims description 3
- 239000001863 hydroxypropyl cellulose Substances 0.000 claims description 3
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 claims description 3
- ZQMHJBXHRFJKOT-UHFFFAOYSA-N methyl 2-[(1-methoxy-2-methyl-1-oxopropan-2-yl)diazenyl]-2-methylpropanoate Chemical compound COC(=O)C(C)(C)N=NC(C)(C)C(=O)OC ZQMHJBXHRFJKOT-UHFFFAOYSA-N 0.000 claims description 3
- HVAMZGADVCBITI-UHFFFAOYSA-N pent-4-enoic acid Chemical compound OC(=O)CCC=C HVAMZGADVCBITI-UHFFFAOYSA-N 0.000 claims description 3
- 229920001223 polyethylene glycol Polymers 0.000 claims description 3
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 3
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 3
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 3
- MNCGMVDMOKPCSQ-UHFFFAOYSA-M sodium;2-phenylethenesulfonate Chemical compound [Na+].[O-]S(=O)(=O)C=CC1=CC=CC=C1 MNCGMVDMOKPCSQ-UHFFFAOYSA-M 0.000 claims description 3
- 229920002197 Sodium polyaspartate Polymers 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 claims 9
- 238000000926 separation method Methods 0.000 abstract description 10
- 102000004169 proteins and genes Human genes 0.000 abstract description 8
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- 238000004458 analytical method Methods 0.000 description 3
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
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- 125000002843 carboxylic acid group Chemical group 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
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- 235000000346 sugar Nutrition 0.000 description 2
- 125000000542 sulfonic acid group Chemical group 0.000 description 2
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- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 229920000805 Polyaspartic acid Polymers 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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- 159000000000 sodium salts Chemical class 0.000 description 1
- 238000012437 strong cation exchange chromatography Methods 0.000 description 1
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- 238000006467 substitution reaction Methods 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F285/00—Macromolecular compounds obtained by polymerising monomers on to preformed graft polymers
-
- 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
-
- 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
- B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
- B01D15/361—Ion-exchange
- B01D15/362—Cation-exchange
-
- 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
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/282—Porous sorbents
- B01J20/285—Porous sorbents based on polymers
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F2/00—Processes of polymerisation
- C08F2/12—Polymerisation in non-solvents
- C08F2/16—Aqueous medium
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F265/00—Macromolecular compounds obtained by polymerising monomers on to polymers of unsaturated monocarboxylic acids or derivatives thereof as defined in group C08F20/00
- C08F265/04—Macromolecular compounds obtained by polymerising monomers on to polymers of unsaturated monocarboxylic acids or derivatives thereof as defined in group C08F20/00 on to polymers of esters
- C08F265/06—Polymerisation of acrylate or methacrylate esters on to polymers thereof
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- G01N30/482—
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/56—Packing methods or coating methods
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/56—Packing methods or coating methods
- G01N2030/562—Packing methods or coating methods packing
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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Abstract
The invention provides a preparation method of a filler for a glycosylated hemoglobin chromatographic column, which comprises the following steps of; the first monomer is primarily polymerized to form microspheres with monodisperse particle sizes, and the second step is carried out; the first monomer is further cross-linked and polymerized to enlarge the particle size of the microsphere, and the third step is carried out; further introducing a second monomer and a third monomer for polymerization, and polymerizing two functional groups on the microspheres, step four; washing is carried out to remove the solvent, dispersant, excess monomer and the like in the reaction system. The invention overcomes the defects of the prior art, has reasonable design and compact structure, and obtains the strong cation exchange chromatographic packing containing the composite functional group by a multi-step dispersion polymerization method. The conditions that the protein separation degree is poor or partial variant protein can not be separated and the column efficiency is low, which are possibly generated by a single functional group in the existing ion exchange chromatographic packing, can be avoided. The preparation method of the filler is simple and easy to operate, and the ion exchange capacity of the filler is controllable.
Description
Technical Field
The invention relates to the technical field of glycosylated hemoglobin layer detection, in particular to a preparation method of a filler for a glycosylated hemoglobin chromatographic column.
Background
Glycated hemoglobin (GHb) is a product of hemoglobin in red blood cells combined with sugars in serum. It is formed by slow, continuous and irreversible glycation reactions, the content of which depends on the blood glucose concentration and the contact time of blood glucose and hemoglobin, and is independent of factors such as the blood drawing time, whether the patient is fasting, whether insulin is used, and the like. GHb consists of HbA1a, HbA1b and HbA1c, wherein HbA1c accounts for about 70%, and the structure is stable, so that HbA1c is a 'gold standard' for evaluating the long-term blood sugar control condition of a diabetic patient at present and has important value in the treatment of diabetes and the monitoring of complications thereof.
The high-efficiency liquid-phase glycated hemoglobin detection system realizes quantitative detection of HbA1 c. According to the working principle of High Performance Liquid Chromatography (HPLC), in the detection and analysis of the glycosylated hemoglobin by the ion exchange method, a high performance liquid analyzer, an elution reagent, a chromatographic column, a calibrator and the like all influence the detection result, and the chromatographic column plays an important role in the separation capability of the glycosylated hemoglobin.
Hemoglobin variant is one of the important factors affecting the detection and interpretation of the results of HbA1c, and if the column has insufficient separation capacity for variants and GHb components, the results may be biased. For example, if the functional groups on the column packing do not completely separate HbF, L-HbA1c and HbA1c, when some clinical samples HbF or L-HbA1c are abnormal, the value of HbA1c may be falsely increased, resulting in inaccurate test results.
Therefore, a preparation method of the filler for the glycosylated hemoglobin chromatographic column is provided.
Disclosure of Invention
It is an object of the present invention to solve or at least alleviate problems in the prior art.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a preparation method of a filler for a glycosylated hemoglobin chromatographic column is characterized by comprising the following steps:
step one; the first monomer is polymerized primarily to form the microsphere with monodisperse particle size, 10-15 parts of the first monomer, 2-5 parts of initiator and 5-10 parts of stabilizer are weighed and added into 70-83 parts of medium, the mixture is stirred and reacts for 2-3h under the condition of heating in water bath at 60-75 ℃ after being dissolved uniformly,
step two; further crosslinking and polymerizing the first monomer, enlarging the particle size of the microspheres, weighing 2-10 parts of the first monomer, 1-5 parts of a crosslinking agent, 2-5 parts of an initiator and 5-10 parts of a stabilizer, dissolving uniformly, adding into the polymerization system obtained in the first step, continuing heating in a water bath, stirring and reacting for 2-3h,
step three; further introducing a second monomer and a third monomer for polymerization, polymerizing two functional groups on the microspheres, weighing 2-10 parts of a mixture of the second monomer and the third monomer and 1-5 parts of an initiator, adding the mixture after dissolving uniformly into the previous reaction system, continuing heating in a water bath and stirring for reaction for 12-16h,
step four; washing, removing solvent, dispersant and redundant monomer in the reaction system, repeatedly centrifuging and washing the precipitated polymerized microspheres with ethanol and purified water for 2-3 times by using a centrifugal device, and drying to obtain the strong cation exchange chromatographic packing containing the composite functional group.
Optionally, the first monomer is one or more of methyl methacrylate, styrene and glycidyl methacrylate, and is used in any combination.
Optionally, the second monomer is one or more of 2-acrylamide-2-methylpropanesulfonic acid, sodium styrene sulfonate and sodium allyl sulfonate.
Optionally, the third monomer is one or more of acrylic acid, 4-pentenoic acid, 3-methyl cinnamic acid, 5-hexenoic acid and 3-butenoic acid which are used in any combination.
Optionally, the crosslinking agent is one or any combination of ethylene glycol dimethacrylate and divinylbenzene.
Optionally, the initiator is one or any combination of azodiisobutyronitrile, dimethyl azodiisobutyrate, benzoyl peroxide and diacyl peroxide.
Optionally, the stabilizer is one or more of polyvinyl alcohol, polyoxyethylene, polyethylene glycol, polyvinylpyrrolidone, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl propyl cellulose and sodium polyaspartate, and is used in any combination.
Optionally, the medium is one or more of ethanol, methanol, toluene and deionized water, and is used in any combination.
Optionally, in the step three mixture, the second monomer: the ratio of the third monomer is 20:80 to 80: 20.
Optionally, when the cleaning treatment in step four is performed, the reaction solution after the reaction in step three is cooled to room temperature.
The embodiment of the invention provides a preparation method of a filler for a glycosylated hemoglobin chromatographic column. The method has the following beneficial effects:
the invention provides a preparation method of glycosylated hemoglobin chromatographic column microsphere filler, wherein two functional groups of carboxylic acid group and sulfonic acid group are introduced on the microsphere in the synthetic process of the filler, and the technical problem of unsatisfactory resolution and peak shape of chromatographic column analysis detection results of the filler is solved by adjusting the proportion of the two functional groups on the microsphere. The filler preparation method provided by the invention can synthesize the microsphere filler with dispersed particle size, and realizes good separation effect and separation column effect of proteins such as HbF, L-HbA1c and HbA1c in the high performance liquid chromatography detection process of glycosylated hemoglobin.
The invention obtains the strong cation exchange chromatographic packing containing the composite functional group by a multi-step dispersion polymerization method. The conditions that the protein separation degree is poor or partial variant protein can not be separated and the column efficiency is low, which are possibly generated by a single functional group in the existing ion exchange chromatographic packing, can be avoided. The preparation method of the filler is simple and easy to operate, and the ion exchange capacity of the filler is controllable.
Drawings
FIG. 1 is a chromatogram generated during detection of a chromatographic column obtained by the preparation method of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A preparation method of a filler for a glycosylated hemoglobin chromatographic column comprises the following steps:
step one; the first monomer is polymerized initially to form the monodisperse particle size microsphere. Weighing 10-15 parts of first monomer, 2-5 parts of initiator and 5-10 parts of stabilizer, adding into 70-83 parts of medium, uniformly dissolving, and stirring for reacting for 2-3h under the condition of heating in water bath at 60-75 ℃.
Step two; the first monomer is further cross-linked and polymerized to enlarge the particle size of the microspheres. Weighing 2-10 parts of first monomer, 1-5 parts of cross-linking agent, 2-5 parts of initiator and 5-10 parts of stabilizer, dissolving uniformly, adding into the polymerization system obtained in the first step, and continuously heating in water bath and stirring for reacting for 2-3 h.
Step three; and further introducing a second monomer and a third monomer for polymerization, and polymerizing two functional groups on the microspheres. Weighing 2-10 parts of a mixture of a second monomer and a third monomer (the ratio of the two monomers is 20: 80-80: 20) and 1-5 parts of an initiator, dissolving uniformly, adding into the reaction system, and continuing to heat in a water bath and stir for reaction for 12-16 h.
Step four; washing is carried out to remove the solvent, dispersant, excess monomer and the like in the reaction system. And (3) cooling the reaction liquid after the reaction in the third step to room temperature, simultaneously carrying out repeated centrifugation and cleaning procedures on the precipitated polymeric microspheres for 2-3 times by using ethanol and purified water through a centrifugal device, and then carrying out vacuum drying to obtain the strong cation exchange chromatographic packing containing the composite functional group.
The first monomer is one or a plurality of methyl methacrylate, styrene and glycidyl methacrylate which are randomly combined for use.
The second monomer is one or more of 2-acrylamide-2-methylpropanesulfonic acid, sodium styrene sulfonate and sodium allyl sulfonate.
The third monomer is one or a plurality of acrylic acid, 4-pentenoic acid, 3-methyl cinnamic acid, 5-hexenoic acid and 3-butenoic acid which are used in any combination.
The cross-linking agent is one or more of ethylene glycol dimethacrylate and divinyl benzene which are used in any combination.
The initiator is one or more of azobisisobutyronitrile, dimethyl azobisisobutyrate, benzoyl peroxide and diacyl peroxide which are used in any combination.
The stabilizer is one or more of polyvinyl alcohol, polyoxyethylene, polyethylene glycol, polyvinylpyrrolidone, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl propyl cellulose, and polyaspartic acid sodium salt.
The medium is one or more of ethanol, methanol, toluene and deionized water.
The invention provides a preparation method of glycosylated hemoglobin chromatographic column microsphere filler, wherein two functional groups of carboxylic acid group and sulfonic acid group are introduced on the microsphere in the synthetic process of the filler, and the technical problem of unsatisfactory resolution and peak shape of chromatographic column analysis detection results of the filler is solved by adjusting the proportion of the two functional groups on the microsphere. The filler preparation method provided by the invention can synthesize the microsphere filler with dispersed particle size, and realizes good separation effect and separation column effect of proteins such as HbF, L-HbA1c and HbA1c in the high performance liquid chromatography detection process of glycosylated hemoglobin, as shown in FIG. 1.
The strong cation exchange chromatographic packing containing the composite functional group is obtained by a multi-step dispersion polymerization method. The conditions that the protein separation degree is poor or partial variant protein can not be separated and the column efficiency is low, which are possibly generated by a single functional group in the existing ion exchange chromatographic packing, can be avoided. The preparation method of the filler is simple and easy to operate, and the ion exchange capacity of the filler is controllable.
Example 1
Step one; methyl methacrylate is polymerized primarily to form monodisperse microballoons with particle size.
Using a three-neck flask to weigh 13 parts of methyl methacrylate, 4 parts of azodiisobutyronitrile, 10 parts of polyvinyl alcohol, 70 parts of ethanol and 3 parts of purified water, placing the mixture in a water bath after ultrasonic mixing uniformly, starting a stirrer to keep the reaction system uniform, introducing nitrogen for 10min to remove oxygen in the device, raising the temperature of the water bath to 70 ℃, and starting the reaction.
Step two; and (3) further crosslinking and polymerizing the methyl methacrylate to enlarge the particle size of the microspheres.
Using a beaker, weighing 7 parts of methyl methacrylate, 3 parts of ethylene glycol dimethacrylate, 4 parts of azobisisobutyronitrile and 10 parts of polyvinyl alcohol, and ultrasonically dissolving the materials uniformly. After the polymerization system in the step one reacts for 3 hours, adding the polymerization system into the step one; the reaction system is continuously reacted for 3 hours, and the water bath heating and stirring are kept.
Step three; 2-acrylamide-2-methylpropanesulfonic acid and acrylic acid are further introduced to carry out polymerization, and two functional groups are polymerized on the microsphere.
Weighing 2 parts of acrylic acid, 4 parts of 2-acrylamide-2-methylpropanesulfonic acid and 3 parts of azobisisobutyronitrile by using a beaker, and performing ultrasonic dissolution uniformly to obtain a second step; the reaction system is continuously heated in a water bath and stirred for reaction for 14 hours.
Step four; washing is carried out to remove the solvent, dispersant, excess monomer and the like in the reaction system. And after the reaction in the third step is finished, taking down the three-neck flask, standing to room temperature, transferring the reaction liquid into a centrifugal tube, centrifuging, removing the supernatant, adding ethanol into the precipitated microspheres, uniformly mixing, and continuously centrifuging to remove the supernatant. And continuously repeating the steps, and cleaning and centrifuging the microspheres by using ethanol. And (3) cleaning the microspheres with water and drying to finally obtain the monodisperse strong cation exchange chromatography filler microspheres.
The obtained filler is prepared into a chromatographic column by using a column loading machine, and a whole blood sample is detected and analyzed to obtain a chromatogram with good separation effect, as shown in figure 1.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A preparation method of a filler for a glycosylated hemoglobin chromatographic column is characterized by comprising the following steps:
step one; the first monomer is polymerized primarily to form the microsphere with monodisperse particle size, 10-15 parts of the first monomer, 2-5 parts of initiator and 5-10 parts of stabilizer are weighed and added into 70-83 parts of medium, the mixture is stirred and reacts for 2-3h under the condition of heating in water bath at 60-75 ℃ after being dissolved uniformly,
step two; further crosslinking and polymerizing the first monomer, enlarging the particle size of the microspheres, weighing 2-10 parts of the first monomer, 1-5 parts of a crosslinking agent, 2-5 parts of an initiator and 5-10 parts of a stabilizer, dissolving uniformly, adding into the polymerization system obtained in the first step, continuing heating in a water bath, stirring and reacting for 2-3h,
step three; further introducing a second monomer and a third monomer for polymerization, polymerizing two functional groups on the microspheres, weighing 2-10 parts of a mixture of the second monomer and the third monomer and 1-5 parts of an initiator, adding the mixture after dissolving uniformly into the previous reaction system, continuing heating in a water bath and stirring for reaction for 12-16h,
step four; washing, removing solvent, dispersant and redundant monomer in the reaction system, repeatedly centrifuging and washing the precipitated polymerized microspheres with ethanol and purified water for 2-3 times by using a centrifugal device, and drying to obtain the strong cation exchange chromatographic packing containing the composite functional group.
2. The method of claim 1, wherein the packing material for a glycated hemoglobin chromatography column comprises: the first monomer is one or a plurality of methyl methacrylate, styrene and glycidyl methacrylate which are used in any combination.
3. The method of claim 1, wherein the packing material for a glycated hemoglobin chromatography column comprises: the second monomer is one or more of 2-acrylamide-2-methylpropanesulfonic acid, sodium styrene sulfonate and sodium allyl sulfonate.
4. The method of claim 1, wherein the packing material for a glycated hemoglobin chromatography column comprises: the third monomer is one or a plurality of acrylic acid, 4-pentenoic acid, 3-methyl cinnamic acid, 5-hexenoic acid and 3-butenoic acid which are used in any combination.
5. The method of claim 1, wherein the packing material for a glycated hemoglobin chromatography column comprises: the cross-linking agent is one or more of ethylene glycol dimethacrylate and divinylbenzene which can be used in any combination.
6. The method of claim 1, wherein the packing material for a glycated hemoglobin chromatography column comprises: the initiator is one or any combination of azodiisobutyronitrile, dimethyl azodiisobutyrate, benzoyl peroxide and diacyl peroxide.
7. The method of claim 1, wherein the packing material for a glycated hemoglobin chromatography column comprises: the stabilizer is one or more of polyvinyl alcohol, polyoxyethylene, polyethylene glycol, polyvinylpyrrolidone, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl propyl cellulose and sodium polyaspartate.
8. The method of claim 1, wherein the packing material for a glycated hemoglobin chromatography column comprises: the medium is one or more of ethanol, methanol, toluene and deionized water, and can be used in any combination.
9. The method of claim 1, wherein the packing material for a glycated hemoglobin chromatography column comprises: a second monomer in the step three mixture: the ratio of the third monomer is 20:80 to 80: 20.
10. The method of claim 1, wherein the packing material for a glycated hemoglobin chromatography column comprises: and when the cleaning treatment in the fourth step is carried out, the reaction liquid after the reaction in the third step is cooled to room temperature.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010236907A (en) * | 2009-03-30 | 2010-10-21 | Sekisui Medical Co Ltd | COLUMN PACKING FOR SEPARATING HEMOGLOBINS, METHOD FOR MEASURING HEMOGLOBIN A1c AND ABNORMAL HEMOGLOBINS, AND METHOD FOR MANUFACTURING COLUMN FILLER FOR SEPARATING HEMOGLOBINS |
| JP2011047859A (en) * | 2009-08-28 | 2011-03-10 | Sekisui Medical Co Ltd | COLUMN PACKING FOR SEPARATING HEMOGLOBIN, METHOD FOR MEASURING HEMOGLOBIN A1c, METHOD FOR MEASURING HEMOGLOBIN A1c AND ABNORMAL HEMOGLOBIN, AND METHOD FOR PRODUCING COLUMN PACKING FOR SEPARATING HEMOGLOBIN |
| JP2011047858A (en) * | 2009-08-28 | 2011-03-10 | Sekisui Medical Co Ltd | COLUMN PACKING FOR SEPARATING HEMOGLOBIN, METHOD FOR MEASURING HEMOGLOBIN A1c, METHOD FOR MEASURING HEMOGLOBIN A1c AND ABNORMAL HEMOGLOBIN, AND METHOD FOR PRODUCING COLUMN PACKING FOR SEPARATING HEMOGLOBIN |
| JP2014222247A (en) * | 2014-07-28 | 2014-11-27 | 積水メディカル株式会社 | Column filler for measuring hemoglobins, method for producing column filler for measuring hemoglobins, and method for measuring hemoglobins by liquid chromatography |
| JP2018017560A (en) * | 2016-07-26 | 2018-02-01 | 東ソー株式会社 | Filler for liquid chromatography for use in separation and measurement of hemoglobin |
| CN110314664A (en) * | 2019-06-05 | 2019-10-11 | 南京亘闪生物科技有限公司 | A kind of partial size monodisperse HbA1C ion-exchange chromatography filler synthetic method and its application |
-
2021
- 2021-07-30 CN CN202110869427.5A patent/CN113549183B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010236907A (en) * | 2009-03-30 | 2010-10-21 | Sekisui Medical Co Ltd | COLUMN PACKING FOR SEPARATING HEMOGLOBINS, METHOD FOR MEASURING HEMOGLOBIN A1c AND ABNORMAL HEMOGLOBINS, AND METHOD FOR MANUFACTURING COLUMN FILLER FOR SEPARATING HEMOGLOBINS |
| JP2011047859A (en) * | 2009-08-28 | 2011-03-10 | Sekisui Medical Co Ltd | COLUMN PACKING FOR SEPARATING HEMOGLOBIN, METHOD FOR MEASURING HEMOGLOBIN A1c, METHOD FOR MEASURING HEMOGLOBIN A1c AND ABNORMAL HEMOGLOBIN, AND METHOD FOR PRODUCING COLUMN PACKING FOR SEPARATING HEMOGLOBIN |
| JP2011047858A (en) * | 2009-08-28 | 2011-03-10 | Sekisui Medical Co Ltd | COLUMN PACKING FOR SEPARATING HEMOGLOBIN, METHOD FOR MEASURING HEMOGLOBIN A1c, METHOD FOR MEASURING HEMOGLOBIN A1c AND ABNORMAL HEMOGLOBIN, AND METHOD FOR PRODUCING COLUMN PACKING FOR SEPARATING HEMOGLOBIN |
| JP2014222247A (en) * | 2014-07-28 | 2014-11-27 | 積水メディカル株式会社 | Column filler for measuring hemoglobins, method for producing column filler for measuring hemoglobins, and method for measuring hemoglobins by liquid chromatography |
| JP2018017560A (en) * | 2016-07-26 | 2018-02-01 | 東ソー株式会社 | Filler for liquid chromatography for use in separation and measurement of hemoglobin |
| CN110314664A (en) * | 2019-06-05 | 2019-10-11 | 南京亘闪生物科技有限公司 | A kind of partial size monodisperse HbA1C ion-exchange chromatography filler synthetic method and its application |
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