CN113549183B - Preparation method of packing for glycosylated hemoglobin chromatographic column - Google Patents
Preparation method of packing for glycosylated hemoglobin chromatographic column Download PDFInfo
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- CN113549183B CN113549183B CN202110869427.5A CN202110869427A CN113549183B CN 113549183 B CN113549183 B CN 113549183B CN 202110869427 A CN202110869427 A CN 202110869427A CN 113549183 B CN113549183 B CN 113549183B
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- 102000017011 Glycated Hemoglobin A Human genes 0.000 title claims abstract description 26
- 108010014663 Glycated Hemoglobin A Proteins 0.000 title claims abstract description 26
- 238000012856 packing Methods 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title abstract description 14
- 239000000178 monomer Substances 0.000 claims abstract description 48
- 239000004005 microsphere Substances 0.000 claims abstract description 30
- 238000006243 chemical reaction Methods 0.000 claims abstract description 26
- 125000000524 functional group Chemical group 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 17
- 238000006116 polymerization reaction Methods 0.000 claims abstract description 11
- 238000004140 cleaning 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
- 230000000379 polymerizing effect Effects 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
- 239000000203 mixture Substances 0.000 claims description 16
- 239000003999 initiator Substances 0.000 claims description 13
- 238000010438 heat treatment Methods 0.000 claims description 10
- 239000003381 stabilizer Substances 0.000 claims description 10
- 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
- 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 8
- 238000003756 stirring Methods 0.000 claims description 8
- VVQNEPGJFQJSBK-UHFFFAOYSA-N Methyl methacrylate Chemical compound COC(=O)C(C)=C VVQNEPGJFQJSBK-UHFFFAOYSA-N 0.000 claims description 7
- MYRTYDVEIRVNKP-UHFFFAOYSA-N 1,2-Divinylbenzene Chemical compound C=CC1=CC=CC=C1C=C MYRTYDVEIRVNKP-UHFFFAOYSA-N 0.000 claims description 6
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims description 6
- PVEOYINWKBTPIZ-UHFFFAOYSA-N but-3-enoic acid Chemical compound OC(=O)CC=C PVEOYINWKBTPIZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000003431 cross linking reagent Substances 0.000 claims description 6
- -1 polyoxyethylene Polymers 0.000 claims description 6
- 238000005303 weighing Methods 0.000 claims description 6
- DZSVIVLGBJKQAP-UHFFFAOYSA-N 1-(2-methyl-5-propan-2-ylcyclohex-2-en-1-yl)propan-1-one Chemical compound CCC(=O)C1CC(C(C)C)CC=C1C DZSVIVLGBJKQAP-UHFFFAOYSA-N 0.000 claims description 5
- 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
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 5
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 5
- DBCAQXHNJOFNGC-UHFFFAOYSA-N 4-bromo-1,1,1-trifluorobutane Chemical compound FC(F)(F)CCCBr DBCAQXHNJOFNGC-UHFFFAOYSA-N 0.000 claims description 4
- 238000001035 drying Methods 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
- 238000005406 washing Methods 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
- 239000002202 Polyethylene glycol Substances 0.000 claims description 3
- 229920002197 Sodium polyaspartate Polymers 0.000 claims description 3
- 235000019400 benzoyl peroxide Nutrition 0.000 claims description 3
- 239000001913 cellulose Substances 0.000 claims description 3
- 229920002678 cellulose Polymers 0.000 claims description 3
- 239000012295 chemical reaction liquid Substances 0.000 claims description 3
- 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
- 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
- 238000004519 manufacturing process Methods 0.000 claims 5
- 239000000945 filler Substances 0.000 abstract description 21
- 238000000926 separation method Methods 0.000 abstract description 12
- 239000002245 particle Substances 0.000 abstract description 8
- 102000004169 proteins and genes Human genes 0.000 abstract description 8
- 108090000623 proteins and genes Proteins 0.000 abstract description 8
- 238000005342 ion exchange Methods 0.000 abstract description 7
- 238000012674 dispersion polymerization Methods 0.000 abstract description 3
- 230000007547 defect Effects 0.000 abstract 1
- 238000001514 detection method Methods 0.000 description 11
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 102000001554 Hemoglobins Human genes 0.000 description 3
- 108010054147 Hemoglobins Proteins 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 125000002843 carboxylic acid group Chemical group 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 125000000542 sulfonic acid group Chemical group 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000012437 strong cation exchange chromatography Methods 0.000 description 1
- 238000002305 strong-anion-exchange chromatography Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
Classifications
-
- 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
-
- 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
-
- G01N30/482—
-
- 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
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Polymers & Plastics (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Physics & Mathematics (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
The invention provides a preparation method of a filler for a glycosylated hemoglobin chromatographic column, which comprises the following steps of; preliminary polymerization is carried out on the first monomer to form microspheres with monodisperse particle size; the first monomer is further crosslinked 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 microsphere, wherein the step four is carried out; and cleaning, namely removing the solvent, the dispersing agent, the excessive 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 groups by a multi-step dispersion polymerization method. Can avoid the conditions that the protein separation degree is poor or partial variant protein can not be separated and the column efficiency is low which possibly occur in the single functional group in the existing ion exchange chromatographic packing. 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 glycosylated hemoglobin chromatographic column.
Background
Glycosylated hemoglobin (GHb) is the product of the binding of hemoglobin in red blood cells to carbohydrates in serum. It is formed by a slow, continuous and irreversible saccharification reaction, and the content of the saccharification reaction depends on the concentration of blood sugar and the contact time of the blood sugar and hemoglobin, and is irrelevant to factors such as blood drawing time, whether a patient is fasting, whether insulin is used or not, and the like. GHb is composed of HbA1a, hbA1b and HbA1c, wherein HbA1c accounts for about 70% and is stable in structure, and HbA1c is currently a 'gold standard' for evaluating long-term blood glucose control conditions of diabetics and has important value in diabetes treatment and complications monitoring.
The high performance liquid glycosylated 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 glycosylated hemoglobin by an ion exchange method, a high performance liquid analyzer, an eluting reagent, a chromatographic column, a calibrator and the like all have an influence on the detection result, and the chromatographic column plays a very important role in the separation capability of glycosylated hemoglobin.
Hemoglobin variant is one of the important factors affecting HbA1c detection and result interpretation, and if the separation capacity of the chromatographic column for each component of variant and GHB is insufficient, the result may deviate. For example, if the functional groups on the column packing cannot completely separate HbF, L-HbA1c and HbA1c, in the case of abnormality of HbF or L-HbA1c in some clinical samples, a false increase in HbA1c measurement results may be caused, resulting in inaccurate measurement results.
For this purpose, we propose a method for preparing a packing for glycosylated hemoglobin chromatographic column.
Disclosure of Invention
The present invention aims to solve or at least alleviate the problems of the prior art.
In order to achieve the above purpose, the invention is realized by the following technical scheme:
a method for preparing a packing for a glycosylated hemoglobin chromatographic column, which is characterized by comprising the following steps:
step one, a step one; the first monomer is polymerized preliminarily to form microsphere with monodisperse grain diameter, 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, after being dissolved evenly, the mixture is stirred and reacted for 2-3 hours under the condition of water bath heating at 60-75 ℃,
step two, a step two is carried out; the first monomer is further crosslinked and polymerized, the grain diameter of the microsphere is enlarged, 2-10 parts of the first monomer, 1-5 parts of the crosslinking agent, 2-5 parts of the initiator and 5-10 parts of the stabilizer are weighed, after being uniformly dissolved, the mixture is added into the polymerization system of the step one to continue the water bath heating and stirring reaction for 2-3 hours,
step three, a step of performing; further introducing a second monomer and a third monomer for polymerization, polymerizing the two functional groups on the microsphere, 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 into the previous reaction system after the mixture is uniformly dissolved, continuing to perform water bath heating and stirring reaction for 12-16 hours,
step four, a step four is carried out; washing, removing solvent, dispersant and excessive monomer in the reaction system, centrifuging the precipitated polymeric 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.
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.
Optionally, the cross-linking agent is one or more of ethylene glycol dimethacrylate and divinylbenzene.
Optionally, the initiator is one or more of azodiisobutyronitrile, dimethyl azodiisobutyrate, benzoyl peroxide and diacyl peroxide.
Optionally, the stabilizer is one or more selected from polyvinyl alcohol, polyoxyethylene, polyethylene glycol, polyvinylpyrrolidone, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl propyl cellulose and sodium polyaspartate.
Optionally, the medium is one or more of ethanol, methanol, toluene and deionized water.
Optionally, the second monomer in the step three mixture: the ratio of the third monomer is 20:80-80:20.
Optionally, in the step four cleaning treatment, the reaction solution after the completion of the step three reaction is required to be cooled to room temperature.
The embodiment of the invention provides a preparation method of a filler for a glycosylated hemoglobin chromatographic column. The beneficial effects are as follows:
the invention provides a preparation method of glycosylated hemoglobin chromatographic column microsphere filler, which is characterized in that in the synthesis process of the filler, two functional groups of carboxylic acid groups and sulfonic acid groups are introduced on microspheres, and the technical problems of non-ideal separation degree and peak shape of chromatographic column analysis and detection results of the filler are solved by adjusting the proportion of the two functional groups on the microspheres. The preparation method of the filler provided by the invention can synthesize microsphere fillers with dispersed particle sizes, and realizes good separation effect and separation column effect of HbF, L-HbA1c, hbA1c and other proteins 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. Can avoid the conditions that the protein separation degree is poor or partial variant protein can not be separated and the column efficiency is low which possibly occur in the single functional group in the existing ion exchange chromatographic packing. 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 shows a chromatogram generated during detection of a chromatographic column obtained by the preparation method of the invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
A preparation method of a packing for a glycosylated hemoglobin chromatographic column comprises the following steps:
step one, a step one; the first monomer is polymerized preliminarily to form the microsphere with monodisperse particle size. Weighing 10-15 parts of a first monomer, 2-5 parts of an initiator and 5-10 parts of a stabilizer, adding the first monomer, the initiator and the stabilizer into 70-83 parts of a medium, uniformly dissolving, and stirring and reacting for 2-3h under the condition of heating in a water bath at 60-75 ℃.
Step two, a step two is carried out; the first monomer is further crosslinked and polymerized to enlarge the particle size of the microsphere. Weighing 2-10 parts of a first monomer, 1-5 parts of a cross-linking agent, 2-5 parts of an initiator and 5-10 parts of a stabilizer, uniformly dissolving, and then adding the mixture into the polymerization system of the step one, and continuing the water bath heating and stirring reaction for 2-3 hours.
Step three, a step of performing; and further introducing a second monomer and a third monomer for polymerization, and polymerizing the two functional groups on the microsphere. 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, adding the mixture into the previous reaction system after the mixture is uniformly dissolved, and continuing to perform water bath heating and stirring reaction for 12-16 hours.
Step four, a step four is carried out; and cleaning, namely removing the solvent, the dispersing agent, the excessive monomer and the like in the reaction system. Cooling the reaction liquid after the reaction to room temperature, centrifuging and cleaning the precipitated polymeric microspheres with ethanol and purified water for 2-3 times by using a centrifugal device, and then drying in vacuum to obtain the strong cation exchange chromatographic packing containing the composite functional groups.
The first monomer is one or more of methyl methacrylate, styrene and glycidyl methacrylate.
The second monomer is 2-acrylamide-2-methylpropanesulfonic acid, and one or more of sodium styrene sulfonate and sodium allyl sulfonate are used in any combination.
The third monomer is one or more of acrylic acid, 4-pentenoic acid, 3-methyl cinnamic acid, 5-hexenoic acid and 3-butenoic acid.
The cross-linking agent is one or more of ethylene glycol dimethacrylate and divinylbenzene.
The initiator is one or more of azodiisobutyronitrile, dimethyl azodiisobutyrate, benzoyl peroxide and diacyl peroxide.
The stabilizer is one or more of polyvinyl alcohol, polyoxyethylene, polyethylene glycol, polyvinylpyrrolidone, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl propyl cellulose and sodium polyaspartate.
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, which is characterized in that in the synthesis process of the filler, two functional groups of carboxylic acid groups and sulfonic acid groups are introduced on microspheres, and the technical problems of non-ideal separation degree and peak shape of chromatographic column analysis and detection results of the filler are solved by adjusting the proportion of the two functional groups on the microspheres. The preparation method of the filler provided by the invention can synthesize microsphere fillers with dispersed particle sizes, and realize good separation effects and separation column effects of HbF, L-HbA1c, hbA1c and other proteins in the high performance liquid chromatography detection process of glycosylated hemoglobin, as shown in figure 1.
The strong cation exchange chromatographic packing containing the composite functional group is obtained by a multi-step dispersion polymerization method. Can avoid the conditions that the protein separation degree is poor or partial variant protein can not be separated and the column efficiency is low which possibly occur in the single functional group in the existing ion exchange chromatographic packing. 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, a step one; methyl methacrylate is polymerized preliminarily to form the microsphere with monodisperse particle size.
13 parts of methyl methacrylate, 4 parts of azodiisobutyronitrile, 10 parts of polyvinyl alcohol, 70 parts of ethanol and 3 parts of purified water are weighed by a three-neck flask, are placed in a water bath after being mixed uniformly by ultrasonic, a stirrer is started to keep a reaction system uniform, nitrogen is introduced for 10min to remove oxygen in the device, the temperature of the water bath is raised to 70 ℃, and the reaction is started.
Step two, a step two is carried out; the methyl methacrylate is further crosslinked and polymerized to enlarge the particle size of the microsphere.
7 parts of methyl methacrylate, 3 parts of ethylene glycol dimethacrylate, 4 parts of azobisisobutyronitrile and 10 parts of polyvinyl alcohol were weighed using a beaker and dissolved uniformly by ultrasonic. After the polymerization system in the first step reacts for 3 hours, adding the polymerization system into the first step; and continuing to react for 3 hours in the reaction system, and keeping the water bath heated and stirred.
Step three, a step of performing; further introducing 2-acrylamide-2-methylpropanesulfonic acid and acrylic acid for polymerization, and polymerizing the two functional groups 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 uniformly dissolving by ultrasonic waves; the water bath heating and stirring reaction are continued in the reaction system for 14h.
Step four, a step four is carried out; and cleaning, namely removing the solvent, the dispersing agent, the excessive monomer and the like in the reaction system. After the three-neck flask is subjected to the three-step reaction, the three-neck flask is taken down and kept stand to room temperature, the reaction liquid is transferred into a centrifuge tube for centrifugation, the supernatant is removed, ethanol is added into the precipitated microspheres for uniform mixing, and the centrifugation is continued to remove the supernatant. And continuing to repeat the steps, and cleaning and centrifuging the microspheres by using ethanol. And (3) washing the microspheres with water and drying to finally obtain the monodisperse strong cation exchange chromatography filler microspheres.
And preparing the obtained filler into a chromatographic column by using a column filling machine, and detecting and analyzing the whole blood sample to obtain a chromatogram with good separation effect, as shown in figure 1.
It is noted that relational terms such as first and second, and the like are 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. Moreover, 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 one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the 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 scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (6)
1. A method for preparing a packing for a glycosylated hemoglobin chromatographic column, which is characterized by comprising the following steps:
step one, a step one; the first monomer is polymerized preliminarily to form microsphere with monodisperse grain diameter, 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, after being dissolved evenly, the mixture is stirred and reacted for 2-3 hours under the condition of water bath heating at 60-75 ℃,
step two, a step two is carried out; the first monomer is further crosslinked and polymerized, the grain diameter of the microsphere is enlarged, 2-10 parts of the first monomer, 1-5 parts of the crosslinking agent, 2-5 parts of the initiator and 5-10 parts of the stabilizer are weighed, after being uniformly dissolved, the mixture is added into the polymerization system of the step one to continue the water bath heating and stirring reaction for 2-3 hours,
step three, a step of performing; further introducing a second monomer and a third monomer for polymerization, polymerizing the two functional groups on the microsphere, 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 into the previous reaction system after the mixture is uniformly dissolved, continuing to perform water bath heating and stirring reaction for 12-16 hours,
step four, a step four is carried out; washing, namely removing solvent, dispersing agent, excessive monomer and the like in a reaction system, repeatedly centrifuging and washing the precipitated polymeric microspheres with ethanol and purified water for 2-3 times through a centrifugal device, and drying to obtain strong cation exchange chromatographic packing containing composite functional groups;
the first monomer is one or more of methyl methacrylate, styrene and glycidyl methacrylate which are used in any combination;
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 more of acrylic acid, 4-pentenoic acid, 3-methyl cinnamic acid, 5-hexenoic acid and 3-butenoic acid;
the second monomer in the mixture of the step three: the ratio of the third monomer is 20:80-80:20.
2. The method for producing a packing for a glycosylated hemoglobin chromatographic column according to claim 1, wherein: the cross-linking agent is one or more of ethylene glycol dimethacrylate and divinylbenzene which are used in any combination.
3. The method for producing a packing for a glycosylated hemoglobin chromatographic column according to claim 1, wherein: the initiator is one or more of azodiisobutyronitrile, dimethyl azodiisobutyrate, benzoyl peroxide and diacyl peroxide.
4. The method for producing a packing for a glycosylated hemoglobin chromatographic column according to claim 1, wherein: the stabilizer is one or more of polyvinyl alcohol, polyoxyethylene, polyethylene glycol, polyvinylpyrrolidone, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl propyl cellulose and sodium polyaspartate.
5. The method for producing a packing for a glycosylated hemoglobin chromatographic column according to claim 1, wherein: the medium is one or more of ethanol, methanol, toluene and deionized water.
6. The method for producing a packing for a glycosylated hemoglobin chromatographic column according to claim 1, wherein: and in the fourth step of cleaning treatment, the reaction liquid after the completion of the third step of reaction is required to be cooled to room temperature.
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