CN102513071B - Novel high performance liquid chromatography medium and preparation method thereof - Google Patents
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- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 23
- 239000012501 chromatography medium Substances 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims abstract description 54
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 29
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 27
- 239000003446 ligand Substances 0.000 claims abstract description 22
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 14
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 13
- 239000002609 medium Substances 0.000 claims abstract description 6
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 6
- 239000000203 mixture Substances 0.000 claims abstract description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 28
- 238000001816 cooling Methods 0.000 claims description 14
- 238000010992 reflux Methods 0.000 claims description 11
- 238000003756 stirring Methods 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N pyridine Substances C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims 5
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims 5
- 229910052799 carbon Inorganic materials 0.000 claims 1
- 238000010792 warming Methods 0.000 claims 1
- 239000004005 microsphere Substances 0.000 abstract description 16
- 102000004169 proteins and genes Human genes 0.000 abstract description 14
- 108090000623 proteins and genes Proteins 0.000 abstract description 14
- 125000004076 pyridyl group Chemical group 0.000 abstract description 12
- 230000002209 hydrophobic effect Effects 0.000 abstract description 11
- 238000000926 separation method Methods 0.000 abstract description 7
- 238000001179 sorption measurement Methods 0.000 abstract description 7
- 230000008901 benefit Effects 0.000 abstract description 4
- 238000010828 elution Methods 0.000 abstract description 4
- 239000011159 matrix material Substances 0.000 abstract description 4
- 239000003795 chemical substances by application Substances 0.000 abstract description 3
- 229910021426 porous silicon Inorganic materials 0.000 abstract description 3
- 238000000746 purification Methods 0.000 abstract description 2
- 235000012239 silicon dioxide Nutrition 0.000 abstract description 2
- 238000001914 filtration Methods 0.000 description 13
- 239000003960 organic solvent Substances 0.000 description 4
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 125000004105 2-pyridyl group Chemical group N1=C([*])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 125000003349 3-pyridyl group Chemical group N1=C([H])C([*])=C([H])C([H])=C1[H] 0.000 description 1
- 125000000339 4-pyridyl group Chemical group N1=C([H])C([H])=C([*])C([H])=C1[H] 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- -1 Hydroxy-ethyltrimethoxysilane Chemical compound 0.000 description 1
- TXUZVXJJXXKPBH-UHFFFAOYSA-N [butyl(dimethoxy)silyl]oxymethanol Chemical compound OCO[Si](OC)(OC)CCCC TXUZVXJJXXKPBH-UHFFFAOYSA-N 0.000 description 1
- GLZPKHIDCKLLFQ-UHFFFAOYSA-N [dimethoxy(propyl)silyl]oxymethanol Chemical compound CCC[Si](OC)(OC)OCO GLZPKHIDCKLLFQ-UHFFFAOYSA-N 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
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- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
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- Pyridine Compounds (AREA)
Abstract
本发明公开了一种新型高效液相色谱介质及其制备方法。介质的组成中基质为多孔二氧化硅微球，功能配基为通过硅烷化试剂引入的吡啶基。制备方法如下，将多孔氧化硅微球充分干燥后，在氮气保护下，于甲苯中与具有吡啶基的硅烷化试剂进行加热反应，即可得到疏水性电荷诱导型高效液相色谱介质。本发明所研制的高效液相色谱介质，具有疏水性电荷诱导分离原理，具有制备方法简单、成本低廉、配基密度高、蛋白吸附量大、特异性强和吸附洗脱条件温和等优点，可用于蛋白质的高效分离纯化。The invention discloses a novel high performance liquid chromatography medium and a preparation method thereof. In the composition of the medium, the matrix is porous silicon dioxide microspheres, and the functional ligand is pyridyl introduced by a silylating agent. The preparation method is as follows. After the porous silicon oxide microspheres are fully dried, under the protection of nitrogen, they are heated and reacted with a pyridyl silylating reagent in toluene to obtain a hydrophobic charge-induced high performance liquid chromatography medium. The high-performance liquid chromatography medium developed by the present invention has the principle of hydrophobic charge-induced separation, and has the advantages of simple preparation method, low cost, high ligand density, large protein adsorption capacity, strong specificity, and mild adsorption and elution conditions. for efficient separation and purification of proteins.
Description
技术领域 technical field
本发明涉及一种新型高效液相色谱介质及其制备方法The invention relates to a novel high-performance liquid chromatography medium and a preparation method thereof
背景技术 Background technique
高效液相色谱(HPLC)是一种强有力并被广泛使用的纯化和分析复杂组分的技术，而传统的高效液相色谱大部分都是基于反相色谱原理的。反相层析(RPC)是根据蛋白质的高表面张力进行吸附，通过降低表面张力进行洗脱的；层析介质上的非极性基团可以和蛋白质或多肽表面的疏水部位进行相互作用；在水溶液中目标物质具有较高的极性而吸附，通过在洗脱液中添加有机溶剂降低极性达到洗脱的目的。传统的高效液相色谱介质大部分都是通过在介质上连接一定长度的碳链，起功能化作用，目标产物吸附后，再用有机溶剂梯度洗脱，具有分辨率高的优点。但是有机溶剂极易造成蛋白质的变性，使高效液相色谱技术在蛋白质的分离应用受到了限制。High-performance liquid chromatography (HPLC) is a powerful and widely used technique for purifying and analyzing complex components, while most of the traditional high-performance liquid chromatography is based on the principle of reversed-phase chromatography. Reverse-phase chromatography (RPC) is based on the high surface tension of proteins for adsorption and elution by reducing surface tension; the non-polar groups on the chromatography medium can interact with the hydrophobic parts on the surface of proteins or polypeptides; The target substance in the aqueous solution has a high polarity and is adsorbed, and the purpose of elution is achieved by adding an organic solvent to the eluent to reduce the polarity. Most of the traditional high-performance liquid chromatography media are functionalized by connecting a certain length of carbon chain to the media. After the target product is adsorbed, it is eluted with an organic solvent gradient, which has the advantage of high resolution. However, organic solvents can easily cause protein denaturation, which limits the application of high performance liquid chromatography in protein separation.
具有吡啶基的疏水性电荷诱导型配基属于弱碱性化合物，在pH 4-10的范围内不带电荷，可以避免静电作用对杂质的吸附，配基可以通过疏水作用与蛋白质相结合，而当pH值降低到配基的pKa以下时，杂环质子化，若此时蛋白质也带有正电荷，可通过静电排斥作用实现洗脱。这种以疏水作用吸附、静电排斥协助解析的过程是HCIC的最大特征，可通过将流动相pH降低到4.0-4.55进行洗脱，避免反相色谱中有机溶剂对蛋白质活性的液相。由此可见，疏水性电荷诱导层析是一种应用广阔的新型层析分离方法。Hydrophobic charge-inducible ligands with pyridyl groups are weakly basic compounds, which are uncharged in the range of pH 4-10, which can avoid the adsorption of impurities by electrostatic interaction. Ligands can bind to proteins through hydrophobic interactions, while When the pH value drops below the pKa of the ligand, the heterocycle is protonated, and if the protein is also positively charged at this time, it can be eluted by electrostatic repulsion. This process of adsorption by hydrophobic interaction and electrostatic repulsion to assist analysis is the biggest feature of HCIC. It can be eluted by reducing the pH of the mobile phase to 4.0-4.55, avoiding the liquid phase where organic solvents are active on proteins in reversed-phase chromatography. It can be seen that hydrophobic charge induction chromatography is a new type of chromatographic separation method with wide application.
然而大部分的疏水性电荷诱导型层析介质，都以琼脂糖、纤维素等水凝胶为基质材料的，很难应用于高效液相色谱中。氧化硅具有机械强度高、孔隙率和比表面积大、极性可调节和表面易修饰等优点，因而被广泛地用作高效液相色谱介质的基质材料，可通过硅烷化试剂引入所需的配基来制备高效液相色谱介质。However, most of the hydrophobic charge-induced chromatographic media are based on agarose, cellulose and other hydrogels as matrix materials, which are difficult to apply in high performance liquid chromatography. Silica has the advantages of high mechanical strength, large porosity and specific surface area, adjustable polarity and easy surface modification, so it is widely used as a matrix material for high-performance liquid chromatography media, and the required ingredients can be introduced through silylating reagents. base for the preparation of high performance liquid chromatography media.
因此，本发明将新型疏水性电荷诱导型配基引入高效液相色谱技术中，以二氧化硅微球为载体，通过硅烷化试剂引入吡啶基，可以提高对蛋白质的选择性和分辨率，并可在较温和的条件下洗脱，形成一种高效的蛋白质分离新方法，有效促进蛋白质分离工艺的改进和提高。Therefore, the present invention introduces novel hydrophobic charge-inducible ligands into high-performance liquid chromatography technology, uses silica microspheres as a carrier, and introduces pyridyl groups through silylating reagents, which can improve the selectivity and resolution of proteins, and It can be eluted under milder conditions, forming a new efficient method for protein separation, and effectively promoting the improvement and improvement of protein separation technology.
发明内容 Contents of the invention
本发明的目的是提供一种新型高效液相色谱介质及其制备方法。The purpose of the present invention is to provide a novel high-performance liquid chromatography medium and a preparation method thereof.
新型高效液相色谱介质，其介质的组成中基质为多孔二氧化硅微球，功能配基为通过硅烷化试剂引入的吡啶基，具有吡啶基的硅烷化试剂的结构为：A new type of high performance liquid chromatography medium, the matrix in the composition of the medium is porous silica microspheres, the functional ligand is a pyridyl group introduced by a silylating reagent, and the structure of the silylating reagent with a pyridyl group is:
所述的多孔二氧化硅微球的粒径为5-30μm。具有吡啶基的硅烷化试剂的吡啶基包括2-吡啶基、3-吡啶基和4-吡啶基。具有吡啶基的硅烷化试剂的结构中的R构成了碳链，R构成单元为CH2或CHOH，长度为0-6个碳。配基密度为5-150μmol/mL介质。The particle size of the porous silicon dioxide microspheres is 5-30 μm. The pyridyl group of the silylating agent having a pyridyl group includes 2-pyridyl, 3-pyridyl and 4-pyridyl. The R in the structure of the pyridyl silylating agent constitutes a carbon chain, the R constituent unit is CH2 or CHOH, and the length is 0-6 carbons. The ligand density is 5-150μmol/mL medium.
新型高效液相色谱介质的制备方法如下：取10-20mL多孔氧化硅微球于110℃烘箱中充分干燥24h，冷却后转移至250mL圆底烧瓶中；再加入100-180mL甲苯、2-10g具有吡啶基的硅烷化试剂，通入氮气；升温至50-150℃，100-250r/min下搅拌回流5-24h；反应完毕后，过滤收集微球，用甲苯和丙酮清洗并烘干。The preparation method of the new high-performance liquid chromatography medium is as follows: take 10-20mL porous silica microspheres and fully dry them in an oven at 110°C for 24 hours, then transfer them to a 250mL round-bottomed flask after cooling; then add 100-180mL toluene, 2-10g For pyridyl silylating reagents, nitrogen gas is introduced; the temperature is raised to 50-150°C, stirred and refluxed at 100-250r/min for 5-24h; after the reaction is completed, the microspheres are collected by filtration, washed with toluene and acetone and dried.
本发明所研制的高效液相色谱介质，具有疏水性电荷诱导分离原理，具有制备方法简单、成本低廉、配基密度高、蛋白吸附量大、特异性强和吸附洗脱条件温和等优点，可用于蛋白质的高效分离纯化。制备工艺的关键是：将多孔氧化硅微球充分干燥后，在氮气保护下，于甲苯中与具有吡啶基的硅烷化试剂进行加热反应，一步即可得到新型疏水性电荷诱导型高效液相色谱介质。The high-performance liquid chromatography medium developed by the present invention has the principle of hydrophobic charge-induced separation, and has the advantages of simple preparation method, low cost, high ligand density, large protein adsorption capacity, strong specificity, and mild adsorption and elution conditions. for efficient separation and purification of proteins. The key to the preparation process is: after the porous silica microspheres are fully dried, under the protection of nitrogen, they are heated and reacted with pyridyl silylating reagents in toluene, and a new type of hydrophobic charge-induced high performance liquid chromatography can be obtained in one step. medium.
具体实施方式 Detailed ways
以下通过实施例对本发明作进一步的描述：The present invention will be further described below by embodiment:
实施例1Example 1
取10mL多孔氧化硅微球于110℃烘箱中充分干燥24h，冷却后转移至250mL圆底烧瓶中；再加入150mL甲苯、2g具有吡啶基的硅烷化试剂2-(2-吡啶基)乙基三甲氧基硅烷，通入氮气；升温至80℃，200r/min下搅拌回流12h；反应完毕后，过滤收集微球，用甲苯和丙酮清洗并烘干，配基密度为77μmol/mL。Take 10mL of porous silica microspheres and fully dry them in an oven at 110°C for 24h, then transfer them to a 250mL round-bottomed flask after cooling; Oxysilane, blown nitrogen; heat up to 80°C, stir and reflux at 200r/min for 12h; after the reaction, collect the microspheres by filtration, wash with toluene and acetone and dry, the ligand density is 77μmol/mL.
实施例2Example 2
取10mL多孔氧化硅微球于110℃烘箱中充分干燥24h，冷却后转移至250mL圆底烧瓶中；再加入150mL甲苯、3g具有吡啶基的硅烷化试剂2-(3-吡啶基)乙基三甲氧基硅烷，通入氮气；升温至85℃，200r/min下搅拌回流10h；反应完毕后，过滤收集微球，用甲苯和丙酮清洗并烘干，配基密度为79μmol/mL。Take 10mL of porous silica microspheres and fully dry them in an oven at 110°C for 24h, then transfer them to a 250mL round bottom flask after cooling; Oxysilane, blown nitrogen; heat up to 85°C, stir and reflux at 200r/min for 10h; after the reaction, collect the microspheres by filtration, wash with toluene and acetone and dry, the ligand density is 79μmol/mL.
实施例3Example 3
取10mL多孔氧化硅微球于110℃烘箱中充分干燥24h，冷却后转移至250mL圆底烧瓶中；再加入150mL甲苯、3g具有吡啶基的硅烷化试剂2-(4-吡啶基)乙基三甲氧基硅烷，通入氮气；升温至90℃，200r/min下搅拌回流10h；反应完毕后，过滤收集微球，用甲苯和丙酮清洗并烘干，配基密度为91μmol/mL。Take 10mL of porous silica microspheres and fully dry them in an oven at 110°C for 24h, then transfer them to a 250mL round-bottomed flask after cooling; Oxysilane, blown nitrogen; heat up to 90°C, stir and reflux at 200r/min for 10h; after the reaction, collect the microspheres by filtration, wash with toluene and acetone and dry, the ligand density is 91μmol/mL.
实施例4Example 4
取10mL多孔氧化硅微球于110℃烘箱中充分干燥24h，冷却后转移至250mL圆底烧瓶中；再加入150mL甲苯、3g具有吡啶基的硅烷化试剂4-(2-吡啶基)丁基三甲氧基硅烷，通入氮气；升温至80℃，250r/min下搅拌回流20h；反应完毕后，过滤收集微球，用甲苯和丙酮清洗并烘干，配基密度为105μmol/mL。Take 10mL of porous silica microspheres and fully dry them in an oven at 110°C for 24h, then transfer them to a 250mL round-bottomed flask after cooling; Oxysilane, blown nitrogen; heat up to 80°C, stir and reflux at 250r/min for 20h; after the reaction, collect the microspheres by filtration, wash with toluene and acetone and dry, the ligand density is 105μmol/mL.
实施例5Example 5
取15mL多孔氧化硅微球于110℃烘箱中充分干燥24h，冷却后转移至250mL圆底烧瓶中；再加入180mL甲苯、6g具有吡啶基的硅烷化试剂6-(3-吡啶基)己基三甲氧基硅烷，通入氮气；升温至80℃，250r/min下搅拌回流24h；反应完毕后，过滤收集微球，用甲苯和丙酮清洗并烘干，配基密度为131μmol/mL。Take 15mL of porous silica microspheres and fully dry them in an oven at 110°C for 24h, then transfer them to a 250mL round bottom flask after cooling; The temperature was raised to 80°C, and stirred and refluxed at 250r/min for 24h; after the reaction was completed, the microspheres were collected by filtration, washed with toluene and acetone and dried. The ligand density was 131μmol/mL.
实施例6Example 6
取15mL多孔氧化硅微球于110℃烘箱中充分干燥24h，冷却后转移至250mL圆底烧瓶中；再加入180mL甲苯、5g具有吡啶基的硅烷化试剂5-(3-吡啶基)戊基三甲氧基硅烷，通入氮气；升温至70℃，250r/min下搅拌回流20h；反应完毕后，过滤收集微球，用甲苯和丙酮清洗并烘干，配基密度为122μmol/mL。Take 15mL of porous silica microspheres and fully dry them in an oven at 110°C for 24h, then transfer them to a 250mL round bottom flask after cooling; Oxysilane, blown nitrogen; heat up to 70°C, stir and reflux at 250r/min for 20h; after the reaction, collect the microspheres by filtration, wash with toluene and acetone and dry, the ligand density is 122μmol/mL.
实施例7Example 7
取20mL多孔氧化硅微球于110℃烘箱中充分干燥24h，冷却后转移至250mL圆底烧瓶中；再加入180mL甲苯、7g具有吡啶基的硅烷化试剂3-(2-吡啶基)丙基三甲氧基硅烷，通入氮气；升温至75℃，250r/min下搅拌回流20h；反应完毕后，过滤收集微球，用甲苯和丙酮清洗并烘干，配基密度为141μmol/mL。Take 20mL of porous silica microspheres and fully dry them in an oven at 110°C for 24h, then transfer them to a 250mL round-bottomed flask after cooling; Oxysilane, blown nitrogen; heat up to 75°C, stir and reflux at 250r/min for 20h; after the reaction, collect the microspheres by filtration, wash with toluene and acetone and dry, the ligand density is 141μmol/mL.
实施例8Example 8
取15mL多孔氧化硅微球于110℃烘箱中充分干燥24h，冷却后转移至250mL圆底烧瓶中；再加入180mL甲苯、7g具有吡啶基的硅烷化试剂3-(3-吡啶基)丙基三甲氧基硅烷，通入氮气；升温至70℃，250r/min下搅拌回流20h；反应完毕后，过滤收集微球，用甲苯和丙酮清洗并烘干，配基密度为121μmol/mL。Take 15mL of porous silica microspheres and fully dry them in an oven at 110°C for 24h, then transfer them to a 250mL round bottom flask after cooling; Oxysilane, blown nitrogen; heat up to 70°C, stir and reflux at 250r/min for 20h; after the reaction, collect the microspheres by filtration, wash with toluene and acetone and dry, the ligand density is 121μmol/mL.
实施例9Example 9
取15mL多孔氧化硅微球于110℃烘箱中充分干燥24h，冷却后转移至250mL圆底烧瓶中；再加入180mL甲苯、8g具有吡啶基的硅烷化试剂3-(4-吡啶基)丙基三甲氧基硅烷，通入氮气；升温至80℃，250r/min下搅拌回流20h；反应完毕后，过滤收集微球，用甲苯和丙酮清洗并烘干，配基密度为97μmol/mL。Take 15mL of porous silica microspheres and fully dry them in an oven at 110°C for 24h, then transfer them to a 250mL round bottom flask after cooling; Oxysilane, blown nitrogen; heat up to 80°C, stir and reflux at 250r/min for 20h; after the reaction, collect the microspheres by filtration, wash with toluene and acetone and dry, the ligand density is 97μmol/mL.
实施例10Example 10
取15mL多孔氧化硅微球于110℃烘箱中充分干燥24h，冷却后转移至250mL圆底烧瓶中；再加入180mL甲苯、8g具有吡啶基的硅烷化试剂3-(4-吡啶基)-2-羟基-丙基三甲氧基硅烷，通入氮气；升温至80℃，250r/min下搅拌回流20h；反应完毕后，过滤收集微球，用甲苯和丙酮清洗并烘干，配基密度为108μmol/mL。Take 15mL of porous silica microspheres and fully dry them in an oven at 110°C for 24h, then transfer them to a 250mL round-bottomed flask after cooling; Hydroxy-propyltrimethoxysilane, blown nitrogen; heat up to 80°C, stir and reflux at 250r/min for 20h; after the reaction, collect the microspheres by filtration, wash with toluene and acetone and dry, the ligand density is 108μmol/ mL.
实施例11Example 11
取15mL多孔氧化硅微球于110℃烘箱中充分干燥24h，冷却后转移至250mL圆底烧瓶中；再加入180mL甲苯、8g具有吡啶基的硅烷化试剂4-(4-吡啶基)-3-羟基-丁基三甲氧基硅烷，通入氮气；升温至80℃，250r/min下搅拌回流20h；反应完毕后，过滤收集微球，用甲苯和丙酮清洗并烘干，配基密度为110μmol/mL。Take 15mL of porous silica microspheres and fully dry them in an oven at 110°C for 24h, then transfer them to a 250mL round bottom flask after cooling; Hydroxy-butyltrimethoxysilane, blowing nitrogen; heating up to 80°C, stirring and refluxing at 250r/min for 20h; after the reaction, collect the microspheres by filtration, wash with toluene and acetone and dry, the ligand density is 110μmol/ mL.
实施例12Example 12
取15mL多孔氧化硅微球于110℃烘箱中充分干燥24h，冷却后转移至250mL圆底烧瓶中；再加入180mL甲苯、8g具有吡啶基的硅烷化试剂2-(2-吡啶基)-1-羟基-乙基三甲氧基硅烷，通入氮气；升温至80℃，250r/min下搅拌回流20h；反应完毕后，过滤收集微球，用甲苯和丙酮清洗并烘干，配基密度为132μmol/mL。Take 15mL of porous silica microspheres and fully dry them in an oven at 110°C for 24h, then transfer them to a 250mL round bottom flask after cooling; Hydroxy-ethyltrimethoxysilane, blown with nitrogen; heated to 80°C, stirred and refluxed at 250r/min for 20h; after the reaction was completed, the microspheres were collected by filtration, washed with toluene and acetone and dried, the ligand density was 132μmol/ mL.
基于本发明精神或主要特征的具体形式并不仅限于上述实施例，还可有多种组合或变化，如具有吡啶基的硅烷化试剂的结构、各种时间、试剂种类和添加量等均可在一定程度上进行调整，因此，无论从哪一点来看，本发明的上述实施方式都只能认为是对本发明的说明而不能限制本发明，在与本发明权利要求书相当的含义和范围内的任何变化，都应认为是包括在权利要求书的范围内。The specific forms based on the spirit or main features of the present invention are not limited to the above-mentioned embodiments, and there are also various combinations or changes, such as the structure of the silylating reagent with pyridyl, various times, reagent types and additions, etc. Adjustment to a certain extent, therefore, no matter from which point of view, the above-mentioned embodiments of the present invention can only be considered as descriptions of the present invention and cannot limit the present invention, within the meaning and scope equivalent to the claims of the present invention Any changes should be considered to be included in the scope of the claims.
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| CN1257891A (en) * | 1998-12-21 | 2000-06-28 | 中国科学院化学研究所 | Cross-linking polyvinyl pyridine microspheres for liquid-phase chromatography and its preparing process |
| CN1598570A (en) * | 2004-08-10 | 2005-03-23 | 武汉大学 | Surface silanization regenerated cellulose microsphere filling and preparation method and use thereof |
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| CN1257891A (en) * | 1998-12-21 | 2000-06-28 | 中国科学院化学研究所 | Cross-linking polyvinyl pyridine microspheres for liquid-phase chromatography and its preparing process |
| CN1598570A (en) * | 2004-08-10 | 2005-03-23 | 武汉大学 | Surface silanization regenerated cellulose microsphere filling and preparation method and use thereof |
Non-Patent Citations (3)
| Title |
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| Lucia M.L.A. Auler et al..New stationary phase for anion-exchange chromatography.《journal of chromatography A》.2005,第1073卷147-153. |
| Lucia M.L.A. Auler.New stationary phase for anion-exchange chromatography.《journal of chromatography A》.2005,第1073卷147-153. * |
| New stationary phase for anion-exchange chromatography;Lucia M.L.A. Auler et al.;《journal of chromatography A》;20051231;第1073卷;147-153 * |
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