CN104324708B - 一种亲水色谱介质的制备方法 - Google Patents
一种亲水色谱介质的制备方法 Download PDFInfo
- Publication number
- CN104324708B CN104324708B CN201410618281.7A CN201410618281A CN104324708B CN 104324708 B CN104324708 B CN 104324708B CN 201410618281 A CN201410618281 A CN 201410618281A CN 104324708 B CN104324708 B CN 104324708B
- Authority
- CN
- China
- Prior art keywords
- add
- stir
- methanol
- ammonia
- water
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000012501 chromatography medium Substances 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 168
- 238000003756 stirring Methods 0.000 claims abstract description 112
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims abstract description 84
- 229910021529 ammonia Inorganic materials 0.000 claims abstract description 42
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims abstract description 28
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 28
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims abstract description 28
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 28
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000005406 washing Methods 0.000 claims abstract description 15
- 229920005654 Sephadex Polymers 0.000 claims abstract description 14
- 239000012507 Sephadex™ Substances 0.000 claims abstract description 14
- 239000004005 microsphere Substances 0.000 claims abstract description 14
- 229910000027 potassium carbonate Inorganic materials 0.000 claims abstract description 14
- 125000005456 glyceride group Chemical group 0.000 claims abstract description 10
- 239000011159 matrix material Substances 0.000 claims abstract description 5
- JGJLWPGRMCADHB-UHFFFAOYSA-N hypobromite Inorganic materials Br[O-] JGJLWPGRMCADHB-UHFFFAOYSA-N 0.000 claims description 13
- 239000002609 medium Substances 0.000 claims description 8
- 229920001503 Glucan Polymers 0.000 claims description 5
- TWNIBLMWSKIRAT-VFUOTHLCSA-N levoglucosan Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@H]2CO[C@@H]1O2 TWNIBLMWSKIRAT-VFUOTHLCSA-N 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 2
- 239000003292 glue Substances 0.000 claims 1
- 238000001228 spectrum Methods 0.000 claims 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 abstract description 12
- 229910017604 nitric acid Inorganic materials 0.000 abstract description 12
- 229920001184 polypeptide Polymers 0.000 abstract description 2
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 2
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 2
- 102000004169 proteins and genes Human genes 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 2
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 abstract 1
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 abstract 1
- 229910052794 bromium Inorganic materials 0.000 abstract 1
- 125000005395 methacrylic acid group Chemical class 0.000 abstract 1
- 239000003643 water by type Substances 0.000 abstract 1
- 229960000074 biopharmaceutical Drugs 0.000 description 4
- 235000009508 confectionery Nutrition 0.000 description 4
- 239000004519 grease Substances 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 229920002307 Dextran Polymers 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 235000008216 herbs Nutrition 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 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
- 238000004519 manufacturing process Methods 0.000 description 2
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 108090000394 Erythropoietin Proteins 0.000 description 1
- 102000003951 Erythropoietin Human genes 0.000 description 1
- 102000002265 Human Growth Hormone Human genes 0.000 description 1
- 108010000521 Human Growth Hormone Proteins 0.000 description 1
- 239000000854 Human Growth Hormone Substances 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 208000035269 cancer or benign tumor Diseases 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229940105423 erythropoietin Drugs 0.000 description 1
- 229940044627 gamma-interferon Drugs 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000002013 hydrophilic interaction chromatography Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- -1 polyphenol compound Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
Classifications
-
- 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
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
-
- 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
- B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
-
- 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
- B01D15/30—Partition chromatography
- B01D15/305—Hydrophilic interaction chromatography [HILIC]
-
- 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
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
- B01J20/28047—Gels
Landscapes
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Dispersion Chemistry (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
本发明公开了一种亲水色谱介质的制备方法，色谱介质以甲基丙烯酸为配体、以葡聚糖凝胶为基体，其制备方法包括如下的步骤：取30wt％浓度、60‑100μm平均粒径葡聚糖凝胶微球200‑400ml，在分液漏斗上用水和甲醇洗涤多次，转入反应瓶或反应器，加400‑600ml甲醇；加入2‑8g甘油酯，搅拌30‑45min；加入20‑35ml的20wt％氨水，搅拌1‑2h；加甲醇搅拌；用2‑8倍体积的甲醇洗15‑30min，用1‑3倍体积的水洗10‑20min；加200‑400ml水，加入5‑15g碳酸钾，搅拌2‑3h；加入10‑30ml的20％氨水，搅拌1‑2h；加入10‑20ml三氯甲烷搅拌6‑8h；用硝酸调pH2‑4；加入10‑45ml溴水，搅拌2‑3h；加入20‑50g甲基丙烯酸，10‑20ml的20％氨水，搅拌5‑8h；用盐酸调pH2‑4；用10‑20倍体积水洗即得亲水色谱介质。此制备方法制备的色谱介质具有高选择性，特别适合用于多酚蛋白质、亲水性多肽和天然有机小分子的分离纯化。
Description
技术领域
本发明涉及一种亲水色谱介质的制备方法。
背景技术
生物药物生产过程中分离纯化的费用占成本的绝大部分，其中使用最普遍的是色谱技术及其组合，目前所用介质主要是凝胶过滤、离子交换、疏水相互作用、亲和、反相介质，其基质材料有琼脂糖、聚苯乙烯和硅胶等。葡聚糖介质是在生物药物生产中使用最为广泛的基质材料。它是一种天然多糖，具有高孔度；具有高的连通性有效保证了分子内物质传递；大比表面积提供很高的吸附能力；易制备成不同葡聚糖浓度、不同尺寸的球形颗粒以满足不同领域的需求；低的非特异性吸附使应用面较广；在氢氧化钠中稳定可满足苛刻的在位清洗步骤；可通过交联使介质具有刚性；可被大量配基取代而应用于多种色谱技术。
葡聚糖介质已经在生物医药领域被使用了40余年，利用葡聚糖基质介质生产的生物药物种类繁多，例如：γ-干扰素、人生长激素、人血清白蛋白、胰岛素、促红细胞生成素、各种单克隆抗体和疫苗等。这些产品的开发和生产有助于保障公共卫生安全、提高人民生活质量。随着生物药物需求的快速增长、新生物药物种类的增加，对葡聚糖基质介质的需求也将迅猛增长。目前国内有一些企业、研究院所及高校从事该介质的研究开发，但产品种类少、质量稳定性较差、市场占有率非常低，是我国生物技术产业发展急待解决的战略问题。
氢键吸附色谱属于亲水相互作用色谱的一种，是近年来在中药活性成分分离纯化研究过程中提出的概念，常用的氢键吸附色谱介质包括葡聚糖凝胶和聚酰胺，已成功应用于多种中药多酚类化合物的分离纯化，对强极性有机小分子化合物能够实现较好的保留，可分离获得高纯度的中药活性成分。
发明内容
本发明的目的在于提出一种亲水色谱介质的制备方法。
为达此目的，本发明采用以下技术方案：
一种亲水色谱介质的制备方法，色谱介质以甲基丙烯酸为配体、以葡聚糖凝胶为基体，其制备方法包括如下的步骤：取30wt％浓度、60-100μm平均粒径葡聚糖凝胶微球200-400ml，在分液漏斗上用水和甲醇洗涤多次，转入反应瓶或反应器，加400-600ml甲醇；加入2-8g甘油酯，搅拌30-45min；加入20-35ml的20wt％氨水，搅拌1-2h；加甲醇搅拌；用2-8倍体积的甲醇洗15-30min，用1-3倍体积的水洗10-20min；加200-400ml水，加入5-15g碳酸钾，搅拌2-3h；加入10-30ml的20％氨水，搅拌1-2h；加入10-20ml三氯甲烷搅拌6-8h；用硝酸调pH 2-4；加入10-45ml溴水，搅拌2-3h；加入20-50g甲基丙烯酸，10-20ml的20％氨水，搅拌5-8h；用盐酸调pH 2-4；用10-20倍体积水洗即得亲水色谱介质。
此制备方法制备的色谱介质具有高选择性，特别适合用于多酚蛋白质、亲水性多肽和天然有机小分子的分离纯化。
具体实施方式
实施例1
取30wt％浓度、60-100μm平均粒径葡聚糖凝胶微球250ml，在分液漏斗上用水和甲醇洗涤多次，转入反应瓶或反应器，加400-600ml甲醇；加入2-8g甘油酯，搅拌30-45min；加入20-35ml的20wt％氨水，搅拌1-2h；加甲醇搅拌；用2-8倍体积的甲醇洗15-30min，用1-3倍体积的水洗10-20min；加200-400ml水，加入5-15g碳酸钾，搅拌2-3h；加入10-30ml的20％氨水，搅拌1-2h；加入10-20ml三氯甲烷搅拌6-8h；用硝酸调pH 2-4；加入10-45ml溴水，搅拌2-3h；加入20-50g甲基丙烯酸，10-20ml的20％氨水，搅拌5-8h；用盐酸调pH 2-4；用10-20倍体积水洗即得亲水色谱介质。
实施例2
取30wt％浓度、60-100μm平均粒径葡聚糖凝胶微球300ml，在分液漏斗上用水和甲醇洗涤多次，转入反应瓶或反应器，加400-600ml甲醇；加入2-8g甘油酯，搅拌30-45min；加入20-35ml的20wt％氨水，搅拌1-2h；加甲醇搅拌；用2-8倍体积的甲醇洗15-30min，用1-3倍体积的水洗10-20min；加200-400ml水，加入5-15g碳酸钾，搅拌2-3h；加入10-30ml的20％氨水，搅拌1-2h；加入10-20ml三氯甲烷搅拌6-8h；用硝酸调pH 2-4；加入10-45ml溴水，搅拌2-3h；加入20-50g甲基丙烯酸，10-20ml的20％氨水，搅拌5-8h；用盐酸调pH 2-4；用10-20倍体积水洗即得亲水色谱介质。
实施例3
取30wt％浓度、60-100μm平均粒径葡聚糖凝胶微球350ml，在分液漏斗上用水和甲醇洗涤多次，转入反应瓶或反应器，加400-600ml甲醇；加入2-8g甘油酯，搅拌30-45min；加入20-35ml的20wt％氨水，搅拌1-2h；加甲醇搅拌；用2-8倍体积的甲醇洗15-30min，用1-3倍体积的水洗10-20min；加200-400ml水，加入5-15g碳酸钾，搅拌2-3h；加入10-30ml的20％氨水，搅拌1-2h；加入10-20ml三氯甲烷搅拌6-8h；用硝酸调pH 2-4；加入10-45ml溴水，搅拌2-3h；加入20-50g甲基丙烯酸，10-20ml的20％氨水，搅拌5-8h；用盐酸调pH 2-4；用10-20倍体积水洗即得亲水色谱介质。
实施例4
取30wt％浓度、60-100μm平均粒径葡聚糖凝胶微球400ml，在分液漏斗上用水和甲醇洗涤多次，转入反应瓶或反应器，加400-600ml甲醇；加入2-8g甘油酯，搅拌30-45min；加入20-35ml的20wt％氨水，搅拌1-2h；加甲醇搅拌；用2-8倍体积的甲醇洗15-30min，用1-3倍体积的水洗10-20min；加200-400ml水，加入5-15g碳酸钾，搅拌2-3h；加入10-30ml的20％氨水，搅拌1-2h；加入10-20ml三氯甲烷搅拌6-8h；用硝酸调pH 2-4；加入10-45ml溴水，搅拌2-3h；加入20-50g甲基丙烯酸，10-20ml的20％氨水，搅拌5-8h；用盐酸调pH 2-4；用10-20倍体积水洗即得亲水色谱介质。
实施例5
取30wt％浓度、60-100μm平均粒径葡聚糖凝胶微球200-400ml，在分液漏斗上用水和甲醇洗涤多次，转入反应瓶或反应器，加400-600ml甲醇；加入2g甘油酯，搅拌30-45min；加入20-35ml的20wt％氨水，搅拌1-2h；加甲醇搅拌；用2-8倍体积的甲醇洗15-30min，用1-3倍体积的水洗10-20min；加200-400ml水，加入5-15g碳酸钾，搅拌2-3h；加入10-30ml的20％氨水，搅拌1-2h；加入10-20ml三氯甲烷搅拌6-8h；用硝酸调pH 2-4；加入10-45ml溴水，搅拌2-3h；加入20-50g甲基丙烯酸，10-20ml的20％氨水，搅拌5-8h；用盐酸调pH 2-4；用10-20倍体积水洗即得亲水色谱介质。
实施例6
取30wt％浓度、60-100μm平均粒径葡聚糖凝胶微球200-400ml，在分液漏斗上用水和甲醇洗涤多次，转入反应瓶或反应器，加400-600ml甲醇；加入4g甘油酯，搅拌30-45min；加入20-35ml的20wt％氨水，搅拌1-2h；加甲醇搅拌；用2-8倍体积的甲醇洗15-30min，用1-3倍体积的水洗10-20min；加200-400ml水，加入5-15g碳酸钾，搅拌2-3h；加入10-30ml的20％氨水，搅拌1-2h；加入10-20ml三氯甲烷搅拌6-8h；用硝酸调pH 2-4；加入10-45ml溴水，搅拌2-3h；加入20-50g甲基丙烯酸，10-20ml的20％氨水，搅拌5-8h；用盐酸调pH 2-4；用10-20倍体积水洗即得亲水色谱介质。
实施例7
取30wt％浓度、60-100μm平均粒径葡聚糖凝胶微球200-400ml，在分液漏斗上用水和甲醇洗涤多次，转入反应瓶或反应器，加400-600ml甲醇；加入6g甘油酯，搅拌30-45min；加入20-35ml的20wt％氨水，搅拌1-2h；加甲醇搅拌；用2-8倍体积的甲醇洗15-30min，用1-3倍体积的水洗10-20min；加200-400ml水，加入5-15g碳酸钾，搅拌2-3h；加入10-30ml的20％氨水，搅拌1-2h；加入10-20ml三氯甲烷搅拌6-8h；用硝酸调pH 2-4；加入10-45ml溴水，搅拌2-3h；加入20-50g甲基丙烯酸，10-20ml的20％氨水，搅拌5-8h；用盐酸调pH 2-4；用10-20倍体积水洗即得亲水色谱介质。
实施例8
取30wt％浓度、60-100μm平均粒径葡聚糖凝胶微球200-400ml，在分液漏斗上用水和甲醇洗涤多次，转入反应瓶或反应器，加400-600ml甲醇；加入8g甘油酯，搅拌30-45min；加入20-35ml的20wt％氨水，搅拌1-2h；加甲醇搅拌；用2-8倍体积的甲醇洗15-30min，用1-3倍体积的水洗10-20min；加200-400ml水，加入5-15g碳酸钾，搅拌2-3h；加入10-30ml的20％氨水，搅拌1-2h；加入10-20ml三氯甲烷搅拌6-8h；用硝酸调pH 2-4；加入10-45ml溴水，搅拌2-3h；加入20-50g甲基丙烯酸，10-20ml的20％氨水，搅拌5-8h；用盐酸调pH 2-4；用10-20倍体积水洗即得亲水色谱介质。
实施例9
取30wt％浓度、60-100μm平均粒径葡聚糖凝胶微球200-400ml，在分液漏斗上用水和甲醇洗涤多次，转入反应瓶或反应器，加400-600ml甲醇；加入2-8g甘油酯，搅拌30-45min；加入20-35ml的20wt％氨水，搅拌1-2h；加甲醇搅拌；用2-8倍体积的甲醇洗15-30min，用1-3倍体积的水洗10-20min；加200-400ml水，加入5-15g碳酸钾，搅拌2-3h；加入10-30ml的20％氨水，搅拌1-2h；加入10-20ml三氯甲烷搅拌6-8h；用硝酸调pH 2-4；加入10-45ml溴水，搅拌2-3h；加入20g甲基丙烯酸，10-20ml的20％氨水，搅拌5-8h；用盐酸调pH 2-4；用10-20倍体积水洗即得亲水色谱介质。
实施例10
取30wt％浓度、60-100μm平均粒径葡聚糖凝胶微球200-400ml，在分液漏斗上用水和甲醇洗涤多次，转入反应瓶或反应器，加400-600ml甲醇；加入2-8g甘油酯，搅拌30-45min；加入20-35ml的20wt％氨水，搅拌1-2h；加甲醇搅拌；用2-8倍体积的甲醇洗15-30min，用1-3倍体积的水洗10-20min；加200-400ml水，加入5-15g碳酸钾，搅拌2-3h；加入10-30ml的20％氨水，搅拌1-2h；加入10-20ml三氯甲烷搅拌6-8h；用硝酸调pH 2-4；加入10-45ml溴水，搅拌2-3h；加入30g甲基丙烯酸，10-20ml的20％氨水，搅拌5-8h；用盐酸调pH 2-4；用10-20倍体积水洗即得亲水色谱介质。
实施例11
取30wt％浓度、60-100μm平均粒径葡聚糖凝胶微球200-400ml，在分液漏斗上用水和甲醇洗涤多次，转入反应瓶或反应器，加400-600ml甲醇；加入2-8g甘油酯，搅拌30-45min；加入20-35ml的20wt％氨水，搅拌1-2h；加甲醇搅拌；用2-8倍体积的甲醇洗15-30min，用1-3倍体积的水洗10-20min；加200-400ml水，加入5-15g碳酸钾，搅拌2-3h；加入10-30ml的20％氨水，搅拌1-2h；加入10-20ml三氯甲烷搅拌6-8h；用硝酸调pH 2-4；加入10-45ml溴水，搅拌2-3h；加入40g甲基丙烯酸，10-20ml的20％氨水，搅拌5-8h；用盐酸调pH 2-4；用10-20倍体积水洗即得亲水色谱介质。
Claims (1)
1.一种亲水色谱介质的制备方法，色谱介质以甲基丙烯酸为配体、以葡聚糖凝胶为基体，其特征在于制备方法包括如下的步骤：取30wt％浓度、60-100μm平均粒径葡聚糖凝胶微球200-400ml，在分液漏斗上用水和甲醇洗涤多次，转入反应器，加400-600ml甲醇；加入2-8g甘油酯，搅拌30-45min；加入20-35ml的20wt％氨水，搅拌1-2h；加甲醇搅拌；用2-8倍体积的甲醇洗15-30min，用1-3倍体积的水洗10-20min；加200-400ml水，加入5-15g碳酸钾，搅拌2-3h；加入10-30ml的20wt％氨水，搅拌1-2h；加入10-20ml三氯甲烷搅拌6-8h；用硝酸调pH 2-4；加入10-45ml溴水，搅拌2-3h；加入20-50g甲基丙烯酸，10-20ml的20wt％氨水，搅拌5-8h；用盐酸调pH 2-4；用10-20倍体积水洗即得亲水色谱介质。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410618281.7A CN104324708B (zh) | 2014-11-05 | 2014-11-05 | 一种亲水色谱介质的制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410618281.7A CN104324708B (zh) | 2014-11-05 | 2014-11-05 | 一种亲水色谱介质的制备方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104324708A CN104324708A (zh) | 2015-02-04 |
| CN104324708B true CN104324708B (zh) | 2016-08-24 |
Family
ID=52399586
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410618281.7A Active CN104324708B (zh) | 2014-11-05 | 2014-11-05 | 一种亲水色谱介质的制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104324708B (zh) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105289542A (zh) * | 2015-11-13 | 2016-02-03 | 无锡清杨机械制造有限公司 | 一种亲水改性膨润土的制备方法 |
| CN105457594A (zh) * | 2015-11-13 | 2016-04-06 | 无锡清杨机械制造有限公司 | 一种亲水磁性活性炭的制备方法 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4794177A (en) * | 1984-12-29 | 1988-12-27 | Ceskoslovenska Akademie Ved | Method for the production of bead dextran materials for gel chromatography |
| US6284250B1 (en) * | 1998-02-05 | 2001-09-04 | The Henry M. Jackson Foundation For The Advancement Of Military Medicine | Simplified method for removing free protein during preparation of protein-polysaccharide conjugates and vaccines using restricted-access media |
| CN1868577A (zh) * | 2005-05-25 | 2006-11-29 | 上海医药工业研究院 | 葡聚糖凝胶介质及其制备方法 |
| CN101252986A (zh) * | 2005-08-31 | 2008-08-27 | 通用电气健康护理生物科学股份公司 | 色谱基体的制造 |
-
2014
- 2014-11-05 CN CN201410618281.7A patent/CN104324708B/zh active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4794177A (en) * | 1984-12-29 | 1988-12-27 | Ceskoslovenska Akademie Ved | Method for the production of bead dextran materials for gel chromatography |
| US6284250B1 (en) * | 1998-02-05 | 2001-09-04 | The Henry M. Jackson Foundation For The Advancement Of Military Medicine | Simplified method for removing free protein during preparation of protein-polysaccharide conjugates and vaccines using restricted-access media |
| CN1868577A (zh) * | 2005-05-25 | 2006-11-29 | 上海医药工业研究院 | 葡聚糖凝胶介质及其制备方法 |
| CN101252986A (zh) * | 2005-08-31 | 2008-08-27 | 通用电气健康护理生物科学股份公司 | 色谱基体的制造 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104324708A (zh) | 2015-02-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104372045B (zh) | 一种高纯度萝卜硫素的制备方法 | |
| CN105001348B (zh) | 一种高得率高岩藻糖比例的岩藻多糖的提取方法 | |
| CN104324708B (zh) | 一种亲水色谱介质的制备方法 | |
| CN103467617A (zh) | 一种连续逆流超声提取高纯度黄芪多糖的方法 | |
| CN110256600A (zh) | 一种分步提取富硒堇叶碎米荠中蛋白质和多糖的方法 | |
| CN104498564A (zh) | 一种低分子量硫酸软骨素的制备方法 | |
| CN104306428B (zh) | 一种从绞股蓝中提取纯化绞股蓝皂苷的方法 | |
| CN101108871A (zh) | 从大枣中提取环磷酸腺苷的工艺方法 | |
| CN102580687A (zh) | 一种多胺改性壳聚糖基膨胀床吸附介质及其制备方法 | |
| CN103342668A (zh) | 一种从鲍鱼内脏中提取天然牛磺酸的简易方法 | |
| CN108299547A (zh) | 一种从虎奶菇菌核中制备糖蛋白的方法 | |
| CN1869055A (zh) | 一种从人参叶中提取分离人参皂苷单体的方法 | |
| CN103787885B (zh) | 一种从金银花地上部分提取高纯度绿原酸的方法 | |
| CN109082452A (zh) | 一种液-液-固体系酶催化水解萃取分离桑椹红色素的方法 | |
| CN104262451B (zh) | 一种从海参加工废液中提取海参皂苷的方法 | |
| CN101504398A (zh) | 一种三聚氰胺配基的琼脂糖亲水吸附色谱介质的合成方法 | |
| CN101530783A (zh) | 一种没食子酸配基的琼脂糖亲水吸附色谱介质的合成方法 | |
| CN102964197A (zh) | 一种氨基酸水解液的脱色处理方法 | |
| CN101559356A (zh) | 一种三羟甲基氨基甲烷配基琼脂糖亲水色谱介质合成方法 | |
| CN103570548B (zh) | 一种丹酚酸a的制备方法 | |
| CN101856382B (zh) | 一种提取分离桔梗皂苷有效成分的方法 | |
| CN104497632B (zh) | 一种利用大孔树脂-活性炭复合吸附剂纯化红继木花红色素的方法 | |
| CN108059673A (zh) | 一种人血清中分离免疫球蛋白IgG的方法 | |
| CN102993267A (zh) | 一种灵芝活性肽的制取方法 | |
| CN110845314A (zh) | 一种生姜次生代谢物的提取方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20170405 Address after: 226100 Guangzhou road Jiangsu city Nantong Province Haimen Economic and Technological Development Zone No. 999 Patentee after: Nantong Construction Engineering Co., Ltd. Address before: Lake town of Xishan District 214000 Jiangsu city of Wuxi Province Green River Dang Village No. 11 Patentee before: Hua Yuye |
|
| TR01 | Transfer of patent right |