CN104324708A - 一种亲水色谱介质的制备方法 - Google Patents
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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。
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| 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 |
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