CN114618451A - 一种弱阳离子交换色谱固定相及其制备及应用 - Google Patents
一种弱阳离子交换色谱固定相及其制备及应用 Download PDFInfo
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/32—Bonded phase chromatography
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B01J2220/00—Aspects relating to sorbent materials
- B01J2220/80—Aspects related to sorbents specially adapted for preparative, analytical or investigative chromatography
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Abstract
本发明涉及液相色谱固定相,键合相末端为羧基基团,键合相中间包括酰胺基团。其结构式如下:其中Silica Gel为硅胶,m=1‑6,R1是氢、苯基、苄基、酰胺、碳数为1~8的烷基链、碳数为1~8的含卤素的烷基链、碳数为2~12的含双键的烷基链中的一种,R2是氢、苯基、苄基、酰胺、碳数为1~8的烷基链、碳数为1~8的含卤素的烷基链、碳数为2~12的含双键的烷基链中的一种。本发明还提供了上述液相色谱固定相的制备方法,首先在硅胶表面引入氨基,然后通过氨基‑酸酐的亲核取代反应,制备得到含酰胺基团和末端可电离的羧基基团的新型固定相。本发明提供的固定相结构新颖,表面带有负电荷,可作为弱阳离子交换色谱固定相,可广泛用于阳离子化合物的分离分析。
Description
技术领域
本发明涉及液相色谱固定相,具体地说是一种键合相末端为羧酸基团并且键合相中间包含酰胺基团的新型阳离子色谱固定相。
技术背景
离子交换色谱利用被分离组分离子交换能力的差别被广泛应用于对离子型物质的分离分析和纯化制备。离子交换色谱的固定相为离子交换剂,常用的有离子交换树脂和化学键合离子交换剂。经典离子交换色谱的固定相为聚合物基质,具有pH适用范围广、稳定性好等优点[Loewus F.A.et al,Analyt.Biochem.1983,130,191-198],其缺点是易于膨胀,传质较慢,柱效低,不耐高压。硅胶基质作为高效液相色谱中最常用的分离材料,具有机械强度高、传质快、柱效高、表面亲水性好、粒径和孔径分布均匀等优点,可以很好的克服聚合物基质的缺陷。目前,硅胶基质在离子交换色谱方面的应用较少。
弱阳离子交换色谱的静电作用较弱,固定相上的酸性基团在pH=4-8之间部分解离,可以通过调控流动相的pH来实现对带正电的离子型化合物或容易离子化的中性化合物的分离;同时在pH<4时,固定相上的酸性基团不解离,可以通过氢键等作用力对中性化合物进行分离分析。
Bai等发展了一种以硅胶为基质、氨基己酸为配基制备了一种新型弱阳离子交换/疏水(WCX/HIC)双功能混合模式色谱固定相,可实现对8种蛋白质的快速分离[Bai,Q.etal,Chin.J.Chromatogr.2016,34,1228-1233]。Sze等报道了一种利用硅胶基键合聚合天冬氨酸的弱阳离子交换色谱柱选择性地保留带正电荷的肽段,同时允许带负电荷的十二烷基硫酸钠在样品加载过程中被冲洗出去,完成在线质谱对蛋白质组的分析研究[Sze,S.K.etal,J.Proteome Res.2018,17,2390-2400]。但是当前尚未出现关于硅胶基质内嵌极性酰胺基团的弱阳离子交换固定相的制备技术。
发明内容
本发明的目的是提供一种新型弱阳离子交换色谱固定相及其制备方法。该固定相末端为羧酸基团,键合相中间包含酰胺基团,其制备方法简单,适用性广泛。
本发明的目技术方案是:弱离子交换色谱固定相,其特征在于结构为:
其中Silica Gel为硅胶的示意(代表硅胶),m=1-6,R1是氢、苯基、苄基、酰胺、碳数为1~8的烷基链、碳数为1~8的含卤素的烷基链、碳数为2~12的含双键的烷基链中的一种,R2是氢、苯基、苄基、酰胺、碳数为1~8的烷基链、碳数为1~8的含卤素的烷基链、碳数为2~12的含双键的烷基链中的一种。
本发明还提供了上述固定相的制备方法,其特征在于包括如下步骤:
a.硅胶表面引入氨基:在氮气或氩气保护下,在乙腈、甲苯或二甲苯有机溶剂中加入硅烷偶联剂和经120-160℃干燥8~18小时的微球形硅胶,在80~130℃下反应8~24小时,过滤,依次用甲醇、甲醇水、甲醇、四氢呋喃洗涤,所得固体于干燥箱中40~80℃条件下干燥8~24小时,既得氨基硅胶;
以每克微球形硅胶计,硅烷偶联剂的用量1-10mmol,有机溶剂4-10mL;
b.羧酸基团键合:在上述制备的氨基硅胶中加入乙腈、甲苯或N,N-二甲基甲酰胺有机溶剂和丁二酸酐,再加入4-二甲氨基吡啶或吡啶,在25~50℃下反应8~24小时,过滤,依次用甲醇、乙酸钠溶液、水、甲醇洗涤,所得固体于干燥箱中40~80℃条件下干燥8~24小时,既得羧酸基固定相。
以每克氨基硅胶计,酸酐的用量0.1-2.4mmol,碱性催化剂的用量0.1-4mmol,有机溶剂4-10mL;
本发明还提供了上述液相色谱固定相的制备方法,首先在硅胶表面引入氨基,然后通过氨基-酸酐的亲核取代反应,制备得到含酰胺基团和末端可电离的羧基基团的新型固定相。本发明提供的固定相结构新颖,表面带有负电荷,可作为弱阳离子交换色谱固定相,可广泛用于阳离子化合物的分离分析。
本发明具有如下优点:
1.结构新颖。本发明首次提出末端为羧酸基团,键合相中间包含酰胺基团的固定相作为弱阳离子交换色谱固定相。该固定相结构中带有羧酸基团,通过pH调控固定相表面可带负电荷,具有静电作用,同时固定相结构中还带有酰胺基团,可以形成氢键作用,十分适合作为阳离子交换色谱固定相。
2.本发明提供的弱阳离子交换固定相对绝大部分阳离子化合物具有很好的分离选择性,可广泛用于各类样品的分离分析及纯化制备。
3.本发明提供的弱阳离子交换固定相制备过程简单可靠,有利于实现产业化。
具体实施方式
下面结合实例,对本发明做进一步说明。实例仅限于说明本发明,而非对本发明的限定。
实施例1
在氮气保护下,向100mL烧瓶中加入10g经160℃干燥16小时的微球形硅胶(粒径为5μm,孔径为10nm)、6mL氨丙基三甲氧基硅烷和60mL二甲苯,在110℃下反应16小时,过滤,依次用甲醇、体积比1:1的甲醇水、甲醇、四氢呋喃洗涤,所得固体在干燥箱中80℃条件下干燥16个小时,既得氨基硅胶。
向250mL烧瓶中加入10g氨基硅胶、3g 4-二甲氨基吡啶、2.4g丁二酸酐(摩尔数为24mmol)和100mL N,N-二甲基甲酰胺在40℃下反应24小时,过滤,依次用甲醇、50mM乙酸钠溶液、水、甲醇洗涤,所得固体在干燥箱中80℃条件下干燥16个小时,既得羧酸基固定相,结构如下:
实施例2
在氮气保护下,向100mL烧瓶中加入10g经160℃干燥16小时的微球形硅胶(粒径为3.5μm,孔径为10nm)、2mL氨丙基三甲氧基硅烷和40mL二甲苯,在80℃下反应16小时,过滤,依次用甲醇、体积比1:1的甲醇水、甲醇、四氢呋喃洗涤,所得固体在干燥箱中60℃条件下干燥24个小时,既得氨基硅胶。
向250mL烧瓶中加入10g氨基硅胶、3mL三乙胺、4g苯基琥珀酸酐(摩尔数为22mmol)和100mL二甲苯在60℃下反应24小时,过滤,依次用甲醇、50mM乙酸钠溶液、水、甲醇洗涤,所得固体在干燥箱中80℃条件下干燥16个小时,既得羧酸基固定相,结构如下:
实施例3
在氮气保护下,向100mL烧瓶中加入10g经160℃干燥16小时的微球形硅胶(粒径为3.5μm,孔径为10nm)、5mL氨丙基三甲氧基硅烷和100mL甲苯,在110℃下反应16小时,过滤,依次用甲醇、体积比1:1的甲醇水、甲醇、四氢呋喃洗涤,所得固体在干燥箱中80℃条件下干燥16个小时,既得氨基硅胶。
向250mL烧瓶中加入10g氨基硅胶、2g 4-二甲氨基吡啶、4.4g十二烯基丁二酸酐(摩尔数为20mmol)和100mL N,N-二甲基甲酰胺在60℃下反应24小时,过滤,依次用甲醇、50mM乙酸钠溶液、水、甲醇洗涤,所得固体在干燥箱中80℃条件下干燥16个小时,既得羧酸基固定相,结构如下:
实施例4
在氮气保护下,向100mL烧瓶中加入10g经160℃干燥16小时的微球形硅胶(粒径为5μm,孔径为10nm)、8mL氨丙基三甲氧基硅烷和60mL二甲苯,在110℃下反应16小时,过滤,依次用甲醇、体积比1:1的甲醇水、甲醇、四氢呋喃洗涤,所得固体在干燥箱中80℃条件下干燥16个小时,既得氨基硅胶。
向250mL烧瓶中加入10g氨基硅胶、2mL吡啶、3.3g氯丙基丁二酸酐(摩尔数为19mmol)和100mL N,N-二甲基甲酰胺在40℃下反应24小时,过滤,依次用甲醇、50mM乙酸钠溶液、水、甲醇洗涤,所得固体在干燥箱中80℃条件下干燥16个小时,既得羧酸基固定相,结构如下:
实施例5
操作过程和条件同实施例4,与实施例4不同之处在于,使用二苯琥珀酸酐(摩尔数为12mmol)代替氯丙基丁二酸酐(摩尔数为19mmol),结构如下:
实施例6
操作过程和条件同实施例4,与实施例4不同之处在于,使用氨丙基三乙氧基硅烷代替氨丙基三甲氧基硅烷,结构与实施例1结构一致。
实施例7
操作过程和条件同实施例4,与实施例4不同之处在于,使用辛基丁二酸酐(摩尔数为17mmol)代替氯丙基丁二酸酐(摩尔数为19mmol),结构如下:
实施例8
操作过程和条件同实施例4,与实施例4不同之处在于,使用苄基丁二酸酐(摩尔数为14mmol)代替氯丙基丁二酸酐(摩尔数为19mmol),结构如下:
实施例9
使用实施例1所得色谱固定相1装填4.6×100mm色谱柱,用于天然药物的分离分析。填料对天然药物具有良好的分离选择性,色谱条件为:
样品:天然药物混标(天麻素1.2mg/mL,咖啡酸1.5mg/mL,栀子苷0.8mg/mL,松果菊苷1.1mg/mL);
溶剂:A:乙腈;B:0.1%甲酸水(V/V);
洗脱:0~5~10min,5%~20%~90%A(V/V);
流速:1.0mL/min;
柱温:30℃;
检测:DAD(190nm-400nm)&254nm;
测试结果:天麻素(保留时间0.428min),咖啡酸(保留时间0.750min,与天麻素分离度3.05),栀子苷(保留时间1.250min,与咖啡酸分离度1.35),松果菊苷(保留时间3.271min,与栀子苷分离度4.79)。
Claims (11)
2.按照权利要求1所述的色谱固定相,其特征在于:每克硅胶上含有0.1-2.4mmol的羧基基团。
3.一种权利要求1或2所述的固定相的制备方法,其特征在于,包括如下步骤:
a.硅胶表面引入氨基:在氮气和/或氩气保护下,在有机溶剂中加入硅烷偶联剂和经80-200℃干燥8~18小时的微球形硅胶,在40~130℃下反应8~24小时,过滤,依次用甲醇、体积比1:1-3的甲醇水、甲醇、四氢呋喃洗涤,所得固体于干燥箱中40~80℃条件下干燥8~24小时,既得氨基硅胶;
b.羧酸基团键合:在上述制备的氨基硅胶中加入有机溶剂和酸酐试剂,再加入碱性催化剂,在25~80℃下反应8~48小时,过滤,依次用甲醇、乙酸钠溶液、水、甲醇洗涤,所得固体于干燥箱中40~80℃条件下干燥8~24小时,既得羧酸基固定相。
5.按照权利要求3所述的制备方法,其特征在于:步骤a所用的有机溶剂为二氯甲烷、乙腈、甲苯、二甲苯、N,N-二甲基甲酰胺中的一种或二种以上。
7.按照权利要求3所述的制备方法,其特征在于:步骤b所用的碱性催化剂为有机碱类化合物选自三乙胺、吡啶或4-二甲氨基吡啶中的一种或二种以上。
8.按照权利要求3所述的制备方法,其特征在于:步骤b所用的有机溶剂为二氯甲烷、乙腈、甲苯、二甲苯、N,N-二甲基甲酰胺中的一种或二种以上。
9.按照权利要求3所述的制备方法,其特征在于:
步骤a所用的硅烷偶联剂的用量为每克硅胶1-10mmol;
步骤a所用的有机溶剂的用量为每克硅胶4-10mL;
步骤b所用的酸酐的用量为每克硅胶0.1-2.4mmol;
步骤b所用的碱性催化剂的用量为每克硅胶0.1-4mmol;
步骤b所用的有机溶剂的用量为每克硅胶4-10mL。
10.一种权利要求1或2所述的固定相在色谱分离过程中的应用。
11.按照权利要求10所述的应用,其特征在于:色谱分离模式为离子交换分离。
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JP2011007731A (ja) * | 2009-06-29 | 2011-01-13 | Fuji Silysia Chemical Ltd | 親水性有機化合物の分離方法、および親水性相互作用クロマトグラフィー用充填剤 |
CN102101047A (zh) * | 2009-12-16 | 2011-06-22 | 中国科学院大连化学物理研究所 | 一种酰胺基色谱固定相及其制备方法 |
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US5439979A (en) * | 1990-02-24 | 1995-08-08 | Merck Patent Gesellschaft Mit Beschrankter Haftung | Separating materials |
JP2011007731A (ja) * | 2009-06-29 | 2011-01-13 | Fuji Silysia Chemical Ltd | 親水性有機化合物の分離方法、および親水性相互作用クロマトグラフィー用充填剤 |
CN102101047A (zh) * | 2009-12-16 | 2011-06-22 | 中国科学院大连化学物理研究所 | 一种酰胺基色谱固定相及其制备方法 |
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