CN114618454A - 一种极性包埋双链反相色谱固定相及其制备和应用 - Google Patents
一种极性包埋双链反相色谱固定相及其制备和应用 Download PDFInfo
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- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
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Abstract
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中含有双烷基疏水链,同时内嵌极性基团的液相色谱固定相。
技术背景
反相液相色谱具有柱效高、分离能力强、保留机理清楚等优点,是液相色谱分离模式中使用最为广泛的一种,广泛应用于生物大分子、蛋白质及酶的分离分析。反相色谱是以表面非极性载体为固定相,以比固定相极性强的溶剂为流动相的一种液相色谱分离模式。反相色谱固定相大多是硅胶表面键合疏水基团,基于样品中的不同组分和疏水基团之间疏水作用的不同而分离。
内嵌极性基团固定相是指将具有极性的化学基团如酰胺基团、醚键等引入硅胶表面烷烃链的根部或侧链,这类色谱固定相除了与待测样品之间具有疏水作用力外,还会存在π-π作用力、氢键作用力、偶极-偶极等作用力。Silva等制备了一种内嵌脲基基团的C18固定相,并将其应用于极性与非极性混合物的分离分析中,同时该固定相对酸性化合物及芳香胺类化合物具有良好的分离效果[Silva.R.C.et al,J.Chromatogra.A,2005,1087,29-37]。Guo等将酰胺基团嵌入到C18烷烃上,实现了对二氢麦角碱甲磺酸不同剂型药品中4种组分的定量检测[Guo,Z.Q.et al,Chinese J.Anal.Chem,2009,32,232-236]。但是当前尚未出现关于含有双烷基疏水链,同时内嵌极性基团的反相色谱固定相的制备技术及其用于天然产物及药物分离分析的相关报道。
发明内容
本发明的目的是提供一种新型含极性基团反相色谱固定相及其制备方法。该键合相包含双烷基疏水链及极性基团,其制备方法简单,适用性广泛。
本发明的技术方案是:液相色谱固定相,其特征在于结构为:
其中Silica Gel为硅胶的示意(代表硅胶),R是带有氨基、酰胺基、酯基、羰基、醚键、脲基中的一种或二种以上官能团的C1-C10的烷基链,m=1-30整数,n=1-30整数。
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.硅胶预处理:硅胶加入质量浓度为5~40wt%的强酸溶液中,加热回流搅拌2~24小时,过滤,用水洗涤至pH=6~7,所得固体于干燥箱中100~160℃条件下干燥8~24小时,得酸化后硅胶;
以每克硅胶计,强酸溶液的用量6-20mL;
b.硅烷化反应:在氮气和/或氩气保护下,在有机溶剂中加入硅烷偶联剂、碱性催化剂和酸化后硅胶,在40~115℃下反应3~24小时,过滤,依次用甲醇、体积比1:1-2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,得到色谱固定相;
以每克硅胶计,硅烷偶联剂的用量为0.5-2.4mmol,有机溶剂的用量为6-10mL,碱性催化剂的用量为1-5mmol。
本发明具有如下优点:
1.结构新颖。本发明首次提出键合相中含有双烷基疏水链,同时内嵌极性基团的液相色谱固定相。该固定相结构中带有脲基、酰胺基团、醚键等极性基团及疏水烷基链,烷基双链结构使得烷基链键合密度高,内嵌极性基团使得固定相具有耐纯水的特点,同时固定相还具有多种作用力如疏水作用力、氢键作用力、偶极-偶极等,对绝大部分天然产物及药物具有很好的分离选择性,可广泛用于天然产物及药物的分离分析及纯化制备。
2.本发明提供的液相色谱固定相制备过程简单可靠,有利于实现产业化。
附图说明
图1为实施例6的色谱图;
图2为实施例7的色谱图;
图3为实施例8的色谱图。
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
向250mL烧瓶中加入10g硅胶,加入100mL质量浓度为38wt%的盐酸溶液中,加热回流搅拌2小时,过滤,用水洗涤至pH=6~7,所得固体于干燥箱中160℃条件下干燥24小时,得酸化后硅胶;
在氮气保护下,向100mL烧瓶中加入10g酸化后硅胶(粒径为5μm,孔径为10nm)、8mL1,1-二辛基-3-[3-(三乙氧基硅烷基)丙基]脲(摩尔数为24mmol)、6g N,N-二甲基吡啶和80mL二甲苯,在115℃下反应16小时,过滤,依次用甲醇、体积比1:2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,得到色谱固定相1,结构如下:
实施例2
操作过程和条件同实施例1,与实施例1不同之处在于,使用1,1-二己基-3-[3-(三乙氧基硅烷基)丙基]脲(摩尔数为22mmol)代替1,1-二辛基-3-[3-(三乙氧基硅烷基)丙基]脲(摩尔数为24mmol),得到色谱固定相2,结构如下:
实施例3
向250mL烧瓶中加入10g硅胶,加入80mL浓度为10wt%的盐酸溶液中,加热回流搅拌48小时,过滤,用水洗涤至pH=6~7,所得固体于干燥箱中200℃条件下干燥12小时,得酸化后硅胶;
在氩气保护下,向100mL烧瓶中加入10g酸化后硅胶(粒径为3.5μm,孔径为30nm)、8mL N,N-二辛基-5-(三甲氧基硅基)戊酰胺(摩尔数为24mmol)、2mL1,5-二氮杂双环[4.3.0]壬-5-烯和60mL异辛烷,在90℃下反应48小时,过滤,依次用甲醇、体积比1:2的甲醇水、甲醇、四氢呋喃洗涤,所得固体在干燥箱中100℃条件下干燥16个小时,得到色谱固定相3,结构如下:
实施例4
操作过程和条件同实施例3,与实施例3不同之处在于,使用N,N-二辛基-3-(3-(三甲氧基硅基)丙氧基)丙酰胺(摩尔数为22mmol)代替N,N-二辛基-5-(三乙氧基硅基)戊酰胺(摩尔数为24mmol),得到色谱固定相4,结构如下:
实施例5
向250mL烧瓶中加入10g硅胶,加入80mL浓度为15wt%的盐酸溶液中,加热回流搅拌48小时,过滤,用水洗涤至pH=6~7,所得固体于干燥箱中200℃条件下干燥12小时,得酸化后硅胶;
在氩气保护下,向100mL烧瓶中加入10g酸化后硅胶(粒径为3.5μm,孔径为30nm)、6mL 1,1-二辛基-3-(3-三氯硅基丙基)氨丙基脲(摩尔数为18mmol)、3mL三乙胺和60mL二甲苯,在90℃下反应48小时,过滤,依次用甲醇、体积比1:2的甲醇水、甲醇、四氢呋喃洗涤,所得固体在干燥箱中100℃条件下干燥16个小时,得到色谱固定相5,结构如下:
实施例6
使用实施例1所得色谱固定相1装填4.6×50mm色谱柱,用于非极性化合物的分离分析。如图1所示,填料对非极性化合物具有良好的分离选择性,色谱条件为:
色谱柱:4.6×50mm;
样品:非极性化合物混标(尿嘧啶1mg/mL,硝基苯1mg/mL,萘2mg/mL,芴2.2mg/mL);
溶剂:A:乙腈;B:水;
洗脱:A:B=60:40(V/V);
流速:1.5mL/min;
柱温:30℃;
检测:DAD(190nm-400nm)&UV(254nm);
实施例7
使用实施例1所得色谱固定相1装填4.6×50mm色谱柱,用于浸润性测试样品的分离分析。如图2所示,填料在纯水浸润后,各化合物仍得到很好的分离。色谱条件为:
色谱柱:4.6×50mm;
样品:浸润性测试混标(尿嘧啶1mg/mL,胞嘧啶1.2mg/mL,胸腺嘧啶2mg/mL);
溶剂:A:乙腈,B:100mM甲酸铵(pH=3.2);
洗脱:A:B=90:10;
流速:1.0mL/min;
柱温:30℃;
检测:DAD(190nm-400nm)&UV(254nm);
实施例8
使用实施例1所得色谱固定相1装填4.6×50mm色谱柱,用于生物碱的分离分析。如图3所示,填料对不同类型的生物碱化合物具有良好的分离选择性,色谱条件为:
色谱柱:4.6×50mm;
样品:碱性混标(青藤碱0.4mg/mL,士的宁0.4mg/mL,荷叶碱0.4mg/mL,小檗碱0.1mg/mL,秋水仙碱1.0mg/mL,辣椒碱1.5mg/mL);
溶剂:A:乙腈;B:0.1%甲酸水;
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。
Claims (10)
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0.5-2.4mmol的R。
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.硅胶预处理:硅胶加入质量浓度为5~40wt%的强酸溶液中,加热回流搅拌2~24小时,过滤,用水洗涤至pH=6~7,所得固体于干燥箱中100~160℃条件下干燥8~24小时,得酸化后硅胶;
b.硅烷化反应:在氮气和/或氩气保护下,在有机溶剂中加入硅烷偶联剂、碱性催化剂和酸化后硅胶,在40~115℃下反应3~24小时,过滤,依次用甲醇、体积比1:1-2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,得到色谱固定相。
4.按照权利要求3所述的制备方法,其特征在于:步骤a所用的强酸为盐酸、硝酸、硫酸中的一种。
6.按照权利要求3所述的制备方法,其特征在于:步骤b所用的有机溶剂为与水不互溶的二氯甲烷、甲苯、二甲苯、正庚烷、异辛烷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二异丙基乙胺、三乙胺、1,8-二氮杂环[5,4,0]十一烯-7、1,5-二氮杂双环[4.3.0]壬-5-烯、N,N-二甲基吡啶、吡啶、咪唑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a所用的强酸溶液的用量为每克硅胶6-20mL;
步骤b所用的有机溶剂的用量为每克硅胶6-10mL;
步骤b所用的硅烷偶联剂的用量为每克硅胶0.5-2.4mmol;
步骤b所用的碱性催化剂的用量为每克硅胶1-5mmol。
9.一种权利要求1或2所述的固定相在色谱分离过程中的应用。
10.按照权利要求9所述的应用,其特征在于:色谱分离模式为反相色谱分离,并对生物碱类化合物具有良好的分离选择性。
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US20050023203A1 (en) * | 2003-07-28 | 2005-02-03 | Vladislav Orlovsky | Universal bonded phase materialfor chromatographic separation |
US6884345B1 (en) * | 1998-11-09 | 2005-04-26 | Knut Irgum | Chromatography method and a column material useful in said method |
CN101306263A (zh) * | 2008-01-22 | 2008-11-19 | 广西师范大学 | 烷基胺硅胶毛细管整体柱及其制备方法与应用 |
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US6884345B1 (en) * | 1998-11-09 | 2005-04-26 | Knut Irgum | Chromatography method and a column material useful in said method |
US20050023203A1 (en) * | 2003-07-28 | 2005-02-03 | Vladislav Orlovsky | Universal bonded phase materialfor chromatographic separation |
CN101306263A (zh) * | 2008-01-22 | 2008-11-19 | 广西师范大学 | 烷基胺硅胶毛细管整体柱及其制备方法与应用 |
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