CN114618455A - 一种多极性官能团包埋的反相色谱固定相及其制备和应用 - Google Patents
一种多极性官能团包埋的反相色谱固定相及其制备和应用 Download PDFInfo
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- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
- B01J20/287—Non-polar phases; Reversed phases
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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,具体地说是一种内嵌多极性官能团的反相色谱固定相。
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。
嵌入极性基团的反相固定相具有许多优点,如降低了峰的拖尾,增强了特定官能团的选择性,同时流动相还可以使用高水相。Schure R.M.比较了内嵌酰胺或醚键的C18固定相与传统的C18固定相间的差异性。结果表明,由于特定的氢键相互作用,含有内嵌基团的C18固定相相比传统的C18固定相对非极性化合物的保留较弱,而对极性化合物则因内嵌基团的氢键作用和固定相内部的有机溶剂的量的增加而延长保留,这导致了极性化合物从流动相中转移到残留硅羟基表面的自由能显著降低,解释了内嵌极性基团的C18固定相对极性化合物峰拖尾减少的原因[Schure,R.M.et al,Anal.Chem.2008,80,6214–6221]。Jiang制备了内嵌咪唑阳离子基团的C18固定相,并使用烷基苯、1-烷基萘和多环芳烃化合物进行色谱表征。结果表明,咪唑阳离子的存在和分布会影响固定相的性质,并且影响C18配体的流动性,咪唑结构的存在提高了芳基选择性,减弱了烷基链的疏水性,同时具有多重相互作用,如氢键和疏水相互作用[Jiang,S.X.et al,Talanta,2014,126,177-184]。
本发明通过氨基-酸酐亲核取代反应制备出一种含有多极性基团,包括氨基、羧基、酰胺基团,同时末端为疏水碳链的新型反相色谱固定相,可对不同类型的样品进行分离分析。当前,尚未出现使用此方法制备内嵌多极性官能团的反相固定相的报道。
发明内容
本发明的目的是提供一种内嵌多极性官能团的反相色谱固定相及其制备方法。该键合相包含氨基、羧基、酰胺基团及疏水碳链,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,R1是C1-C10的烷基链,R2是C1-C10的烷基链,R3是C1-C20的烷基链或带有双键的C2-C20的烷基链。
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.硅烷化: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,制备得到多氨基硅胶;
以每克硅胶计,硅烷偶联剂的用量为1-10mmol,有机溶剂的用量为4-15mL;
b.氨基-酸酐亲核取代反应:在氮气和/或氩气保护下,在上述制备的多氨基硅胶中加入有机溶剂和酸酐,再加入碱性催化剂,在25~110℃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,制备得到色谱固定相。
以每克多氨基硅胶计,酸酐的用量为0.6-2.4mmol,有机溶剂的用量为4-15mL,碱性催化剂的用量为0.1-10mmol。
本发明具有如下优点:
1.结构新颖。本发明首次提出内嵌多官能基团的反相色谱固定相。该固定相结构中带有氨基、羧基、酰胺基团等极性基团及疏水烷基链,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,可广泛用于各类样品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。
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3.5μm,孔径为10nm)、6mL N-(2-氨乙基)-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、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,制备得到N-(2-氨乙基)-氨丙基硅胶。
在氮气保护下,向250mL烧瓶中加入10g氨基硅胶、3g 4-二甲氨基吡啶、10.5g十二烯基丁二酸酐(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,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N-(2-氨丙基)-氨丙基三甲氧基硅烷(摩尔数为23mmol)代替N-(2-氨乙基)-氨丙基三甲氧基硅烷(摩尔数为24mmol),得到色谱固定相2,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)、6mL N-(2-氨乙基)-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、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,制备得到N-(2-氨乙基)-氨丙基硅胶。
在氮气保护下,向250mL烧瓶中加入10g氨基硅胶、3g咪唑、12g十八烯基丁二酸酐(摩尔数为22mmol)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,得到色谱固定相3,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)、6mL N-(2-氨丙基)-氨丙基三甲氧基硅烷和80mL甲苯,在110℃下反应24小时,过滤,依次用甲醇、甲醇水、甲醇、四氢呋喃洗涤,所得固体在干燥箱中80℃条件下干燥24个小时,制备得到N-(2-氨丙基)-氨丙基硅胶。
在氮气保护下,向250mL烧瓶中加入10g氨基硅胶、3mL 1,8-二氮杂环[5,4,0]十一烯-7、10.5g十二烷基丁二酸酐(摩尔数为24mmol)和100mL N,N-二甲基甲酰胺在40℃下反应16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,得到色谱固定相4,结构如下:
实施例5
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2.5μm,孔径为10nm)、8mL N-(2-氨丙基)-氨己基三甲氧基硅烷和80mL甲苯,在110℃下反应24小时,过滤,依次用甲醇、甲醇水、甲醇、四氢呋喃洗涤,所得固体在干燥箱中80℃条件下干燥24个小时,制备得到N-(2-氨丙基)-氨丙基硅胶。
在氩气保护下,向250mL烧瓶中加入10g氨基硅胶、3mL吡啶、6g辛基丁二酸酐(摩尔数为24mmol)和100mL二甲亚砜在60℃下反应12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,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(V/V);
洗脱:A:B=60:40;
流速:1.5mL/min;
柱温:30℃;
检测:DAD(190nm-400nm)&UV(254nm);
实施例7
使用不同类型的色谱固定相装填4.6×50mm色谱柱,用于苯甲酸类和苯丙素类化合物的分离分析。测试结果见下表,色谱条件为:
色谱柱:4.6×50mm;
样品:混标(没食子酸0.067mg/mL,新绿原酸0.33mg/mL);
溶剂:A:乙腈,B:0.1%甲酸水;
洗脱:梯度0~10~15min,5%~30%~90%A;
流速:1.0mL/min;
柱温:30℃;
检测波长:PDA(190nm-400nm)&UV(254nm);
实施例1中制备的固定相A由于存在疏水碳链,固定相的疏水性更强,对含有疏水骨架的新绿原酸的保留更强,因此对两种化合物的分离选择性更好。
实施例8
使用不同类型的色谱固定相装填4.6×50mm色谱柱,用于双环氧木脂素和蒽醌类化合物的分离分析。测试结果见下表,色谱条件为:
色谱柱:4.6×50mm;
样品:混标(连翘苷0.33mg/mL,茜草素0.067mg);
溶剂:A:乙腈,B:0.1%甲酸水;
洗脱:梯度0~10~15min,15%~60%~90%A;
流速:1.0mL/min;
柱温:30℃;
检测波长:PDA(190nm-400nm)&UV(254nm);
实施例1中制备的固定相A由于存在疏水碳链,固定相的疏水性更强,对含有糖基等极性基团的连翘苷的保留较弱,对极性小的茜草素的保留较强,因此对两种化合物的分离选择性更好。
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0.6-2.4mmol烷基链R3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.硅烷化: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,制备得到多氨基硅胶;
b.氨基-酸酐亲核取代反应:在氮气和/或氩气保护下,在上述制备的多氨基硅胶中加入有机溶剂和酸酐,再加入碱性催化剂,在25~110℃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,制备得到色谱固定相。
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、甲苯、二甲苯、正庚烷、异辛烷中的一种或二种以上;
步骤b所用的有机溶剂为N,N-二甲基甲酰胺、甲苯、二甲苯、二甲亚砜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。
8.按照权利要求2所述的制备方法,其特征在于:
步骤a所用的有机溶剂的用量为每克硅胶4-15mL;
步骤a所用的硅烷偶联剂的用量为每克硅胶1-10mmol;
步骤b所用的有机溶剂的用量为每克多氨基硅胶4-15mL;
步骤b所用的酸酐的用量为每克多氨基硅胶0.6-2.4mmol;
步骤b所用的碱性催化剂的用量为每克多氨基硅胶0.1-10mmol。
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,对含有疏水骨架的化合物具有良好的保留及分离选择性。
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