CN103301822B - 一种极性液相色谱填料及其制备方法 - Google Patents

一种极性液相色谱填料及其制备方法 Download PDF

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CN103301822B
CN103301822B CN201210412474.8A CN201210412474A CN103301822B CN 103301822 B CN103301822 B CN 103301822B CN 201210412474 A CN201210412474 A CN 201210412474A CN 103301822 B CN103301822 B CN 103301822B
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polarity
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bisamide
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silane
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CN103301822A (zh
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文爱东
孙晓莉
王海波
贾艳艳
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Fourth Military Medical University FMMU
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Abstract

本发明公开了一种含有双酰胺键极性高效液相色谱填料，其化学结构通式如下所示，，其中R1为C1～C20的烷基，R2为C1～C8烷基或苯基，a为0,1,2，X为卤素，烷氧基，酰氧基或胺基。本发明利用新型极性双酰胺键官能团作为硅胶表面键合相，能与硅胶基质上残留的硅羟基形成氢键或离子对，更好地屏蔽硅羟基活性，消除残留硅羟基的影响。与传统C18色谱柱相比，这些新的色谱固定相具有更优良的选择性和分离度，柱效高，应用范围宽。

Description

一种极性液相色谱填料及其制备方法
技术领域
本发明属于高效液相色谱分离材料制备技术领域，特别涉及一种极性液相色谱填料及其制备方法，适用于分离极性和碱性化合物，用于有机合成、食品、环境和制药行业中多组分混合物的分离提纯。
背景技术
高效液相色谱（HPLC）是20世纪70年代发展起来的一项高效、快速的分离分析技术，现已成为化学化工、生命科学、生物技术、食品卫生、药物检测、环境监测等诸多领域中最常用的分离分析手段。色谱分析的基本要求是实现混合物各组分的分离，分离的实质则是基于被分析溶质与流动相和固定相之间的相互作用的差异。其中，高选择性固定相的研制是分离分析的核心，是各种HPLC分离模式赖以建立和发展的基础。在众多色谱固定相中，以硅胶为基质的固定相具有不可替代的作用，这是由于硅胶除了具有良好的机械强度、容易控制的孔结构和比表面积、较好的化学稳定性和热稳定性以外，其表面还含有高反应活性的硅羟基，通过化学修饰能获得多种功能固定相。反相液相色谱（RPLC）是一种应用极为广泛的分离分析技术，因其具有柱效高、重现性好、分离效率高以及与质谱检测器兼容性好等优点，目前大多数的应用是采用反相液相色谱方法实现的。烷基键合硅胶固定相是反相液相色谱分析方法应用的主要填料，但是在反相色谱填料制备的过程中，由于位阻原因，硅胶表面的硅羟基不可能全部与硅烷试剂反应，残留的硅羟基在分离一些极性、碱性化合物时，色谱峰将严重拖尾变形，柱效下降。封尾作用是用含短链烷基的硅烷化试剂再次进行硅烷化反应，以去除未反应的硅羟基，但封尾反应不能完全消除硅羟基的影响。为了改善碱性化合物的色谱分离分析，色谱工作者制备了含有极性基团封端或嵌入的新型色谱填料。Kirkland等制备了侧链含异丙基和异丁基的C18单齿（J.Chromatogr.Sci.1994,32,473）和双齿（Anal.Chem.1998,70,4344）硅烷键合固定相，由于侧链的位阻效应阻碍了其它基团对残余硅羟基的进攻，该填料在pH7时对碱性化合物的分离有良好的柱效，且色谱峰形对称。Buszewski等（J.Chromatogr.A1994,763,11）制备了酰胺型电荷屏蔽键合固定相，用于碱性化合物的分离，但两步合成法重现性差，同时未反应的氨基易与分析物进行离子交换，造成色谱峰拖尾。现有技术的主要不足之处在于：1、两步键合过程副反应多；2、批次间重现性差；3、填料耐酸碱性差。
发明内容
针对上述缺陷，本发明提供一种含有双酰胺键的极性固定相，以满足在简单的色谱条件下，实现包括极性、碱性化合物在内的大多数有机化合物的分离分析，并能够有效地改善碱性化合物的色谱峰形和在高水流动相条件下的运行能力。
本发明的另一目的是提供上述含有双酰胺键的极性固定相的制备方法。
本发明实现过程如下：
含有双酰胺键极性高效液相色谱填料，其化学结构通式为：
其中R1为C1～C20的烷基；
R2为C1～C8烷基或苯基；
a为0,1,2；
X为卤素，烷氧基，酰氧基或胺基。
所述硅胶基质是球形多孔硅胶，粒径范围从1mm到60mm，孔径范围从50?到1000?，比表面积范围从50m2/g到500m2/g，金属杂质含量小于30ppm的高纯硅胶。
上述的液相色谱填料的制备方法如下：
利用含有双酰胺键的极性硅烷试剂对硅胶基质进行表面处理，对所得材料进行水解、干燥，进一步与硅烷封端试剂反应得到含有双酰胺键极性高效液相色谱填料。
所述的硅烷封端试剂是单，二，三，四或五硅烷中的一种或几种，所述的单硅烷为三甲基氯硅烷，N,N-二甲基三甲基硅胺，三甲基硅基咪唑，甲基三氯硅烷，二甲基二氯硅烷，二甲氧基二甲基硅烷，三甲基硅醇或N-三甲基硅基乙酰胺；所述的二硅烷为六甲基二硅氮烷或1,3-二甲氧基四甲基二硅氧烷；所述的三硅烷是六甲基环三硅氧烷；所述的四硅烷是八甲基环四硅氧烷；所述的五硅烷是十甲基环五硅氧烷。
本发明的优点与积极效果：
本发明利用极性双酰胺键官能团作为硅胶表面键合相，能与硅胶基质上残留的硅羟基形成氢键或离子对，更好地屏蔽硅羟基活性，消除残留硅羟基的影响。与传统C18色谱柱相比，这些新的色谱固定相具有更优良的选择性和分离度，柱效高，应用范围宽。
本发明极性双酰胺键官能团既具有偶极-偶极作用，同时又有疏水作用等多种作用机理，能有效地同时分离检测酸性，中性和碱性化合物，特别是对极性、碱性化合物具有很强的分离能力，可与含氧，氮，磷，硫的有机化合物形成氢键，因而具有很好的应用潜力。
本发明的色谱柱可用于正相、反相和亲水色谱分离，并适用于等度或梯度分析，即流动相的组分比例在整个分离过程中可以保持恒定或按一定规律改变。流动相中可含有0-100%水或0-100%有机溶剂。当含水时，其它成份应与水互溶。常用的有机溶剂有（但不限于）甲醇，乙腈，异丙醇，乙醇，四氢呋喃等。流动相中可加入0-100mmol/L可溶性酸，碱或其它缓冲盐。流动相pH范围在pH2-8之间以保证一定的色谱柱稳定性。使用的温度范围可以在5-600C，最好在20-400C。在LC-MS联机时，使用高有机流动相能够加大离子化过程，从而提高检测灵敏度。
附图说明
图1是实施例3中本发明色谱柱对硫脲-苯胺-甲苯胺(o-,m-,p-)-苯酚-N,N-二甲基甲酰胺-苯甲酸乙酯-甲苯-乙苯分离的色谱图；1代表硫脲，2代表苯胺，3代表o-,m-,p-甲苯胺，4代表苯酚，5代表N,N-二甲基苯胺，6代表苯甲酸乙酯，7代表甲苯，8代表乙苯。
图2是实施例3中本发明色谱柱在pH1.5和pH11条件下的稳定性测试。
图3是实施例3中本发明色谱柱对头孢他啶-头孢羟氨苄-头孢呋辛酯-头孢唑啉-头孢克洛-头孢氨苄混合物连续进样1000针的测试结果；1代表头孢他啶，2代表头孢羟氨苄，3代表头孢呋辛酯，4代表头孢唑啉，5代表头孢克洛，6代表头孢氨苄。
图4是实施例4中本发明色谱柱在高pH条件下对β-受体阻滞剂混合物分离的色谱图；1代表吲哚洛尔，2代表美托洛尔，3代表比索洛尔，4代表普萘洛尔，5代表阿普洛尔。
图5是实施例4中本发明色谱柱在低pH条件下对β-受体阻滞剂混合物分离的色谱图；1代表纳多洛尔，2代表吲哚洛尔，3代表美托洛尔，4代表拉贝洛尔，5代表普萘洛尔，6代表阿普洛尔。
图6是实施例5中本发明色谱柱对咖啡因代谢物分离的色谱图；1代表尿酸，2代表黄嘌呤，3代表7-甲基黄嘌呤，4代表1-甲基尿酸，5代表3-甲基黄嘌呤，6代表1,3-二甲基尿酸，7代表可可碱，8代表1,7-二甲基黄嘌呤，9代表茶碱。
图7是实施例6中C18柱与本发明色谱柱对生育酚异构体分离的色谱图；1代表δ-生育酚，2代表γ-生育酚，3代表α-生育酚。
具体实施方式
为了更好地理解本发明的技术，通过实例给予进一步的说明。
实施例1硅烷和极性色谱固定相的合成
1.1十一碳酰甘氨酸的制备
在250mL三颈瓶中加入CH2Cl2（50毫升），十一碳酰氯（4.49克）和三乙胺（5毫升），冰浴下剧烈搅拌，滴加含有甘氨酸（1.5克)的CH2Cl2溶液（20毫升）。滴加完毕后室温搅拌反应2小时。粗产品经柱层析纯化，乙酸乙酯/石油醚（3:1）为洗脱剂，得无色油状液体产物（4.53克）。1HNMR(500MHz,CDCl3)δ0.84(t,3H),1.29(m,14H),1.58(m,2H),2.15(m,2H),4.16(s,2H).Calc.C%64.16,H%10.36,N%5.76;FoundC%64.10,H%10.38,N%5.71。
1.2含有双酰胺键硅烷的制备
在三颈瓶中加入十一碳酰甘氨酸（2.4克），N,N’-二环己基碳二亚胺（2.1克），4-二甲氨基吡啶（70毫克）和CH2Cl2（100毫升）。机械搅拌下用恒压滴液漏斗加入3-氨丙基三甲氧基硅烷（1.8毫升），在室温下搅拌反应3小时。反应结束后过滤，滤液用3×20毫升20%的碳酸钠溶液洗涤，有机层用无水硫酸镁干燥。减压除去溶剂后用柱层析纯化（石油醚/乙酸乙酯，3:7），得白色固体产物（3.54克）。m.p.58-590C.1HNMR(500MHz,CDCl3)δ0.57(m,2H),0.86(t,3H),1.29(m,14H),1.60(m,2H),2.09(t,2H),3.21(q,2H),3.60(s,9H),4.12(b,2H).Calc.C%56.40,H%9.96,N%6.92;FoundC%56.33,H%9.87,N%6.98。
1.3键合相的制备
（1）在圆底烧瓶中加入10克球形硅胶（5mm,100?,400m2/g,金属杂质含量小于30ppm）和浓盐酸（50毫升），混合物在1000C下回流16小时后降至室温，过滤，滤饼用水洗至中性，硅胶在1400C真空下干燥8小时。
（2）将步骤（1）硅胶冷却后置入反应器中，通入干燥氩气，安装冷凝管和干燥管，加入正癸烷（50毫升），吡啶4毫升，机械搅拌均匀，然后加入步骤1.2合成的硅烷15克，加热至回流并恒温反应48小时。停止反应，真空抽滤，依次用甲苯，二氯甲烷，四氢呋喃，丙酮，甲醇-水（1：1，v/v），甲醇洗涤。
（3）将步骤（2）键合硅胶（10克）放入反应器中，加入50毫升0.1%三氟醋酸甲醇水溶液（5：1，v/v），在室温下反应24小时，停止反应，真空抽滤，依次用丙酮，甲醇-水（1：1，v/v），甲醇洗涤，800C干燥24小时。
（4）取步骤（3）键合硅胶（10克）放入反应器中，通入干燥氩气，安装冷凝管和干燥管，加入正癸烷（50毫升），吡啶（2毫升），六甲基二硅氮烷（8毫升），机械搅拌均匀，加热至回流并恒温反应16小时，停止反应，真空抽滤，依次用甲苯，二氯甲烷，四氢呋喃，丙酮，甲醇-水（1：1，v/v），甲醇洗涤，800C干燥24小时。元素分析：C%21.49,H%3.62，N%2.64。键合相密度：3.63mmolm-2。
实施例2
采用与实施例1类似的合成方法合成得到了如下系列含有双酰胺键极性高效液相色谱填料。
上述合成的物质通过红外、NMR和元素分析等表征确定了结构。
实施例3色谱柱性能评价
3.1分析柱的装填
用实施例1制备的填料，匀浆法填充于150x4.6mmI.D.不锈钢柱管中，填装压力以40-80MPa为最好。所得色谱柱用于分离混合物样品。
3.2Engelhardt测试
用实施例3.1制备的色谱柱，分离了1硫脲，2苯胺，3o-,m-,p-甲苯胺，4苯酚，5N,N-二甲基苯胺，6苯甲酸乙酯，7甲苯，8乙苯混合物，图1为其色谱图。色谱条件如下：流动相，甲醇:水=55:45(v/v);流速，1mL/min;柱温，250C；检测波长，UV254nm。
3.3稳定性测试
用实施例3.1制备的色谱柱，测定了本发明色谱柱在不同pH下的稳定性（图2）。酸性流动相冲洗条件为：乙腈:1%三氟乙酸（pH1.5,1:1,v/v）;碱性流动相冲洗条件为:乙腈:20mM磷酸盐缓冲液（pH11,1:1,v/v）。色谱条件如下：流动相,乙睛:20mM磷酸缓冲液(pH7)=60:40(v/v);流速,1mL/min;柱温，250C；检测波长,UV254nm.样品，1尿嘧啶，2吡啶，3苯酚，4苯。
3.4重现性测试
用实施例3.1制备的色谱柱，分离了1头孢他啶，2头孢羟氨苄，3头孢呋辛酯，4头孢唑啉，5头孢克洛，6头孢氨苄混合物，图3为其色谱图。色谱条件如下：流动相，甲醇:0.1%三氟醋酸水溶液=30:70(v/v)；流速，1mL/min；柱温，250C；检测波长,UV230nm。
实施例4对β-受体阻滞剂的分离
用实施例3.1制备的色谱柱，分离了1吲哚洛尔，2美托洛尔，3比索洛尔，4普萘洛尔，5阿普洛尔混合物，图4为其色谱图。色谱条件如下：流动相，甲醇:5mM碳酸氢铵水溶液(pH10)=70:30(v/v)；流速，1mL/min；柱温，250C；检测波长,UV220nm。
用实施例3.1制备的色谱柱，分离了1纳多洛尔，2吲哚洛尔，3美托洛尔，4拉贝洛尔，5普萘洛尔，6阿普洛尔混合物，图5为其色谱图。色谱条件如下：流动相，0.1%三氟醋酸乙睛溶液:0.1%三氟醋酸水溶液=30:70(v/v)；流速，1mL/min；柱温，250C；检测波长,UV220nm。
实施例5对咖啡因代谢物异构体的分离
用实施例3.1制备的色谱柱，分离了1尿酸，2黄嘌呤，37-甲基黄嘌呤，41-甲基尿酸，53-甲基黄嘌呤，61,3-二甲基尿酸，7可可碱，81,7-二甲基黄嘌呤，9茶碱混合物，图6为其色谱图。色谱条件如下：流动相，甲醇:1%醋酸水溶液=10:90(v/v)；流速，1mL/min；柱温，250C；检测波长,UV254nm。
实施例6对生育酚异构体的分离
用实施例3.1制备的色谱柱，分离了1δ-生育酚，2γ-生育酚，3α-生育酚混合物，图7为其色谱图。色谱条件如下：流动相，甲醇；流速，1mL/min；柱温，250C；检测波长,UV295nm。

Claims (5)

1.含有双酰胺键极性高效液相色谱填料，其化学结构通式为：
其中R1为C1～C20的烷基；
R2为C1～C8烷基或苯基；
a为1或2；
X为卤素，烷氧基，酰氧基或胺基。
2.根据权利要求1所述的含有双酰胺键极性高效液相色谱填料，其特征在于：所述硅胶基质是球形多孔硅胶，粒径为1mm到60mm，孔径为50?到1000?，比表面积为50m2/g到500m2/g，金属杂质含量小于30ppm的高纯硅胶。
3.权利要求1所述的含有双酰胺键极性高效液相色谱填料的制备方法，其特征在于：利用含有双酰胺键的极性硅烷试剂对硅胶基质进行表面处理，对所得材料进行水解、干燥，进一步与硅烷封端试剂反应得到含有双酰胺键极性高效液相色谱填料。
4.权利要求3所述的含有双酰胺键极性高效液相色谱填料的制备方法，其特征在于：所述的硅烷封端试剂是单，二，三，四或五硅烷中的一种或几种，所述的单硅烷为三甲基氯硅烷，N,N-二甲基三甲基硅胺，三甲基硅基咪唑，甲基三氯硅烷，二甲基二氯硅烷，二甲氧基二甲基硅烷，三甲基硅醇或N-三甲基硅基乙酰胺；所述的二硅烷为六甲基二硅氮烷或1,3-二甲氧基四甲基二硅氧烷；所述的三硅烷是六甲基环三硅氧烷；所述的四硅烷是八甲基环四硅氧烷；所述的五硅烷是十甲基环五硅氧烷。
5.装填有权利要求1所述含有双酰胺键极性高效液相色谱填料的色谱柱。
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