CN105561958A - 一种用于贝类毒素富集纯化的离子液体键合硅胶及其制备方法 - Google Patents
一种用于贝类毒素富集纯化的离子液体键合硅胶及其制备方法 Download PDFInfo
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Abstract
本发明涉及一种用于贝类毒素富集纯化的离子液体键合硅胶,是将N-苯基苯并咪唑加入到γ-氯丙基甲基二乙氧基硅烷的甲苯溶液中,均匀搅拌,之后在氮气气氛下回流48h;冷却后加入活化硅胶粉,继续回流反应24h。本发明首次制备了N-苯基苯并咪唑离子液体键合硅胶材料,与普通离子液体负载硅胶材料和常规离子液体键合硅胶材料相比,通过疏水和离子交换两种模式与目标物发生作用,可实现样品中多种贝类毒素富集纯化的目标,改变了分离对象单一的缺陷。
Description
技术领域
本发明涉及固相萃取用离子液体及其制备技术领域,特别涉及一种用于贝类毒素富集纯化的离子液体键合硅胶及其制备方法。
背景技术
固相萃取(SolidPhaseExtraction,SPE)以其操作简单、重复性好、富集率高等特点被广泛应用到样品前处理过程。商用固相萃取柱根据吸附剂性质的不同可分为正相萃取、反相萃取、离子交换萃取和吸附萃取等几种,其主要通过固相填料和分离对象间的静电作用力、离子交换作用力和氢键作用等,实现对样品组分的分离、纯化和富集,主要目的在于减少杂质干扰,提高痕量组分的检出能力。然而,目前常用商用固相萃取柱存在如下问题:与萃取对象作用模式单一,分离纯化组分较少,针对含有酸性、碱性、两性等多种不同性质组分的复杂样品,分别提取纯化步骤繁多,耗时费力,同时使用大量有机试剂造成严重环境污染;另外,常规固相萃取柱不可反复使用,成本较高。因此,急需研发生产一类含多种官能团的高选择性固相萃取材料,实现对样品中痕量多组分同时富集纯化的目标,提高分离检测效率,将具有较广泛的商用化前景。
离子液体(ILs)已成为多个领域研究的热点(PooleC.F.,J.Chromatogr.A2004,1037(1-2):49-82)。自建立基于离子液体的萃取技术以来,其在色谱样品前处理中的应用得到迅速发展。离子液体与固体材料以一定的物理或化学方式相结合,将得到负载型离子液体或表面具有离子液体结构的固体新材料,形成新型固相萃取吸附剂,新材料在保留原有固相载体性质的基础上,又能很好的发挥离子液体的特异选择性和高极性等优点。集采样、分离、富集和纯化于一体,既缩短了分析时间又减少了样品损失,在痕量组分的分离富集中具有良好的优越性。
在国内外研究中,固定化的离子液体多用作液相色谱柱(SJLiu,FZhou,XHXiao,ChemistryLetters,2004,33(5):496-497)、气相色谱柱(XJSun,YLZhu,PWang,JLi,CYWu,JXing.JournalofChromatographyA,2011,1218:833–841)以及毛细管电色谱柱的固定相(WDQin,SFYLi.JournalofChromatographyA,2004,1048(2):253-256),而极少作为固相萃取吸附剂。另外,将离子液体固定于硅胶表面作为固相萃取材料应用于样品前处理的报道多是通过物理吸附方式(JDLi,YQCai,YLShi,Talanta,2008,74(4):498-504),而由于绝大多数的离子液体不具备与硅胶表面硅羟基缩合的性质,将离子液体化学键合于硅胶上作为固相吸附材料的报道极少。少数与硅胶键合的离子液体为简单咪唑类结构,与目标物作用模式单一,分离纯化组分较少。且常规离子液体键合硅胶材料只用于水样等简单基质,应用范围较窄。
因而,制备新型的离子液体固相萃取材料,是发展离子液体萃取技术的必要途径。针对目标化合物的结构性质合成含不同官能团的功能化离子液体,制备具有混合模式的功能化离子液体键合硅胶材料,实现对样品中多性质组分分离纯化的目标,有效增强吸附剂的选择性。从而有利于将ILs-SPE技术应用到样品中痕量多组分的富集纯化研究。
我国是世界上最大的贝类养殖国,贝类养殖有效的促进国民经济发展,并给人类生活带来丰富蛋白。然而,双壳贝类中检测出多种贝类毒素,已成为发达国家制定贸易和技术壁垒的重要指标。由于贝类毒素含官能团较多、结构复杂,具有酸性、碱性、两性等多种不同性质组分,常用商用化固相萃取柱(MCX或MAX)多为阴离子交换或阳离子交换一种模式,富集纯化多种贝类毒素时难以同时达到较好的效果,需要多个不同类型小柱分别萃取,耗时费力、成本较高,成为限制其准确定量的主要瓶颈。本发明选用具有多个苯环功能基的离子液体键合硅胶作为萃取材料,通过疏水和离子交换两种模式与目标物发生作用,可实现对样品中多性质组分分离纯化的目标,有效增强吸附剂的选择性,期待对芳香类目标物有更好的萃取效果。
发明内容
针对现有技术中存在的缺陷,本发明的目的在于提供一种用于贝类毒素富集纯化的离子液体键合硅胶及其制备方法。
为达到以上目的,本发明采取的技术方案是:
一种用于贝类毒素富集纯化的离子液体键合硅胶,结构式如下:
一种如上所述的用于贝类毒素富集纯化的离子液体键合硅胶的制备方法,具体步骤如下:
(1)在室温下,将甲酸加入到浓度为0.0368gmL-1的2-氨基-二苯胺的浓盐酸悬浮液中,在氮气气氛下回流搅拌90min,其中:2-氨基-二苯胺与甲酸的摩尔比为:1:1;再用氨水(28%)调节pH至8;经乙酸乙酯三次萃取之后,无水硫酸钠干燥,室温下旋转蒸发去溶剂,最后真空干燥得到淡黄色N-苯基苯并咪唑液体。
合成路线如下:
1H-NMR(DMSO;600MHz;δppm):8.68(s,1H,NCHN),8.12(m,1H,PhNCCH),7.22-7.79(m,8H,ArH)。
(2)将40-60μm的硅胶粉末在3mol/L盐酸中,浸泡24h后,用纯净水洗至中性,120℃真空干燥5h。
(3)将将步骤(1)制得的N-苯基苯并咪唑加入到浓度为0.525gmL-1的γ-氯丙基甲基二乙氧基硅烷的甲苯溶液中,搅拌均匀;其中:γ-氯丙基甲基二乙氧基硅烷与N-苯基苯并咪唑的摩尔比为:1:1,反应体系在氮气气氛下回流48h;冷却至室温后加入活化硅胶粉,其中:γ-氯丙基甲基二乙氧基硅烷与活化硅胶粉的摩尔比为>3:1,继续回流反应24h后停止;之后将反应液过滤,用乙腈和去离子水冲洗去除残留物,80℃真空干燥5h,得到淡黄色粉末,C%=13.5,N%=1.85(2.7mmol/g)。结构如下:
本发明的技术优势:1)本发明首次制备了N-苯基苯并咪唑离子液体键合硅胶材料,与普通离子液体负载硅胶材料和常规离子液体键合硅胶材料相比,通过疏水和离子交换两种模式与目标物发生作用,可实现样品中多种贝类毒素富集纯化的目标,改变了分离对象单一的缺陷;2)本发明通过2-氨基-二苯胺与甲酸在酸性条件下成环反应生成N-苯基苯并咪唑,可灵活制备多种功能基取代的苯并咪唑。后与烷氧基氯代硅烷和活化硅胶先后键合,制备具有多个苯环功能基的离子液体键合硅胶作为萃取材料,具有较好的设计灵活性;3)本发明填制的固相萃取小柱重现性较好,操作简单,成本较低,易于实现产业化;4)常规离子液体键合硅胶材料只用于水样等简单基质,本发明的N-苯基苯并咪唑离子液体键合硅胶材料对海水和贝类中的贝类毒素都具有较好的富集净化效果,可提高检测灵敏度并有效排除基质干扰。
附图说明
本发明有如下附图:
图1为本发明实施例2中贝类中未经固相萃取柱净化的两种贝类毒素的分离色谱图;
图2为本发明实施例2中贝类中经固相萃取柱净化的两种贝类毒素的分离色谱图;
图3为本发明实施例3中海水中未经固相萃取柱富集的贝类毒素分离色谱图;
图4为本发明实施例3中海水中经固相萃取柱富集的贝类毒素分离色谱图。
具体实施方式
实施例1
一种用于贝类毒素富集纯化的离子液体键合硅胶的制备方法,具体步骤如下:
(1)在室温下,将甲酸加入到浓度为0.0368gmL-1的2-氨基-二苯胺的浓盐酸悬浮液中,在氮气气氛下回流搅拌90min,其中:2-氨基-二苯胺与甲酸的摩尔比为:1:1;再用氨水(28%)调节pH至8;经乙酸乙酯三次萃取之后,无水硫酸钠干燥,室温下旋转蒸发去溶剂,最后真空干燥得到淡黄色N-苯基苯并咪唑液体,产率约85%;
(2)将40-60μm的硅胶粉末在3mol/L盐酸中,浸泡24h后,用纯净水洗至中性,120℃真空干燥5h;
(3)将将步骤(1)制得的N-苯基苯并咪唑加入到浓度为0.525gmL-1的γ-氯丙基甲基二乙氧基硅烷的甲苯溶液中,搅拌均匀;其中:γ-氯丙基甲基二乙氧基硅烷与N-苯基苯并咪唑的摩尔比为:1:1,反应体系在氮气气氛下回流48h;冷却至室温后加入活化硅胶粉,其中:γ-氯丙基甲基二乙氧基硅烷与活化硅胶粉的摩尔比为>3:1,继续回流反应24h后停止;之后将反应液过滤,用乙腈和去离子水冲洗去除残留物,80℃真空干燥5h,得到淡黄色粉末,产率约80%。C%=13.5,N%=1.85(2.7mmol/g)。
实施例2
利用实施例1中制备的离子液体键合硅胶制作固相萃取柱,用来富集纯化贝类中的贝类毒素。
具体如下:
固相萃取小柱填制
选用固相萃取柱的柱管体积为1mL,柱管为聚丙烯材料制成,进出口筛板选用高纯度聚乙烯材料(直径6.4mm,孔径20um,厚度1.5mm),内部填充固相萃取填料选用本发明合成的烯基咪唑聚合离子液体键合硅胶(填料为50mg,粒径40-60um)。先在空柱管底部放入出口筛板,然后装入离子液体键合硅胶层,其上压入进口筛板,最后用甲醇试剂冲洗填实,并保证所有小柱填充后,填料高度保持(5±0.05)mm。
仪器配置
TSQQuantumAccessTM液相色谱-串联质谱仪(ThermoFisherScientific公司,美国);C18色谱柱(4μm,2.1mm×150mm);XW-80A型旋涡混合器(上海医大仪器厂);Milli-Q型超纯水仪(美国Millipore公司);ThermoSorvallBiofugePrimo型离心机(美国ThermoFisherScientific公司)。
仪器条件
柱温:35℃;流速:0.3mL/min;进样量:10μL;流动相:A:2mmol/L碳酸氢铵溶液(pH=11),B:乙腈+2mmol/L碳酸氢铵(9+1,v/v)溶液(pH=11),梯度洗脱程序:0min,30%B;0-3.00min,30%-90%B;3.00-6.00min,90%B;6.00-6.01min,90%-30%B;6.01-8.00min,30%B。
贝类毒素的质谱分析参数如表1所示。
表1贝类毒素的质谱扫描参数
扇贝中贝类毒素的富集纯化应用
(1)取称2.0克扇贝样品,添加贝类毒素混合标液[大田软海绵酸毒素(OA)和鳍藻毒素-1(DTX-1)],10mL甲醇,5mL4%的NaCl水溶液,超声提取5min后,以8000rpm离心5min,取上清液;再向残渣中加入10mL甲醇重复提取一次,合并上清液;40℃旋转浓缩至近干,加入2mL蒸馏水涡旋溶解残渣,待净化。
(2)分别用1mL甲醇和1mL蒸馏水预活化本发明固相萃取柱,流出液弃去。将2mL提取液加入固相萃取柱中,用2mL0.3%的氨水甲醇进行洗脱。氮吹后用流动相溶解并定容至1mL,经0.22μm滤膜过滤后供液相色谱串联质谱测定。
将未经过本发明固相萃取柱净化的扇贝提取液(1)在氮吹近干后,用1mL流动相定容并过0.22μm滤膜。将此溶液和经过固相萃取柱净化后的溶液分别进行液相色谱测定。如图1和2显示,表明经本发明固相萃取柱净化后,干扰明显减少。
实施例3
利用实施例1中制备的离子液体键合硅胶制作固相萃取柱,用来富集纯化海水中的贝类毒素。
固相萃取小柱填制、仪器条件和仪器配置
与实施例2相同,另外,仪器配置中添加全自动上样装置AUQALOADER698(岛津公司)。
海水中贝类毒素的富集纯化应用
首先,分别用1mL甲醇和1mL水活化本发明的固相萃取小柱,流出液弃去。然后用0.45μm水系滤膜净化海水,除去悬浮颗粒,取100mL已添加贝类毒素混合标液(OA、DTX-1和DTX-2)的海水样品,以5.0mL/min流速通过活化过的固相萃取小柱进行富集。后用2mL水淋洗,减压抽干后用2mL0.3%的氨水甲醇洗脱,洗脱液在40℃下氮气吹干,用1mL流动相定容,充分涡旋溶解残渣,滤液经0.22μm滤膜过滤,供液相色谱串联质谱测定。
将未经过本发明固相萃取柱处理的海水加标样品和经过固相萃取柱富集净化后的溶液分别进行液相色谱测定。如图3和4显示,表明经本发明固相萃取柱富集后,检测灵敏度明显提高,富集倍数约100。
上述实施例表明本发明固相萃取小柱对样品中多种贝类毒素检测具有较好的富集纯化效果。
上述实施方案为本发明最佳的实施方案,但本发明的实施方案并不受上述实施方案的限制,其他的任何不违背本发明原理的条件下,可以通过改变参数的形式所产生的实施例,都包含于本发明的保护范围之内。
Claims (10)
1.一种用于贝类毒素富集纯化的离子液体键合硅胶,其特征在于,结构式如下:
2.根据权利要求1所述的用于贝类毒素富集纯化的离子液体键合硅胶,其特征在于,由以下方法制得:
将N-苯基苯并咪唑加入到γ-氯丙基甲基二乙氧基硅烷的甲苯溶液中,均匀搅拌,之后在氮气气氛下回流48h;冷却至室温后加入活化硅胶粉,继续回流反应24h。
3.根据权利要求2所述的用于贝类毒素富集纯化的离子液体键合硅胶,其特征在于,所述γ-氯丙基甲基二乙氧基硅烷与N-苯基苯并咪唑的摩尔比为:1:1。
4.根据权利要求2或3所述的用于贝类毒素富集纯化的离子液体键合硅胶,其特征在于,所述γ-氯丙基甲基二乙氧基硅烷的甲苯溶液中γ-氯丙基甲基二乙氧基硅烷的浓度为:0.525gmL-1。
5.根据权利要求4所述的用于贝类毒素富集纯化的离子液体键合硅胶,其特征在于,所述γ-氯丙基甲基二乙氧基硅烷与活化硅胶粉的摩尔比为>3:1。
6.根据权利要求2-5任一项所述的用于贝类毒素富集纯化的离子液体键合硅胶,其特征在于,
所述N-苯基苯并咪唑由以下方法制得:将甲酸加入到2-氨基-二苯胺的浓盐酸悬浮液中,在氮气气氛下回流搅拌90min。
7.根据权利要求6任一项所述的用于贝类毒素富集纯化的离子液体键合硅胶,其特征在于,
所述活化硅胶粉是将40-60μm的硅胶粉末在3mol/L盐酸中,浸泡24h后,用纯净水洗至中性,120℃真空干燥5h后得到。
8.一种根据权利要求7所述的用于贝类毒素富集纯化的离子液体键合硅胶的制备方法,其特征在于,具体步骤如下:
(1)在室温下,将甲酸加入到浓度为0.0368gmL-1的2-氨基-二苯胺的浓盐酸悬浮液中,在氮气气氛下回流搅拌90min,其中:2-氨基-二苯胺与甲酸的摩尔比为:1:1;再用氨水(28%)调节pH至8;经乙酸乙酯三次萃取之后,无水硫酸钠干燥,室温下旋转蒸发去溶剂,最后真空干燥得到淡黄色N-苯基苯并咪唑液体;
(2)将40-60μm的硅胶粉末在3mol/L盐酸中,浸泡24h后,用纯净水洗至中性,120℃真空干燥5h;
(3)将将步骤(1)制得的N-苯基苯并咪唑加入到浓度为0.525gmL-1的γ-氯丙基甲基二乙氧基硅烷的甲苯溶液中,搅拌均匀;其中:γ-氯丙基甲基二乙氧基硅烷与N-苯基苯并咪唑的摩尔比为:1:1,反应体系在氮气气氛下回流48h;冷却至室温后加入活化硅胶粉,其中:γ-氯丙基甲基二乙氧基硅烷与活化硅胶粉的摩尔比为>3:1,继续回流反应24h后停止;之后将反应液过滤,用乙腈和去离子水冲洗去除残留物,80℃真空干燥5h,得到淡黄色粉末。
9.根据权利要求1-7任一项所述的用于贝类毒素富集纯化的离子液体键合硅胶,其特征在于,所述贝类毒素为:大田软海绵酸毒素(OA)、鳍藻毒素-1(DTX-1)、鳍藻毒素-2(DTX-2)。
10.一种使用权利要求1-7任一项所述的离子液体键合硅胶作为固相萃取填料用于贝类毒素富集纯化的固相萃取柱。
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