芹菜素与曲昔匹特的分子印迹研究及色谱定量分析
摘要
分子印迹技术又称为分子烙印技术,它是指制备对某一特定的目标分子(印迹分子)具有预定选择性和特殊亲合性的技术。它在印迹分子存在的情况下,功能性单体与交联剂共聚制得高交联的聚合物网络,移去印迹分子后获得在三维空间构象和结合位点上与某一特定分子(模板分子,印迹分子)完全互补的聚合物。近年来,该技术已广泛应用于色谱分离、抗体或受体模拟、生物传感器以及生物酶模拟和催化合成等诸多领域,并因此成为化学和生物学交叉的新兴领域之一,得到世界注目并迅速发展。研究的主要内容有:
(1)介绍了分子印迹技术及高效液相色谱法的原理、方法、应用及展望;
(2)采用本体聚合法合成芹菜素分子印迹聚合物。紫外光度法证明了模板分子与功能单体之间存在相互作用,并据此选择了聚合反应时合适的溶剂并推测出聚合物的印迹机理。傅立叶红外光谱仪测定了聚合物的结构特征。平衡结合实验研究了聚合物的吸附特性,结果表明,在所研究的浓度范围内(0.1 mmol·L~(-1)～2.5 mmol·L~(-1)),MIP的吸附量大于BP的吸附量,由Scatchard分析得聚合物对芹菜素的识别主要有两类结合位点,其离解常数KD和最大表观结合量Qmax分别为:2.52×10~(-4)和0.54×10~(-3) mol·L~(-1);2.65和18.89μmol·g~(-1)。分子印迹聚合物呈现出空白聚合物没有的高选择性。
(3)建立了新型胃药曲昔匹特胶囊的反相高效液相色谱分析方法。比较了内标法和外标法,内标法以茶碱对照品作为内标物,曲昔匹特在10～160μg·mL~(-1)的范围内质量浓度与峰面积比呈现良好的线性关系,回收率在98.61%～99.91%之间,精密度相对标准偏差为1.35%。外标法中曲昔匹特在40～110μg·mL~(-1)的范围内质量浓度与峰面积比呈现良好的线性关系,回收率在98.84%～100.00%之间,精密度相对标准偏差为1.33%。该方法准确可靠、灵敏度高、重现性好。
(4)建立了凌宵花中芹菜素含量的测定方法。采用C18柱(5μm,150mm×4.6 mm),流动相为甲醇-0.4%磷酸水溶液(50:50),室温操作,检测波长336nm。在1.60～12.80 mg·L~(-1)范围内,芹菜素进样量与峰面积响应值呈良好的线性关系,r=0.9998;回收率为98.19%～101.64%,RSD为1.46% (n=5)。方法简便,灵敏度高,重复性好,可用于凌宵花中黄酮类成分的含量测定。
Molecular imprinting technique (MIT) is a polymerizing technique to produce molecular recognition functional materials with high selectivity and affinity.Polymerization is occurred between monomers and crosslinker around a template.Extration of the template forms polymer with molecular memory to the template.It has mrany applications on the fields of analytical consisted chemistry,catalysis and organic synthesis. In this paper, four chapters were consisted.
(1) The infomation of molecular imprinted polymers (MIPs) and High performance liquid chromatography (HPLC) were introduced.
(2) An apigenin molecularly imprinted polymer was prepared by bulk polymerization using apigenin as template,acrylamide (AA) as a functional mono- mer,ethylene glycol dimethacrylate (EDMA) as a crosslinking agent,acetonitrile and N,N-dimethylformamide as porogenic solvents, and 2,2′-azobisisobutyronitrile (AIBN) as an initiator.The interaction between template and functional monomer was proved by ultraviolet visible (UV) spectrophotometry and based on the results, appropriate reaction solvent was selected and the synthesizing process was estimated. the molecularly imprinted polymer structure was analyzed by Fourier Transform Infrared (FT-IR) spectrophotometry.The molecularly imprinted polymer was investigated in equilibrium binding experiment to evaluate its adsorption property,the results showed that the adsorption of apigenin on MIP is higher than that on blank polymer in the studied concentration range(0.1 mmol·L~(-1)～2.5 mmol·L~(-1)). Scathcard analysis showed that two classes of binding sites existed in the apigenin imprinted polymers,with their KDandQmax estimated to be 2.52×10~(-4), 0.54×10~(-3)mol·L~(-1); 2.65, 18.89μmol·g~(-1) respectively.MIP showed the higher affinity than BP.
(3) A high performance liquid chromatography method for determination of Troxipide was established.Contrast the internal standard method and external standard method in quantitative analysis.There were good linear relationship between the mass concentration and the peak area of Troxipide in the range of 10～160μg·mL~(-1) in the internal standard method.The recovery were found to be in the range of 98.61%～99.91% and the relative standard deviation of precise experiments is 1.35%.The linear relationship in the external standard method was in the range of 40～110μg·mL~(-1),The recovery rate was between 98.84%～100.00% and the relative standard deviation of precise experiments is 1.33%.The two methods are rapid and simple with good accuracy and reproducibility.
(4) This paper reports the determination of Apigenin in Flos Campsis by RP-HPLC method.Chromatograph conditions:column C18(5μm,150mm×4.6mm);mobile phase: methanol -0.4% phosphoric acid(50:50),at ambient temperaturet,UV detection at 336nm.The Linearity ranges were1.60～12.80 mg·L~(-1),r=0.9998.The recovery ranges were 98.19～101.64,the RSD was 1.46%(n=5).This is a simple,convenient and accurate method.
(1)介绍了分子印迹技术及高效液相色谱法的原理、方法、应用及展望;
(2)采用本体聚合法合成芹菜素分子印迹聚合物。紫外光度法证明了模板分子与功能单体之间存在相互作用,并据此选择了聚合反应时合适的溶剂并推测出聚合物的印迹机理。傅立叶红外光谱仪测定了聚合物的结构特征。平衡结合实验研究了聚合物的吸附特性,结果表明,在所研究的浓度范围内(0.1 mmol·L~(-1)～2.5 mmol·L~(-1)),MIP的吸附量大于BP的吸附量,由Scatchard分析得聚合物对芹菜素的识别主要有两类结合位点,其离解常数KD和最大表观结合量Qmax分别为:2.52×10~(-4)和0.54×10~(-3) mol·L~(-1);2.65和18.89μmol·g~(-1)。分子印迹聚合物呈现出空白聚合物没有的高选择性。
(3)建立了新型胃药曲昔匹特胶囊的反相高效液相色谱分析方法。比较了内标法和外标法,内标法以茶碱对照品作为内标物,曲昔匹特在10～160μg·mL~(-1)的范围内质量浓度与峰面积比呈现良好的线性关系,回收率在98.61%～99.91%之间,精密度相对标准偏差为1.35%。外标法中曲昔匹特在40～110μg·mL~(-1)的范围内质量浓度与峰面积比呈现良好的线性关系,回收率在98.84%～100.00%之间,精密度相对标准偏差为1.33%。该方法准确可靠、灵敏度高、重现性好。
(4)建立了凌宵花中芹菜素含量的测定方法。采用C18柱(5μm,150mm×4.6 mm),流动相为甲醇-0.4%磷酸水溶液(50:50),室温操作,检测波长336nm。在1.60～12.80 mg·L~(-1)范围内,芹菜素进样量与峰面积响应值呈良好的线性关系,r=0.9998;回收率为98.19%～101.64%,RSD为1.46% (n=5)。方法简便,灵敏度高,重复性好,可用于凌宵花中黄酮类成分的含量测定。
Molecular imprinting technique (MIT) is a polymerizing technique to produce molecular recognition functional materials with high selectivity and affinity.Polymerization is occurred between monomers and crosslinker around a template.Extration of the template forms polymer with molecular memory to the template.It has mrany applications on the fields of analytical consisted chemistry,catalysis and organic synthesis. In this paper, four chapters were consisted.
(1) The infomation of molecular imprinted polymers (MIPs) and High performance liquid chromatography (HPLC) were introduced.
(2) An apigenin molecularly imprinted polymer was prepared by bulk polymerization using apigenin as template,acrylamide (AA) as a functional mono- mer,ethylene glycol dimethacrylate (EDMA) as a crosslinking agent,acetonitrile and N,N-dimethylformamide as porogenic solvents, and 2,2′-azobisisobutyronitrile (AIBN) as an initiator.The interaction between template and functional monomer was proved by ultraviolet visible (UV) spectrophotometry and based on the results, appropriate reaction solvent was selected and the synthesizing process was estimated. the molecularly imprinted polymer structure was analyzed by Fourier Transform Infrared (FT-IR) spectrophotometry.The molecularly imprinted polymer was investigated in equilibrium binding experiment to evaluate its adsorption property,the results showed that the adsorption of apigenin on MIP is higher than that on blank polymer in the studied concentration range(0.1 mmol·L~(-1)～2.5 mmol·L~(-1)). Scathcard analysis showed that two classes of binding sites existed in the apigenin imprinted polymers,with their KDandQmax estimated to be 2.52×10~(-4), 0.54×10~(-3)mol·L~(-1); 2.65, 18.89μmol·g~(-1) respectively.MIP showed the higher affinity than BP.
(3) A high performance liquid chromatography method for determination of Troxipide was established.Contrast the internal standard method and external standard method in quantitative analysis.There were good linear relationship between the mass concentration and the peak area of Troxipide in the range of 10～160μg·mL~(-1) in the internal standard method.The recovery were found to be in the range of 98.61%～99.91% and the relative standard deviation of precise experiments is 1.35%.The linear relationship in the external standard method was in the range of 40～110μg·mL~(-1),The recovery rate was between 98.84%～100.00% and the relative standard deviation of precise experiments is 1.33%.The two methods are rapid and simple with good accuracy and reproducibility.
(4) This paper reports the determination of Apigenin in Flos Campsis by RP-HPLC method.Chromatograph conditions:column C18(5μm,150mm×4.6mm);mobile phase: methanol -0.4% phosphoric acid(50:50),at ambient temperaturet,UV detection at 336nm.The Linearity ranges were1.60～12.80 mg·L~(-1),r=0.9998.The recovery ranges were 98.19～101.64,the RSD was 1.46%(n=5).This is a simple,convenient and accurate method.
引文
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