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液相色谱常用柱前衍生化
使用液相色谱最多的是紫外检测器,为了使一些没有紫外吸收或紫外吸收很弱的化合物能被紫外检测器检测,往往要通过衍生化反应在这些化合物的分子中引入有强紫外吸收的基团。这些衍生物可被紫外检测器检测。

    大多数紫外衍生反应来自经典的广度分析和有机定量分析,新的衍生化反应和衍生试剂随液相色谱的发展而发展,这些反应的原理都来自有机合成,所以就要求操作者对有机合成有所了解。但是,由于柱前衍生化是为色谱分析准备样品,需要衍生处理的样品的含量水平和所参与的衍生化反应过程与常量有机合成反应相比有着明显的不同,而是类似于近年来发展的微量有机合成,紫外衍生化反应要求选择反应产率高、重复性好的反应。过量试剂和试剂中的杂质如果会干扰下一步的色谱分离和检测,则在色谱进样前要进行纯化分离。还要注意反应介质对紫外吸收的影响。下面分别介绍一些常用的反应。

    (1)苯甲酰化反应  苯甲酰氯及其衍生物一对硝基苯甲酰氯,3,5-二硝基苯甲酰氯和对甲氧摹苯甲酰氯都可以同胺、醇和酚类化合物反应,生成强紫外吸收的苯甲酸酯类衍生物,过量试剂可以通过水解除去,反应产物可用有机溶剂提取后直接进样。

    (2) 2,4-二硝基氟代苯 (DNFB) 的反应  DNFB与醇的反应产率很低,但可与大多

数伯胺、仲胺和氨基酸反应,生成强紫外吸收的苯胺类衍生物。

    (3) 苯基异硫氰酸酯 (PITC) 的反应  PITC可与氨基酸反应,生成苯基己内酰硫脲衍生物PTH-氨基酸。

    苯基异氰酸酯与醇类反应生成苯基甲酸酯。

(4) 苯基磺酰氯的反应  苯基磺酰氯可与伯胺和仲胺反应。

    甲苯磺酰氯可与多氨基化合物反应,不仅能提高其检测是敏度,还可改变HPLC的分离度。

    (5) 有机酸的酯化反应  有机酸很容易与酰溴基反应生成酯,常用的酰溴基试剂有苯甲酰溴、甲氧基苯甲酰演、对溴基苯甲酰溴和对硝基苯甲酰溴等。酯化反应在极性溶剂(如乙腈、丙酮或四氢呋喃)中进行,有时需加催化剂,如冠醚加钾离子、三乙胺或N-二异丙基胺等。

    (6) 羰基化合物的反应  醛类和酮类中的羰基可与2,4-二硝基苯肼 (DNPH) 反应, 生成苯腙衍生物,反应在弱酸条件下进行。

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