Research Center for Analytical Sciences, College of Chemistry, Nankai University, Tianjin 300071, China.
J Agric Food Chem. 2010 Jun 9;58(11):6556-61. doi: 10.1021/jf1000138.
A simple colorimetric method for the differentiation of indoleacetic acid (IAA) and indolebutyric acid (IBA) in plant samples is described. The color change is based upon the reaction between the auxins and p-(dimethylamino)benzaldehyde (PDAB, Ehrlich reagent) following the electrophilic substitution reaction mechanism at the indole ring. Using their different response to reaction temperature and time, the selective determination of IBA in the presence of IAA is achieved by controlling the incubation time of 40 min at 25 degrees C. The total absorbance of IAA and IBA is determined after they react to PDAB for 150 min at 70 degrees C. The concentration of IAA can then be calculated using the difference between their total absorbance and the calculated absorbance of IBA. The detection limits (3sigma) of IAA and IBA were 0.10 microM and 0.28 microM, respectively. The precisions for five replicate measurements of 10 microM IAA and IBA were less than 5% (RSD). The recovery from mung bean sprout samples varied from 87.5% to 108% for the two auxins. Moreover, the Ehrlich reaction conditions are compatible with the methanol-hydrochloric acid extraction procedure. All of the above results indicate that this protocol provides a rapid, simple, convenient and practical method for detection and differentiation of IAA and IBA. From the color changes of IAA and IBA after Ehrlich reaction, the identification of auxin at the microM level can be achieved even with the naked eye. The method was successfully used to investigate the auxin changes of mung bean sprout during the growth procedure.
一种用于区分植物样品中吲哚乙酸（IAA）和吲哚丁酸（IBA）的简单比色法。颜色变化基于植物激素与对二甲氨基苯甲醛（PDAB，Ehrlich 试剂）之间的反应，该反应遵循吲哚环上的亲电取代反应机制。通过控制在 25°C 下孵育 40 分钟的时间，可以利用它们对反应温度和时间的不同反应，实现 IBA 在 IAA 存在下的选择性测定。将 IAA 和 IBA 与 PDAB 反应 150 分钟后，测定它们的总吸光度。然后，可以使用它们的总吸光度与计算出的 IBA 吸光度之间的差异来计算 IAA 的浓度。IAA 和 IBA 的检测限（3sigma）分别为 0.10 microM 和 0.28 microM。10 microM IAA 和 IBA 的 5 次重复测量的精密度小于 5%（RSD）。两种植物激素在绿豆芽样品中的回收率在 87.5%到 108%之间。此外，Ehrlich 反应条件与甲醇-盐酸提取程序兼容。所有这些结果表明，该方案提供了一种快速、简单、方便和实用的检测和区分 IAA 和 IBA 的方法。通过 Ehrlich 反应后 IAA 和 IBA 的颜色变化，即使在肉眼下也可以实现对植物激素的 microM 水平的鉴定。该方法成功地用于研究绿豆芽在生长过程中的植物激素变化。
