Selective Differentiation of Indoleacetic Acid and Indolebutyric Acid Using Colorimetric Recognition after Ehrlich Reaction

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Abstract

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.

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