Colorimetric Estimation of Indoleacetic Acid.

The wide use of the auxin, indoleacetic acid, in physiological and biochemical experiments has promoted interest in methods for its colorimetric estimation. MITCHELL and BRUNSTETTER (1) have proposed both the nitrite and the ferric chloride-sulphuric acid tests for the quantitative estimation of indoleacetic acid (IAA) in aqueous solutions, basing their suggested procedures upon a study of optimal reaction conditions for these two reagents. According to them, the nitrite method is sensitive to 10 ug. IAA/ml. and develops a red color that is stable after two hours. In several attempts to duplicate their nitrite method using solutions of IAA varying from 20 to 45 pg./ml., we could not obtain a stable red color with IAA at the two hours proposed, or at any other time. A faint pink develops almost immediately which rapidly fades to orange or yellow, depending on IAA concentrations, within j hour. If the concentration of nitrite is reduced, the red color becomes sufficiently persistent to be read. Indole likewise gives a strong, relatively stable, red color in this test (cf. table II)-a reaction which is sometimes used as a qualitative test for indole (Nitroso-Indole reaction). TANG and BONNER (2) have modified the ferric chloride-sulphuric acid method for IAA, combining the iron and sulphuric acid as a single reagent to yield improved sensitivity. However, the color produced is also unstable, rapidly developing and then fading. We have found, as have these workers, that the fading color can be practically dealt with by adopting a standard time between addition of reagent and reading of absorbancy or transmittance. Both of the methods discussed above possess disadvantages, lacking either specificity, sensitivity, or stability of color complex formed. During a study of the inactivation of IAA in aqueous solutions, it was frequently necessary to assay at one time many samples where the IAA concentrations were low, or where the degree of significance of small differences in concentrations between experimental units required evaluation. Hence, we considered it desirable to re-examine the ferric chloride-sulphuric acid procedure. Several alterations have been made which produce a more stable color, of increased specificity, which changes in density more rapidly with variation in IAA concentration. 1. The procedure of Tang and Bonner can be improved somewhat by reading at 15 minutes after addition of reagent (instead of 30 minutes as they suggest), since the transient color reaches a maximum at the former time. Maximum absorption was found to occur at 530 m,u.