How to prepare Salkowski reagent for the quantification of indole-3-acetic acid?

Estimation of the indole-3-acetic acid using Salkowski reagent

What is a Salkowski reagent

Salkowski reagent is a formulation that researchers have developed for the determination of the concentration of indole-3-acetic acid. It consists of ferric chloride dissolved in a concentrated oxidizing acid. It was originally developed by Salkowski in 1885 for the colorimetric determination of compounds containing indole ring. Currently, Salkowski reagent is primarily used for the determination of the concentration of indole-3-acetic acid.

Please take note, there is another Salkowski reagent that is used for the estimation of the total cholesterol. The same person (Salkowski E.) had developed these two reagents but for different purposes. Though these two reagents have same ingredients; ferric chloride and concentrated sulfuric acid, they have different formulations and mechanism of actions.

Original Salkowski reagent

The original formulation of Salkowski reagent contains 0.5 M FeCl3, distilled water and concentrated H2SO4 in a proportion of 1:50:30 (v/v/v). However, the original formulation forms a pink color reaction product that is stable only for short time. Once the color forms, it starts fading away after 15 minutes. Therefore, we need to finish taking absorbance at 530 nm wavelength as soon as possible.

In order to increase the sensitivity as well as specificity for the indole acetic acid, researchers have developed many formulations by modifying the original Salkowski reagent. These formulations basically differ in the Ferric chloride concentration and the type of oxidizing acids used.

One of such modification is Fe-HClO4 and it includes; use of the 35% perchloric acid in place of the sulfuric acid. The Fe-ClO4 formulation was developed by Gordon and Weber in 1950. It contains 0.5 M FeCl3 in 35% HClO4 in a proportion of 1:50 (v/v). The color developed is stable up to three hours but we need to measure the absorbance after 25 minutes of incubation for better results. We can measure the absorbance at the same wavelength; 530 nm.

Salkowski reagent preparation

We need two things for the Salkowski reagent preparation and these are; 0.5 M ferric chloride and 35 % perchloric acid. You can easily prepare the 0.5 M ferric chloride solution using molarity formula, while you can purchase the ready-made 35 % solution of perchloric acid. Alternatively, you can dilute the higher concentration of perchloric acid to 35 % using the C1V1 = C2V2 equation.

Now, add 1 ml of 0.5 M ferric chloride solution to a 50 ml of 35 % perchloric acid and mix it well. Please do the mixing under the fume hood. After mixing, let the reagent be cool and store it in a brown bottle with proper labeling. That’s all, now, your reagent is ready for the quantification of indole-3-acetic acid.

Estimation of IAA by Salkowski reagent

• Prepare the standards of indole-3-acetic acid in acetone or ethanol ranging from 5 to 50 μg per mL of concentration. Note: IAA is least soluble in water and highly soluble in organic solvent
• Take 1 ml of each indole-3-acetic acid standard in separate test tubes and add 2 ml of Salkowski reagent in each.
• Also, make a blank in which use the solvent with no IAA and add the same amount of reagent.
• Do the same thing for the samples containing an unknown amount of indole-3-acetic acid.
• Mix the reaction mixture well by gentle vortex and incubate it in the dark for 25 minutes.
• After incubation, read the absorbance at 530 nm wavelength. Note: don’t forget to autozero the blank.
• In the next step, plot a standard calibration graph for IAA standards by plotting a graph of absorbance versus IAA concentrations.
• Now, determine the concentration of indole-3-acetic acid by using a linear regression equation.

Reference: Gordon, Solon A., and Robert P. Weber. “Colorimetric estimation of indoleacetic acid.” Plant physiology 26.1 (1951): 192