IAA quantification by Salkowski Reagent
To quantify the indole-3-acetic acid (IAA) content of a sample solution, Salkowski reagent is most frequently used. Salkowski test for IAA quantification was originally developed by Salkowski E. in 1885.
Original Salkowski reagent was formulated by mixing 1.0 ml of o.5 M ferric chloride (FeCl3), 50 ml of distilled water and 30 ml of concentrated sulfuric acid (H2SO4). However, later on, Salkowski test of IAA quantification was slightly modified in which concentrated sulfuric acid is replaced by 35 % perchloric acid (Gordon SA, 1951).
Because the original Salkowski reagent-IAA complex is only stable up to 15 minutes Gordon modified it and replaced the concentrated sulfuric acid by 35 % perchloric acid.
Preparation of Salkowski reagent
To prepare Salkowski reagent, you need to prepare 0.5 M ferric chloride solution (approximately 10-15 ml ) as per needed by dissolving required amount of ferric chloride in distilled water (use the formula of molarity to prepare 0.5 M ferric chloride solution) and store it in a reagent bottle. Now, prepare 35 % perchloric acid solution using the C1V1 = C2V2 equation.
Now, add 1 ml of 0.5 M ferric chloride solution in a 50 ml of 35 % perchloric acid and mix it well and store it in a brown bottle and label it as a Salkowski Reagent. That’s all, now, your reagent is ready to use for the IAA quantification.
- Prepare standard solutions of IAA in acetone or ethanol (IAA is least soluble in water and highly soluble in organic solvent) of different concentrations from a lower value to a higher value.
- Take 1 ml of IAA standard solutions each in a test tube and add 2 ml of Salkowski reagent in each of the test tubes containing 1 ml of the standard IAA solutions and mix it by gentle vortex.
- Incubate the reaction mixture for 25 minutes and read the absorbance at 530 nm wavelength by setting the auto zero absorbance for the control (solvent without IAA) and plot an IAA standard curve of absorbance versus IAA concentration.
- Now, take 1 ml of the IAA solution (sample) and add 2 ml of the reagent and incubate for 25 minutes and read the absorbance. Use this IAA standard curve and calculate the IAA content of the sample solution.