To each individual test tube place 0.25 mL of the reactant solution, 0.6 mL of tryptophan 0.016 M solution and 2 mL of fresh ferric chloride 0.025 M solution. Mix thoroughly, then pour 5 mL of concentrated sulphuric acid divided in around 1 mL portions over 30 minutes under continuous stirring in a cold bath of running tap water to prevent any increase in temperature beyond 50 °C. Then read absorbance at 560 nm within 10-20 minutes. If to allow samples to stand elongated time, secondary transformations involving Fe +2 ion might cause a color change and test failure.