For this demonstration, 10 mL of 50 mM equine heart myoglobin was prepared using deionized water. The protein solution was transferred to a separatory funnel. Then, 100 μL 2 M HCl was added to the protein solution and mixed well, followed by 10 mL of water-saturated 2-butanone. The solution was mixed gently by inversion with occasional venting and allowed to settle down. The bottom aqueous layer was discarded by draining. 1 mL of 5 M NaOH was added dropwise to the remaining layer to observe the formation of the green solution. This solution was mixed further by inverting the separatory funnel several times to obtain a green aqueous layer at the bottom and a colorless butanone layer on top (Figure , right panel).