The effect of different concentration of phosphate buffer solution with pH=7.4 on hydrocarbonylation of 10-phenanthroline-Fe2+ to determine free radicals system was studied.The phosphate buffer solution of 0.1,0.15 and 0.2 mol/L were added to the system of 10-phenanthroline-Fe2+,with the increasing of buffer solution,ΔA536 decreased continually.When the buffer solution is more than 3 mL,ΔA536 remained stably.The system of 10-phenanthroline-Fe2+which contained the phosphate buffer solution with different concentration was eliminated by ascorbic acid.The result showed the effect of removal hydroxyl radicals changed with the concentration and addition of buffer solution.Therefore,the concentration and addition of phosphate buffer solution should be known on hydrocarbonylation of 10-phenanthroline-Fe2+ to determine free radicals system.The effectively comparison could be happened in one system.