The excess iron is added to the serum to bind all the transferrin in the serum, and the excess iron is adsorbed by adding the iron adsorbent. The iron bind with the transferrin is separated from the protein by the action of acid solution and reductant. Fe<sup>3+</sup> in serum is reduced to Fe<sup>2+</sup>, Fe<sup>2+</sup> binds with bipyridine to form pink complex. In a certain range, the amount of iron ions is positively correlated with the color, and the measured iron content is TIBC. Subtracting serum iron value from TIBC is called unsaturated iron binding capacity (UIBC).