Determination of Iron in Biological Material

Abstract

A method is proposed for the determination of iron in biological material which makes use of the reaction between iron and thiogly-collic acid in an alkaline solution with the production of a purple color.

Method for blood. Measure 0.2 cc. of blood into a pipette accurately calibrated to contain 0.2 cc. and deliver it into a Pyrex test tube, 20×180 mm., calibrated at 10, 15 and 20 cc. Add 1 cc. of 10 N sulfuric acid and introduce a small piece of silica or a glass bead to prevent bumping. Heat, preferably over a hot plate to evaporate the water. As soon as the material chars, allow to cool for 45 seconds and add 4 drops of 30% hydrogen peroxide drop by drop. Continue the heating for 5 minutes after the solution becomes colorless. Allow it to cool and add 10 cc. water. Into a similarly calibrated Pyrex test tube accurately measure 1 cc. of standard iron solution containing 0.1 mg. iron and dilute with distilled water to about the 15 cc. mark. Add one drop (0.05 cc.) of thioglycollic acid to both standard and unknown, mix by lateral shaking. Run in concentrated ammonium hydroxide from a burette until the permanent purple color makes its appearance, pH of 8 to 10. Mix contents of both tubes, make up to volume of 20 cc. with distilled water and mix again by inversion. Comparison is then made in the colorimeter. The simplified calculation is 1000/R = mg. iron per 100 cc. blood. This is converted to volumes per cent of oxygen by multiplying the mg. iron by 0.4. To convert this value to grams hemoglobin, divide the volume per cent of oxygen by 1.34.