Abstract: Method of separating A.sub.14 -.sup.125 I-insulin from heterogeneously labeled insulin molecules for biological studies which involves iodating insulin in a controlled manner, selectively absorbing the insulin from the iodination procedure in a tightly packed bed of octadecylsilane bonded to silica employing reverse phase liquid chromatography principles, thereafter eluting large and small radioactive and nonradioactive materials such as damaged insulin, various contaminants and polar materials from the bed with dilute trifluoroacetic acid, thereafter eluting the intact .sup.125 I-insulin from the bed with a mixture of acetonitrile and dilute trifluoroacetic acid and then separating the A.sub.14 -insulin by high performance liquid chromatography or ion exchange chromatography.