Reversed-phase high pressure liquid chromatography of globin chains: its application for the prenatal diagnosis of beta-thalassemia.

The separation of alpha, beta, G gamma and A gamma globin chains by reversed phase high pressure liquid chromatography has been studied using columns of octadecylsilyl-silica. All separations were performed in solvent mixtures of water and acetonitrile acidified with phosphoric acid or the hydrophobic ionpairing reagent trifluoroacetic acid. The addition of trifluoroacetic acid or the chaotropic agent NaClO4 to the mobile phase increased the resolution and the peak sharpness of the eluted globin chains. The use of decreasing gradients of TFA or NaClO4 and chromatography at 40 degree were useful steps for the separation of alpha and beta globin chains, a prerequisite for the successful application of this method for the prenatal diagnosis of beta-thalassemia. The beta/gamma synthetic ratio obtained from blood samples taken by fetoscopy from normal fetuses and fetuses with beta-thalassemia trait were measured simultaneously by CM-cellulose chromatography and high pressure liquid chromatography. The values obtained by HPLC were very similar but slightly lower than those obtained by CM-cellulose chromatography. The new method is fast and accurate and will prove to be useful for prenatal diagnosis involving globin chain separation.