A New High-performance Liquid Chromatographic Procedure for the Separation and Quantitation of Various Hemoglobin Variants in Adults and Newborn Babies

A new cation-exchange HPLC method is described for the separation and quantitation of abnormal human Hbs. The method makes use of Synchropak CM 300, a silica support with carboxylic acid residues, and Bis-Tris-KCN-Na-acetate developers and allows a completion of the chromatogram in 50 to 90 min depending on the sample to be analyzed. Specific chromatographic profiles have been obtained for several beta, alpha, and delta chain variants. The method is useful not only for the quantitation of Hb F and Hb A2 (except in the presence of Hb E) but also provides easy differentiation between simple heterozygotes for a specific abnormality and persons with the same abnormality together with an additional beta-thalassemia heterozygosity. Probably the most important application of the new procedure is in the quantitation of Hb A and the beta chain variants S, C, O-Arab, and E in cord blood samples, thus facilitating the diagnosis of conditions such as AE, EE, AS, SS, S-beta +thal, AC, CC, C-beta +thal, SC, and SO in newborn babies.