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High-performance liquid chromatography (HPLC) was used to separate tryptic peptides of the normal a, β, γ, and δ chains of human hemoglobins A, F, and A2 and of the abnormal chains of 25 hemoglobin variants. HPLC has several advantages over conventional methods used for the separation of proteolytic fragments of hemoglobin chains. The method is fast, and reproducible, and requires only small quantities of material.