Separation of human globin chains by micellar electrokinetic capillary chromatography

Abstract

A new separation method of human globin chains by micellar electrokinetic capillary chromatography (MECC) is described. In this method, a 25 m M phosphate buffer (pH 2.5) containing 7 M urea and 1% (w/v) reduced Triton X-100 buffer system was used. All experiments were performed in a 47 cm×50 μm I.D. uncoated fused-silica capillary. The separation voltage was set at 19 kV. Normal globin chains derived from normal adults and newborns, α, β, δ, Gγ and Aγ globin chains as well as common variant globin chains were successfully separated within 20 min. High reproducible migration times of globin chains (CVs of intra- and inter-assay were less than 1% and 2% respectively), and quantification of Gγ and Aγ chains (CVs for intra- and inter-assay were less than 5% and 10%, respectively) were obtained. This new MECC method provides primary information on structural modification of globin chains. It can be an important diagnostic tool in clinical laboratory practice in the field of hemoglobinopathies.