Perfusion chromatography on reversed-phase column allows fast analysis of human globin chains

Human globin chain analysis provides important information on the genetics and molecular pathophysiology of hemoglobinopathies. We propose using perfusion chromatography on the reversed-phase stationary phase to perform these studies. The technique, herein described, involves a high-velocity flow of the mobile phase through a porous chromatographic stationary phase made of microspheres of poly(styrene-divinylbenzene) having throughpores of 6000-8000 A diameter with short diffusive pores of 500-1000 A diameter connected to them. The composition of fetal hemoglobin (Ggamma:Agamma ratio) is determined, using this method, as an order of magnitude faster than with conventional HPLC. Elution is performed by developing a linear gradient of acetonitrile at a flow rate of 3 ml/min (or more), easily obtained on any HPLC machine. Analyses may be done on samples containing as low as 3.0% Hb F. Results are similar to those obtained with the reference HPLC technique, which uses a C4 column. In addition, reversed-phase perfusion chromatography, using a shallow curvilinear gradient, may help in the characterization of Hb variants. This technique allowed us to discriminate several alpha and beta chain mutants from variants that have closely similar patterns of electrophoretic mobilities.