3] Detection of globin chains by reversed-phase high-performance liquid chromatography

Abstract

Publisher Summary This chapter discusses the principal for the detection of globin chains by reversed-phase high-performance liquid chromatography (HPLC). Reversed-phase high-performance liquid chromatography (RP-HPLC) has greatly influenced and stimulated research in detection, quantitation, and identification of normal and abnormal globin chains in human newborn and adult red blood cells. In fact, RP-HPLC methodology has a number of advantages: it is fast and accurate, minute amounts of material are sufficient for each determination, complete automation is possible, and, most importantly, identification of electrophoretically silent mutant hemoglobins caused by neutral-to-neutral amino acid substitutions is feasible and reliable. It also greatly facilitates the separation and isolation of even microquantities of a mutant globin chain, and of globin proteolytic fragments, for subsequent characterization of the substitution within the abnormal peptide. This is possible by means of HPLC techniques for the determination of the amino acid composition at the picomole or subnanomole level, and microsequencing techniques, fast atom bombardment mass spectrometry (FAB mapping), electrospray mass spectrometry, and capillary electrophoresis. The chapter describes several different HPLC procedures. Some use cation-exchange systems whereas others use the reversed phase with different columns, such as Waters μBondapack C18, Merck LiChrospher 100 CH-8/2, Aquapore RP-300, and Partisil C18.