High performance liquid chromatography of hemoglobins: factors affecting resolution

We have investigated the effect of various factors on resolution of hemoglobin mixtures by ion exchange HPLC. Small changes in the pH of start buffer had a considerable effect on the ability to separate HBs A2 and S. Complete separation of Hbs A2, S, A, Hb A1 fraction and Hb F required 30 min. gradient elution programs. Aging of the hemolysate obscured the presence of small amounts of Hb F. Deterioration in column performance was attributed to dissolution of some of the column packing material as well as irreversible binding of hemoglobin component(s) to the ion exchanger. Substantial differences in elution time were observed between different columns requiring modifications in the gradient programs with some columns. The resolution achieved makes the HPLC technique a powerful new tool for hemoglobin analysis.