Chromatofocusing and isoelectric focusing in immobilized pH gradients compared for characterization of human hemoglobin variants.

We compared the performance of two highly resolving methods, chromatofocusing (CRF) and isoelectric focusing in immobilized pH gradients (IPGF), for the separation of human hemoglobin variants. Lysates containing 13 different hemoglobins, including variants of clinical and geographical importance, and four electrophoretically "silent" variants (Hb Brockton, Hb Cheverly, Hb Köln, and Hb Waco) were analyzed. Both techniques showed a good intrarun precision (CV = 0.87% for CRF, 0.27% for IPGF) and high and similar resolving power (0.010 pH units, with the pH gradients used in this work). The use of an ultranarrow IPGF range (pH 7.15-7.35; pH gradient = 0.019 pH/cm) allowed the resolution between Hb Brockton, Hb Köln, and Hb A. In some cases (Hb D-Los Angeles, Hb F, Hb Waco), the variants were separated from Hb A in different orders, depending on which technique was used, probably because of the different analytical principles of the two methods. As a second-level test, both procedures are informative for characterization of human hemoglobin variants.