An analysis of electrophoretic and microcolumn methods for the separation of hemoglobins A and A2

Parameters of three techniques for quantitating hemoglobin A2 were studied in order to identify problems affecting repeatability and then to compare intertechnique results under selected conditions. Satisfactory repeatability with cellulose acetate electrophoresis and scanning densitometry required an applicator that delivers a constant volume of sample. For cellulose acetate electrophoresis/elution and chromatographic assays a sophisticated absorption spectrophotometer and pH meter are necessary. Even with the most carefully chosen conditions significant intertechnique variation occurs. Although the colums are the most repeatable, trailing (a problem usually associated with hemoglobin electrophoresis) has also been demonstrated with column chromatography. Isoelectric focusing demonstrated the copresence of hemoglobin A and hemoglobin A2 in all trail fractions between the two major peaks and in some fractions of each peak. Standards of low protein concentration could be prepared from column fractions identified by isoelectric focusing as containing only hemoglobin A or hemoglobin A2. Such standards would be useful for assessing the accuracy of hemoglobin A2 quantitation.