Separation of haemoglobins using a monodisperse cation exchanger

In order to separate haemoglobin variants in one step, a chromatographic method using Mono S (Pharmacia) monodisperse cation exchanger was developed The separation of haemoglobins (A, S, E, D, C and F) on a short analytical column (50 X 5 mm I.D.) was accomplished in 30 min, including the regeneration, using linear buffer gradient elution (lithium chloride in 0.01 M sodium malonate, 0 to 100%). For haemoglobin A, two peaks, probably corresponding to different molecular forms, were found. This systematic double peak affected the recognition and the measurement of haemoglobins. By filling the test-tube with carbon monoxide before injecting the haemolysate, we obtained reproducible results with regard to both retention times and peak shape, and also great stability. The long-term stability and the high speed of the separation seem to be adequate for automation, making the method suitable for routine clinical laboratory-use.