This paper concerns the mechanism of separation of polyelectrolytes by steric exclusion chromatography (SEC). It is demonstrated that the fractionation follows universal calibration when the electrostatic exclusion effect, that is, the long-range electrostatic repulsions, are screened: a minimum concentration of 1–1 electrolyte around 10−2 M is necessary. In addition. SEC with multidetection is convenient to fully characterize a poly electrolyte and to demonstrate the role of the ionic concentration on chain dimensions and to determine the intrinsic persistence lengths, a convenient characteristic for polysaccharides, which are considered as wormlike chains. The analysis is developed on a sample of hyaluronan.