A method for the quantitation of charge by size exclusion chromatography demonstrated with components of ficoll 400

Commercial ficoll, rather heterogeneous in molecular weight but generally c onsidered to be nonionic, is demonstrated to contain negatively charged res idues on about 50% of its macromolecular components. We have fractionated t his material by size exclusion chromatography on Sephacryl S-400 HR (under non-adsorbing conditions) according to size and charge, taking advantage of the influence of ionic strength on elution. We subsequently applied the sa me strategy for physical characterization of some of these fractions on Sup erose-6. These measurements illustrate a new chromatographic approach for e stimating the charge and zeta-potential of macroionic solutes. We also cons ider the electro-osmotic swelling of Superose-6, and the possible implicati ons of hydration and charge induction effects for the use: of universal cal ibration. Lastly, we provide a detailed analysis of the sources of error in the measurement of retention volume in SEC and the related limitations wit h respect to the determination of apparent molecular size.