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A Method for the Quantitation of Charge by Size Exclusion Chromatography Demonstrated with Components of Ficoll 400
Author(s) -
Zhu Yuxin,
Potschka Martin,
Dubin Paul L.,
Cai Chunhua
Publication year - 2001
Publication title -
macromolecular chemistry and physics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.57
H-Index - 112
eISSN - 1521-3935
pISSN - 1022-1352
DOI - 10.1002/1521-3935(20010101)202:1<61::aid-macp61>3.0.co;2-h
Subject(s) - size exclusion chromatography , chromatography , gel permeation chromatography , ficoll , chemistry , polymer chemistry , materials science , organic chemistry , polymer , peripheral blood mononuclear cell , biochemistry , in vitro , enzyme
Commercial ficoll, rather heterogeneous in molecular weight but generally considered to be non‐ionic, is demonstrated to contain negatively charged residues on about 50% of its macromolecular components. We have fractionated this 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 same strategy for physical characterization of some of these fractions on Superose‐6. These measurements illustrate a new chromatographic approach for estimating the charge and zeta‐potential of macroionic solutes. We also consider the electro‐osmotic swelling of Superose‐6, and the possible implications of hydration and charge induction effects for the use of universal calibration. Lastly, we provide a detailed analysis of the sources of error in the measurement of retention volume in SEC and the related limitations with respect to the determination of apparent molecular size.