Use of high‐performance size‐exclusion chromatography to measure protein molecular weight and hydrodynamic radius

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Abstract
We have conducted a study of the TSK 3000 SW high‐performance size‐exclusion column to define under what conditions proteins would migrate most consistently with their known hydrodynamic properties. Our findings include the following: 1) the residual negative charge of the column does cause charge‐exclusion or charge‐retention effects at low ionic strengths; with elution in deionized water several anionic proteins elute approximately in the void volume; 2) at μ > 0.5, protein migration is not only independent of ionic strength, but consistent with protein molecular weight and hydrodynamic volume; 3) small hydrophobic peptides are retarded by the column; and 4) very asymmetric proteins and other hydrodynamic particles are likely to be retarded by an “end‐on insertion” mechanism.