The chromatographic analysis of polyethylene glycols (PEG) and their amino-substituted derivatives, with an average molecular mass of 2000 and 3350 is described. The PEG derivatives were perfectly separated by TSK-GEL G4000PWXL column, which was widely used in high-performance size-exclusion chromatography (SEC), at low ionic strength with refractive index detection. It was shown that ion-exchange interactions were mainly involved in the retention mechanism of these compounds on TSK-GEL G4000PWXL column, since retention volume decreased when salt concentration were added to the mobile phase and varied with the pH of the eluent. Additionally, size exclusion for PEG chains plays a role. Organic modifiers also had effect on chromatographic behaviors of PEG compounds.