Separation of polyethylene glycols and their fluorescein-labeled compounds depending on the hydrophobic interaction by high-performance liquid chromatography

The separation and characterization of fluorescein-labeled polyethylene glycols (PEG) is described. Firstly, the polyethylene glycols labeled with fluorescein isothiocyanate (FITC) were synthesized and separated using Sephadex™ LH-20 medium by a step gradient. Secondly, a TSK GEL G4000 PWXL column was developed for determining the FITC derivatives of PEG. The retention mechanism is based on the hydrophobic interaction between the FITC derivatives of PEG and the packing material of the TSK GEL G4000PWXL column. The retention time of the PEG compounds increased by adding of salts in the mobile phase and decreased by adding of organic modifier. In addition, various salts in the eluent can also change the chromatographic behavior of these compounds. Finally, the pH of the mobile phase can have an impact on the retention time of the PEG compounds.