Characterization of fluorescein isothiocyanate-dextrans used in vesicle permeability studies

Fluorescein Isothiocyanate-dextrans of various weight average molecular masses (4,400-487,000) were analyzed in buffer solution for pH, osmolarity, fluorescence intensity as a function of the polymer concentration, average molecular masses, and radii of gyration. Labeling of polymers and conformation of the polymers were characterized by high-performance gel exclusion chromatography (HPLC-GEC) and small-angle X-ray scattering. The fluorescence measurements evidence the absence of fluorescence quenching of the FITC chromophores but the existence of an inner filter effect at high polymer concentration. The conformation of the polymers in buffer is very likely of random coil type, as shown by the relationship between the radii of gyration and the weight-average molecular masses of the dextrans (Mw). The medium used to analyze the FITC-dextrans by HPLC-GEC strongly influences their elution behavior. In buffer medium, they are sieved over the TSK G4000 PW column through a single population according to their Mw. whereas in pure water, they are separated into several species by an exclusion mechanism that depends on the number of labeled sites per dextran molecule. A Monte Carlo simulation was used to analyze the distribution of the fluorescent labels. HPLC-GEC in water could interestingly be applied to yield labeled polymers bearing a known number of functionalized groups.