1515 gpc system

GPC was used to measure the molecular weights of parental and FA‐conjugated polymers. The Waters 1515 GPC system is equipped with an ultrahydrogel 250 column (Waters Corporation, MA) and a refractive index detector (Waters 2410). An aqueous solvent containing 0.1% trifloroacetic acid and 40% acetonitrile was used as the mobile phase. The mobile phase in the column was operated at a flow rate of 0.5 mL/min, and the column temperature was set at 35°C. Poly(2‐vinylpyridine) standards (MWs: 3,300, 7,600, 12,800, 35,000, and 70,000 Da) were employed for molecular weight (MW) calibration. All chromatograms were analyzed using the Waters Millennium 32 GPC software. To further investigate the MW range, the parental polymers were first dialyzed through a 3.5 kDa MWCO membrane to remove any unreacted monomers. Following this first step for 48 hr, the retentate was dialyzed through a 10 kDa MWCO membrane. Samples of retentates from the 3.5 and 10 kDa MWCO dialysis membranes were subjected to ninhydrin analysis which reports for reactive amine content. A calibration using glycine as a standard was used to determine the amine content of polymers, and the relative amounts of amines left behind in the retentates were compared to determine the MW range of the synthesized polymers.

The homogeneity and molecular weight of processed DhPs were analyzed using HPGPC conducted on a Waters 1515 GPC system equipped with a Waters 2410 refractive index detector (Waters, Milford, MA, USA) and a Shodex OHpak SB-804 HQ column (8 × 300 mm, 7μm). The following are the chromatographic conditions: a 45 min isocratic elution was applied with the flow rate of 0.6 mL/min; mobile phase was NaCl solution (0.05 M) with the column temperature set to 40 °C. NaCl solution (0.05 M) was used to dissolve processed DhPs whose final concentration was made to 5 mg/mL for analysis. Dextran polymer standards (Sigma-Aldrich, USA) with different molecular weights (1, 5, 12, 25, 50, 80 and 670 kDa) were used for molecular weight estimation. Data acquisition and determination were performed utilizing Empower Software (Waters, Milford, MA, USA).