Zetapals

Size exclusion chromatography (SEC) was employed to determine the difference in molecular weight between HA and HA–tyrosine. Samples were analyzed at a concentration of 5 mg/mL. The system used was a Waters e2695 separation module, Waters 2414 refractive index detector, and Waters 2489 UV/vis detector with two columns in series: a PL Aquagel-OH 60 Analytical SEC (300 × 7.5 mm) and then a PL Aquagel-OH 40 Analytical SEC (300 × 7.5 mm) (Santa Clara, California). Samples (80 µL of 5 mg/mL) were dissolved and run using an isocratic 0.5 mL/min mobile phase of 0.1 M ammonium acetate with 0.136 M sodium chloride at pH 5. Chromatograms were analyzed using EMPower 3.
Dynamic light scattering (DLS) of HA was executed to determine the approximate physical size of the HA in solution. DLS was performed using a ZetaPALS (Brookhaven Instruments Corp., ZetaPALS, Holtsville, New York). HA was dissolved in 1× PBS at a concentration of 1 mg/mL and filtered through a 0.45 µm filter. Measurements were performed in a glass cuvette using ZetaPALS software (Holtsville, New York).

Hydrodynamic diameters were determined through room temperature
dynamic light scattering (DLS), using a particle size analyzer (Zeta PALS,
Brookhaven Instruments). Aqueous nanoparticle samples (500 µL) with
concentration between 0.1 and 1 mg/mL were filtered using a 0.2 µm
nylon syringe filter prior to measurements. The log-normal distribution of
the volume-weighted measurements was used to obtain the mean hydrodynamic
diameter and geometric deviation.