3.6. Gel Filtration Chromatography (GFC)

GFC analyses were performed using a Merck-Hitachi L4000 apparatus (Tokyo, Japan) equipped with a UV detector set at λ of 254 nm. A hydroxylated acrylic polymer GFC column (Polysep-GFC-P 2000, 300 × 7.8 mm, Phenomenex, Aschaffenburg, Germany) was used with a water-based eluent (sodium borate pH 10 buffer 10 mM plus 300 mM NaCl) at 0.5 mL/min flow rate.

The samples (5 mg) were completely dissolved in a small amount of NaOH. The pH of the solution was adjusted to pH 10 with HCl 6 M, and diluted to the final concentration of 2 mg/mL with sodium borate buffer 10 mM pH 10. The sample was then centrifugated, filtered, and analyzed.