A modified version of the method proposed by Rajapakse et al. [78 (link)] was used. A total of 15.0 g of sephadex G25 fine was weighed and processed. The processed gel was then packed into a column (Φ2.6 cm × 10.0 cm). The detection wavelength was set at 215 nm, and the flow rate was maintained at 5 mL/min. The column was equilibrated with a Gly-NaOH buffer for 5 column volumes (CVs) until a stable baseline was obtained. The sample was then injected with a sample volume of 10 mL per run at a flow rate of 10 mL/min. Desalting was performed using ultrapure water as the eluent for 3 CVs. Fractions were automatically collected at a rate of 3 min/tube, and collection was stopped when conductivity exceeded 1 mS/cm (i.e., each desalting fraction had a volume of 15 mL). The collected desalted solution was freeze-dried to obtain purified protamine.
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