Fig. 1

SEC chromatographic isolation of SEC-NPs from porcine bone soup. Bone soup was fractioned by a Sephacryl S-1000 SF column (1.0 × 100 cm) equilibrated with 0.02 M phosphate buffer (pH 7.4). The column was eluted with the same buffer at a flow rate of 0.50 mL/min at 40 °C with UV and Zetasizer Nano-ZS detector (Malven, UK). The purity and molar mass of SEC-NPs was determined by a SEC-MALLS (Size-Exclusion HPLC coupled with Multi-Angle Laser Light Scattering). A TSK gel G6000PWxl HPLC column (0.78 × 300 cm, Tosoh Bioscience, Japan) was equilibrated and eluted with phosphate buffer (0.1 M, pH7.4) at flow rate 0.80 mL/min. a: SEC chromatogram (Sephacryl S-1000 SF column) of the porcine bone soup is presented as the Derived Count Rate (solid line) and UV absorptance at 280 nm (dash line). The fraction of NPs was collected and named SEC-NPs, as indicated by two dot line. b: The SEC-MALLS chromatogram (TSK gel G6000PWxl HPLC column) of SEC-NPs fraction is presented as the light scattering intensity at 90° of MALLS (solid line) and UV absorptance at 280 nm (dash line). c: The solid line is the SEC-MALLS chromatogram wherein the dash line represents the geometric radius distributions (22–49 nm) of the light scattering peak