Agilent 1260 infinity refractive index detector

The freeze-dried initial WPH, peptide recovery fraction and final WPH lactose content was analyzed by high-performance liquid chromatography (HPLC) [23 ]. Briefly, to perform the analysis, 0.2 g of the sample was solubilized in HPLC-grade water before being treated with Biggs–Szijarto solution. The solution was then centrifuged (5000× g, 5 min, 10 °C). The supernatant was recovered and diluted 10X in HPLC-grade water before being filtered with a 0.45 mm nylon filter. For the HPLC measurements, an Agilent 1100 Series chromatograph (Santa Clara, CA, USA) equipped with an Agilent 1260 Infinity refractive index detector (Santa Clara, CA, USA), a column oven and a cooled 717 Plus autosampler were used. The previously prepared samples were injected onto an ICSep-ICE-ION-300 column (Transgenomic, Omaha, NE, USA), and the mobile phase was a solution of H₂SO₄ (180 μL/L) at a flow rate of 0.4 mL/min. The column was kept under a constant temperature of 40 °C, and the run time was 45 min. To perform the quantification, a standard of lactose anhydrous (Sigma Company, Saint-Louis, MO, USA) was used.

Gel permeation chromatography (GPC) was used to determine the weight average (M_w) and number average (M_n) relative molecular weight and dispersity of the mPCL-HA scaffolds. Sections of the mPCL-HA scaffolds were cut and dissolved in tetrahydrofuran (THF) at a concentration of 1 mg/mL. All samples were passed through 0.22 μm PTFE membrane filters. Measurements were conducted on a PSS SECurity2 system consisting of a PSS SECurity Degasser; PSS SECurity TCC6000 column oven (35°C); PSS SDV column set (8 × 150 mm; 5 μm precolumn; 8 × 300 mm; 5 μm analytical columns; and 100,000 Å, 1,000 Å, and 100 Å); and an Agilent 1260 Infinity isocratic pump, Agilent 1260 Infinity standard autosampler, Agilent 1260 Infinity diode array and multiple wavelength detector (A: 254 nm, B: 360 nm), and Agilent 1260 Infinity refractive index detector (35°C). HPLC-grade THF—stabilized with BHT—was used as an eluent at a flow rate of 1 mL/min. Narrow-disperse linear poly (methyl methacrylate) (M_n: 202 g/mol to 2.2 × 10⁶ g/mol) standards (PSS ReadyCal) were used as calibrants. Molecular weight and dispersity analyses were performed using PSS WinGPC UniChrom software (version 8.2).

Gel permeation chromatography (GPC) was carried out to study the molecular weight reduction of PCL scaffolds during degradation. All samples were dissolved in tetrahydrofuran (THF) and passed over 0.22 µm PTFE membrane filters. GPC measurements were performed on a PSS SECurity2 system consisting of a PSS SECurity Degasser, PSS SECurity TCC6000 Column Oven (35 °C), PSS SDV Column Set (8 × 150 mm 5 µm precolumn, 8 × 300 mm 5 µm analytical columns, 100,000 Å, 1000 Å, and 100 Å) and an Agilent 1260 Infinity Isocratic Pump, Agilent 1260 Infinity Standard Autosampler, Agilent 1260 Infinity Diode Array and Multiple Wavelength Detector (A: 254 nm, B: 360 nm), Agilent 1260 Infinity Refractive Index Detector (35 °C). HPLC grade THF, stabilized with BHT, was used as eluent at a flow rate of 1 mL/min. Poly (methyl methacrylate) (M_n: 202 g·mol⁻¹ to 2.2 × 10⁶ g·mol⁻¹) standards (PSS ReadyCal) were used as calibrants. Molecular weight and dispersity analyses were performed in PSS WinGPC UniChrom software (version 8.2).