Lc 20at solvent delivery pump

Protein concentration in extrudates was determined by HPLC (LC20AT Solvent Delivery Pump, CBM-40lite system controller, SIL-10AF Autosampler, CTO-10A column oven, Shimadzu, Kyoto, Japan) using a C18 reversed phase column (NUCLEOSIL 120–3, C18, 5 μm, 125 × 4 mm, MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany). Approximately 50 mg of the extrudates were accurately weighed and dissolved in Milli-Q® water (n = 3). Samples vials were cooled at 5 °C in the autosampler and 10 μL of each sample was injected. The solvent system consisted of water/acetonitrile/trifluoroacetic acid (A: 100/0/0.1, B: 0/100/0.1, V/V). A linear gradient method was applied (0–5-7 min 5–95-5%B) at a flow rate of 1 mL/min for 7 min and a column temperature of 30 °C.
Chromatograms were obtained using an UV-detector (SPD-40 UV Detector, Shimadzu, Japan) at 220 nm.

Semipreparative HPLC was carried out on a Shimadzu system consisting of DGU-20A5R degassing unit, LC-20AT solvent delivery pump, SIL-10AF autosampler, CBM-20A controller, CTO-20AC column oven, SPD-M20A diode array detector and FRC-10A fraction collector (all Shimadzu, Kyoto, Kinki, Japan). Stationary phase was a Luna C-10(2) column with 250 × 10 mm and 10 µm particle size (Phenomenex). Mobile phase consisted of water (A) and acetonitrile (B). Elution at 4 mL/min and 35 °C column temperature started at 40% B, rising to 100% B at 20.0 min. All compounds of interest eluted within this gradient, followed by varying plateaus of 100% B (0–5 min duration) for column cleaning and re-equilibration at 40% B. Injection volume was 200 µL.

Semipreparative HPLC was carried out on a Shimadzu system consisting of DGU-20A5R degassing unit, LC-20AT solvent delivery pump, SIL-10AF autosampler, CBM-20A controller, CTO-20AC column oven, SPD-M20A diode array detector and FRC-10A fraction collector (all Shimadzu, Kyoto, Kinki, Japan). Stationary phase was a Luna C-10(2) column with 250 × 10 mm and 10 µm particle size (Phenomenex). Mobile phase consisted of water (A) and acetonitrile (B). Elution at 4 mL/min and 35 °C column temperature started at 40% B, rising to 100% B at 20.0 min. All compounds of interest eluted within this gradient, followed by varying plateaus of 100% B (0–5 min duration) for column cleaning and re-equilibration at 40% B. Injection volume was 200 µL.