Agilent dad detector

Method was made according to Bolarinwaa et al. [9 (link)], with some corrections. Briefly, 2 g of protein cakes and PI were measured into a round-bottom flask (100 mL), and then added 50 mL ethanol and the mixture was boiled under reflux during 120 min at 78.5 °C. In the end of extraction, the extracts were filtered and evaporated under vacuum to remove ethanol. On this way samples were dissolved in the water andanalyzed by High-Performance Liquid Chromatography HPLC (Agilent 1290 Infinity I HPLC system with an Agilent DAD detector, Santa Clara, CA, USA. The column for separation was used a SupelcoAnalitycal HS-C18 column (4.6 × 250 mm, 5 µm) Sigma-Aldrich (St Louis, MO, USA). The chromatographic conditions were: flow rate 1 mL/min, temperature 20 °C, injection volume 20 µL and UV detection (Agilent DAD detector, Santa Clara, United States) at 210 nm. Mobil phase consisted of distillation water: methanol (75:25 v/v).

An Agilent 1260 series HPLC instrument (Santa Clara, CA, USA) equipped with an Agilent DAD detector (All Tech, Lexington, KY, USA) was used to obtain HPLC fingerprints. A Thermo C₁₈ column (250 mm × 4.6 mm, i.d., Agilent) was used for analyses. For separation, a column temperature of 30°C, a flow rate of 1.0 mL/min, and an injection volume of 10 μL were used.
The optimized elution conditions were as follows: (C) acetonitrile and (D) 0.3% formic acid in water were used as the mobile phase. A linear gradient of 0%–7% (C) (0–15 min), 7%–20% (C) (15–20 min), and 20%–43% (C) (20–40 min) with wavelength conditions of 273 nm (0–20 min), 360 nm (21–30 min), and 270 nm (31–40 min) was applied.