Luna omega 5μ c18 2 100

Sunflower protein powders (0.1 g) were each dissolved with 10 ml 0.1 M phosphate buffer (pH 7), following method from Albe Slabi, Mathé, et al., (2020) (link) and vortexed for 10 sec before filtering through a 0.2 µm syringe filter (Thermo Fisher Scientific, Hampton, NH). All samples were further diluted (samples: HPLC-grade water 1:4) before HPLC analysis (Figure 4).
Chlorogenic acid (CGA) was quantified as outlined by Y. Liang & Were, (2020) using an external standard curve of 0 to 0.1 mM CGA solutions diluted from 0.1 mM CGA solution (0.0354 g CGA in 10 ml HPLC water).
Figure 4 Quantification and identification of free chlorogenic acid using HPLC Free soluble CGA was quantified using Agilent 1100 series HPLC instrument (Santa Clara, CA, USA) with a G1315B diode array detector. A Phenomenex® Luna Omega 5μ C18 (2) 100 Å (150 × 2 mm, 1.5 μm particle size) column was set at 30 °C. Wavelengths of 320 nm and a flow rate of 0.3 mL/min were used. HPLC-grade water with 0.1% formic acid and HPLC-grade acetonitrile with 0.1% formic acid were used for mobile phases A and B, respectively. The gradient was 0 min, 0% B; 1-1.5 mins, linear gradient from 0% to 15% B; 1.5 -4.5 mins, linear gradient from 15% to 30%; 4.5 to 6.0 mins 30% to 45% B; 6 -8 mins, linear gradient 45% to 100% B; 8 -9 mins isocratic on 100% and 9 -10 mins 0%.

Organic sunflower flour (Heliaflor ® 55) manufactured by excluding oxygen with CO2
extraction to obtain a protein content of 55% was purchased from Austrade Inc. (Palm Beach Gardens, Fla., USA.). We purchased a PageRuler™ Plus prestained protein ladder (10 to 250 kDa), chlorogenic acid (98%), HPLC grade acetonitrile, and HPLC water from Thermo Fisher Scientific (Hampton, NH) . A Luna Omega® 5μ C18 (2) 100 Å (150 × 2 mm, 1.5 μm particle size) column was purchased from Phenomenex ® (Torrance, CA), L-cysteine hydrochloride of 98% purity instead of L-cysteine was used due to its high solubility. Thiols of 98% purity were purchased from Acros Organics (New Jersey, USA).