Waters 996 pda detector

After tryptic digestion, peptides were separated in a first dimension based on hydrophobicity at high pH by using a reversed phase C18 column (X!Select, CSH, RP-C18, 2.1 x 150 mm, 3.5 μm, Waters) connected to a Waters Alliance e2695 HPLC bio-system and a Waters 996 PDA detector (Waters Corporation, Milford, MA, USA). Solvent A contains 200 mM ammonium formate at pH 10, while solvent C contains 100% water and solvent D 100% acetonitrile (ACN) (LC-MS grade, Biosolve, Valkenswaard, Netherlands). During the chromatographic run, an ACN gradient was performed, while continuously 10% of solvent A was added to become an overall pH of 10 during the entire run. The following gradient was used at a constant flow rate of 200 μL/min: 5% to 15% D over the first 5 min, 15% to 40% D over 80 min, 40% to 90% D over 8 min, 5 min 90% D, and 90% to 5% D over 2 min. In total, 30 fractions were collected starting from 10 to 100 min with an interval of 3 min/fraction. The peptide concentration of the different fractions was determined based on the area under the curve (AUC at 214 nm). Fractions were pooled in a concatenated way (e.g. fractions 1, 11 and 21) to obtain optimal orthogonality, yielding in total 10 fractions for further analysis. Collected fractions were lyophilized and re-suspended in RP mobile phase (97% water, 3% ACN, 0.1% FA).

HPLC analysis have been carried out according to Pudziuvelyte et al. [34 (link)] method. For analysis a Waters 2695 chromatography system (Waters, Milford, CT, USA), equipped with a Waters 996 PDA detector was used. Data was collected and analyzed using the Empower-2 chromatographic manager system (Waters Corporation, Milford, MA, USA). For determination of polyphenols, an ACE 5C₁₈ 250 × 4.6 mm (Advanced Chromatography Technologies, Aberdeen, Scotland, UK) column was used. The mobile phase consisted of solvent A (phosphoric acid/acetonitrile/water) (1:19:80 v/v/v) and solvent B (phosphoric acid/methanol/acetonitrile) (1:40:59 v/v/v). The linear gradient elution profile was as follows: 100% A—at 0 min, 55% A/45% B—at 20 min, 100% B—from 25 to 26 min, 100% A—from 30 to 31 min. The flow rate was 1.2 mL/min and the injection volume was 10 μL. Absorption was measured at 330 nm. Quantification of phenolic compounds was performed using reference standards of apigenin, rosmarinic acid, and chlorogenic acid. The linear calibration curves were constructed (apigenin R² = 0.999979, rosmarinic acid R² = 0.999551, chlorogenic acid R² = 0.999914), the peak areas were used for quantification. The contents were expressed as μg/g dry weight.

Preparative HPLC was generally carried out using Quaternary Gradient Module 2545, Photodiode Array Detector 2998, Autosampler 2707, Waters Prep Degasser and Waters Fraction Collector III. Software: Waters ChromScope v1.40 Beta (Waters, Milford, MA, USA). Stationary phase: Nucleodur^® C18 HTec, 5 µm, 250 × 21 mm, mobile phase: binary gradient of water (A) and acetonitrile (B) at a flow rate of approx. 15.5 mL/min.
Separation of fraction SE6 yielded 1 (118 mg) and 2 (1.2 mg). 3 (11 mg) was obtained from SE7, 4 (5.3 mg), 5 (13 mg) and 6 (0.65 mg) from fraction SE8. Subfractionation of MPLC fraction M2 and M3 led to 15 (39 mg). 16 (0.5 mg) was obtained from M4, and M5 yielded 17 (0.6 mg), 18 (0.9 mg) and 19 (0.3 mg).
Fractions N3 to N5 obtained from FCPC and SE3 were further purified using the following system: Two Waters 515 HPLC Pumps, Waters Pump Control Module II, Degasys DG-2410 (Uniflows, Tokyo, Japan), Waters 996 PDA Detector, software: Waters ChromScope v1.40 Beta Software (Waters, Milford, MA, USA), stationary phase: Eurospher 100 C18, 250 × 21 mm; 7 µm (VDS Optilab, Germany), mobile phase gradient: water (A), acetonitrile (B), flow: 10 mL/min, injection volume: 1 mL. 7 (0.12 mg), 8 (0.9 mg) and 9 (1.7 mg) were isolated from N3, N5 and N4 resp. Compound 14 (11 mg) was isolated from SE3 along with 10 (3.9 mg), 11 and 12 as a mixture (4.3 mg) and 13 (2.7 mg).