2998 photodiode array pda detector

HPLC-UV clean-up and enrichment of the enzyme free samples were performed on a 4.6 mm × 150 mm Atlantis^® dC18 100 Å column (5 μm particle size, Waters, Milford, MA, USA), loaded with a 4.6 mm × 20 mm Guard Column 2 pK (Atlantis^® dC18 5 μm) on a Waters Alliance^® HPLC System (Waters e2695Separations Module, including a Waters 2998 Photodiode Array (PDA) detector as previously described [22 (link)]. The gradient program used an eluent composed by 2 mM ammonium formate, acetonitrile and methanol, while the fractions containing the lesions were collected, freeze-dried, pooled, freeze-dried again, and redissolved in Milli-Q water before been injected for LC-MS/MS analysis [21 (link),67 (link),68 (link)].

Analyses of sample compositions were performed using an Alliance e2695 HPLC system (Waters, Milford, CT, USA) equipped with a 2998 Photodiode Array (PDA) detector (Waters, Milford, CT, USA) and a Zorbax Eclipse Plus C18 column (250 × 4.6 mm, 5 μm, Agilent, Palo Alto, CA, USA). The temperature of the column oven was set at 30 °C. A mixture of H₃PO₄ solution (pH 2.5, phase A) and acetonitrile (phase B) were used as the mobile phase using the following gradient program: 0 min, 80% A, 20% B; 25 min, 20% A, 80% B; 35 min, 20% A, 80% B; 36 min, 80% A, 20% B; 41 min, 80% A, 20% B. The PDA was set at 200–600 nm, and the flow rate of the mobile phase was 0.8 mL/min.

Analyses of pigment compositions were performed using an Alliance e2695 HPLC system (Waters, Milford, CT, USA) equipped with a 2998 Photodiode Array (PDA) detector (Waters, Milford, CT, USA) and a Zorbax Eclipse Plus C18 column (250 × 4.6 mm, 5 μm, Agilent, Palo Alto, CA, USA). The temperature of the column oven was set at 30 °C. A mixture of H₃PO₄ solution (pH 2.5, phase A) and acetonitrile (phase B) were used as the mobile phase using the following gradient program: 0 min, 80% A, 20% B; 25 min, 20% A, 80% B; 35 min, 20% A, 80% B; 36 min, 80% A, 20% B; 41 min, 80% A, 20% B. The PDA was set at 200–600 nm, and the flow rate of the mobile phase was 0.8 mL/min [21 (link)].

Chemical profiling of main constituents in the W. ganpi extract were determined with HPLC-PDA. The HPLC-PDA analysis was performed using a Waters e2695 separation module equipped with a 2998 photodiode array (PDA) detector (Waters Corporation, Milford, MA, USA) and a SunFire^® C18 column (4.6 × 250 mm², 5 μm, Waters) maintained at 30 ± 2 °C. Solvent A (0.1% formic acid-acetonitrile) and solvent B (0.1% formic acid-water) were used as the mobile phases (0–5 min, 5–10% A; 5–20 min, 10–30% A; 20–25 min, 30–50% A; 25–30 min, 50% A). The injection volume was 10 µL, the flow rate was set at 1.0 mL/min, and detection was performed at 340 nm.

Isocratic elution method was applied for products analysis of luteolin homo-coupling. DI water with 0.1% formic acid was choose as mobile phase A while acetonitrile with 0.1% formic acid was applied for mobile phase B. The column was equilibrated with 71.5% mobile phase A for 10 min before isocratic elution of the same percentage mobile phase A from 0 to 35 min at a flow rate of 1.0 mL/min, 10 μL from reaction solution was injected into the HPLC system for analysis.
The LC–MS system was equipped with a C18 column (Phenomenex, Luna 5u C18, 250 × 4.6 mm) guard column (4 × 3.0 mm) was applied for product characterizations. The samples (10 μL) were filtered through 0.2 μm membrane (Merck Millipore, USA) before being injected into the HPLC system. The Waters 2998 Photodiode Array (PDA) Detector was connected to the HPLC system with detection wavelengths from 190 and 800 nm. Bruker AmaZon-X is applied for LCMS and LCMSMS for characterization and fragments analysis of unknowns. The LC conditions for LC−MS analysis were similar to those mentioned above. All mass spectra were acquired in both positive and negative ion modes using electrospray ionization. The parent ion was selected with a width of ±2.5 Da and fragmented with 50% setting.