2998 photodiode array

Gas Chromatography (Agilent 6890N Network) associated with a Mass Spectrometer (Agilent 5975 Inert Mass Selective Detector) and Flame Ionization Detector with the separation column DB 5MS 29.8 m × 0.32 mm × 0.5 µm was used for concentration determinations. Helium was used as carrier gas at a flow rate of 1 mL∙min⁻¹ and oven program was: Forty degrees celsius during 2 min; heating ramp of 5 °C∙min⁻¹ up to 320 °C and hold for 20 min. The FID detector was operated at 320 °C. The mass spectrometer was operated at 220 °C; scans were carried out between 35 and 620 m/z.
For large solutes, liquid chromatography (Agilent) associated with a UV detector (Waters 2998 Photodiode Array) with the separation column Poroshell 120 EC-C18 2.7 µm, 4.6 mm × 100 mm was used. An isocratic program with a mixture of 5 % of tetrahydrofuran and 95 % of acetonitrile was applied for 20 min with a flow rate of 1 mL∙min⁻¹ at 40 °C_.

The mobile phases that were used for the HPLC analysis are A: H₂O + 0.1% TFA and B: CH₃CN + 0.1% TFA. The crude peptide is purified by a preparative method on a Waters device (pump module: 2535 Quaternary Gradient module; UV detector: Waters 2998 Photodiode Array), with an XBridge BEH 300A Prep C18 column OBD 5 μm 19 × 250 mm, at a flow rate of 13 mL/min. Analytical HPLC is performed on a Waters device (pump module: e2695 separation module; UV detector: Waters 2998 Photodiode Array) with an XBridge BEH 300A C18 column (5 μm, 4.6 mm × 250 mm, Waters). The indicated retention times are given in minutes. Gradient used was 0–50% B in 50 min with a wash phase at 80% B for 10 min and a column equilibration phase at 100% A for 10 min.

Analysis was performed on a Waters platform, composed of a Waters 2695 HPLC unit equipped with a Waters 2998 photodiode array (PDA) detector and a Luna silica column (4.6 mm i.d. ×250 mm, 5 µm particle size; Phenomenex, Torrance, CA, USA). The unit was coupled with a Bruker Esquire 3000 Plus ion trap mass spectrometer (Bruker–Franzen Analytik GmbH, Bremen, Germany) equipped with ESI. The PCBLs was dissolved in methanol to prepare a 20 mg/mL solution. The mobile phases consisted of hexane/methanol/ethyl acetate (10∶3∶1, v/v/v) (A) and hexane/methanol/ethyl acetate (1∶3∶1, v/v/v) (B). Separations were done by linear gradient at 37°C at 1 mL/min flow rate as follows: 0–90 min, 83.6% A; 90–130 min, 83.6–19.4% A; 130–150 min, 19.4% A. The PDA detector was set to 280 nm and scanning was done from 200 to 400 nm.
Preparation was performed on a Phenomenex (Torrance, CA, USA) Luna silica preparative column (21.2 mm i.d. ×250 mm) with a 5 µm particle size at 37°C. A Shimadzu system equipped with a CBM-20A module, an SIL-10AP autosampler, an SPD-20A UV–vis detector, and two LC-8A pumps was used. Elution was the same as the method described above. The flow rate was 21.6 mL/min and the absorption wavelength was at 280 nm. On a given run, 1 mL (200 mg/mL) extract was applied. The fractions were collected and evaporated under vacuum.

The HPLC analyses were carried out on a Waters e2695 HPLC system equipped with a 2998 photo diode array (PDA) detector (Waters Corp., Milford, MA, USA) using a Phenomenex Luna C18 column (5 μm, 250 × 4.6 mm). Mobile phase A was acetonitrile and mobile phase B was 0.01% (v/v) phosphoric acid aqueous solution. Linear gradient elution was used as follows: 8% A in 0–20 min, 8–24% A in 20–50 min, 50% A in 50–60 min, 50–90% A in 60–68 min. The flow rate was 1.0 mL/min and the column temperature was 27°C. The injection volume was 10 μL. The UV wavelengths were 274 nm for gallic acid, catechin, albiflorin, paeoniflorin, ethyl gallate, galloylpaeoniflorin, pentagalloylglucose, benzoic acid, benzoylpaeoniflorin, and paeonol and 257 nm for oxypaeoniflorin.