Waters 996 photodiode array detector

Chemical components in the needle extracts of the two conifers were analyzed using reversed-phase HPLC (Waters 2695 Alliance HPLC; Waters Inc., Milford, MA, USA), coupled with a 250 × 4.6 mm octadecylsilane column (Prontosil 120-5-C18-SH 5.0 μm; Bischoff, Leonberg, Germany). Two solvents were used as mobile phases; water with 0.1% formic acid (solvent A) and MeOH with 0.1% formic acid (solvent B). The gradient flow was as follows: 0–5% of solvent B for 0–10 min, 5–10% of solvent B for 10–20 min, 10–20% of solvent B for 20–30 min, 20–40% of solvent B for 30–50 min, and 40–70% of solvent B for 50–62 min. Furthermore, the flow rate of the mobile phases was 1.0 mL min⁻¹, and the sample injection volume was 5 μL. Then, resulting peaks were monitored at 278 nm using a Waters 996 photodiode array detector.

The 0.2 mL of the sample extract was diluted with 0.8 mL of 80% ethanol. The diluted extract was filtered through a 0.45 μm syringe filter. The filtrate was analyzed using reverse-phase HPLC (Waters 2695 Alliance HPLC; Bischoff, Leonberg, Germany) with a prontosil column (120-5-C18-SH, 5 μm, 150 × 4.6 mm; Bischoff, Leonberg, Germany) as previously described methods [18 (link),24 (link)]. The mobile phase consisted of (A) water with 0.1% formic acid and (B) acetonitrile. The gradient elution was as follows: 0–23 min, 1–20% B; 23–45 min, 20–60% B; 45–46 min, 60% B; 46–47 min, 60–1% B; and 47–49 min, 1% B. The flow rate of the mobile phase was 1.0 mL·min⁻¹ and the injection volume of the sample was 10 μL. The peaks were detected at 280 nm using the Waters 996 photodiode array detector (Waters Inc., Milford, MA, USA).

The optical rotations were obtained using a Jasco P-1020 polarimeter (Jasco, USA). The nuclear magnetic resonance (NMR) spectra were obtained using a Varian UNITY INOVA 800 NMR spectrometer operating at 800 MHz (¹H) and 200 MHz (¹³C), with chemical shifts given in ppm (δ). Preparative HPLC was performed using a Waters 1525 Binary HPLC pump with a Waters 996 Photodiode Array Detector (Waters Corporation, USA). Semi-preparative HPLC was conducted using a Shimadzu Prominence HPLC System with SPD-20A/20AV Series Prominence HPLC UV-Vis Detectors (Shimadzu, Japan). LC/MS analysis was carried out on an Agilent 1200 Series HPLC System (Agilent Technologies, USA) equipped with a diode array detector and a 6130 Series ESI mass spectrometer by using an analytical Kinetex column (4.6 × 100 mm, 3.5 μm). Precoated Merck silica gel F254 plates and RP-18 F254s plates were used for thin-layer chromatography (TLC). Spots were detected by TLC under UV light or by heating after spraying with anisaldehyde-sulfuric acid.

The molecular weight distribution was determined by High performance size exclusion chromatography (SEC-HPLC, Waters Chromatography Division, Milford, MA, USA). Four walnut proteins (5 mg/mL) were extracted by sodium phosphate buffer (0.05 M, pH 8.0) containing sodium chloride (0.3 M) for 4 h at 25 °C under constant magnetic stirring and then were centrifuged at 10,000× g for 10 min (25 °C). The supernatant was filtered through a cellulose acetate membrane with a pore size of 0.45 μm (Sartorius Co., Ltd., Gottingen, Germany). A Waters 2690 liquid chromatogram system (Waters Chromatography Division, Milford, MA, USA) equipped with a Shodex protein KW-804 column (Shodex Separation and HPLC Group, Tokyo, Japan) and a Waters 996 photodiode array detector were used to determine the molecular weight distribution. The flow rate was 1 mL/min using phosphate buffer (0.05 mol/L, 0.3 mol/L NaCl, pH 7.0) as the mobile phase. About 10 μL protein solutions were injected into the column and the eluent was monitored at 280 nm. All samples were measured in triplicate and the representative examples were selected for discussion. A calibration curve of 10 standard proteins was used for interpreting the results.

The amount of propranolol in all samples was quantified by high performance liquid chromatography (HPLC) using a Waters liquid chromatograph (Waters 600 Controller and Pump) which included a diode-array detector (Waters 996 Photodiode Array Detector) (Waters, Barcelona, Spain), set to 291 nm and an analytical Kromasil^® C₁₈ column (250 × 4 mm, 5 µm) (Análisis Vínicos, Tomelloso, Spain). A mixture of monobasic ammonium phosphate water solution (0.05 M, pH 3.7)-acetonitrile (69:40, v/v) was used as mobile phase, at a flow rate of 1 mL/min injection volume was 50 µL. The method had been previously validated [31 (link)]. The limits of detection and of quantification were 0.171 and 0.115 µM, respectively.