Uv vis detector

Chromatographic analysis was carried out on an Agilent 1220 Infinity high-performance liquid chromatography (HPLC) system equipped with a UV/Vis detector (Agilent Technologies, Santa Clara, CA, USA) using an Eclipse plus C-18 column (5 μm, 4.6 × 150 mm, Agilent). The mobile phase comprised water containing 0.1% acetic acid (A) and acetonitrile containing 0.1% acetic acid (B), and flow rate was 0.7 mL/min. Gradient elution of the mobile phase was applied as follows: 0–5 min, 10% B; 5–15 min, 10–20% B; 15–25 min, 20–30% B; 25–35 min, 30–70% B; 35–40 min, 70–90% B; 40–50 min, 90%; 50–55 min, 90–10% B, 55–65 min, 10% B. The injection volume was 10 μL, and the detection wavelength was 340 nm. SLE and pedaliin standard were dissolved with methanol to prepare 10 and 1 mg/mL of stock solution, respectively, filtered using a 0.45 mm syringe membrane filter, and then diluted to a series of concentrations. Pedaliin in SLE was quantified by integrating the peak area of pedaliin and the calibration curve for pedaliin standard.

Before analysis, the finely ground (<500 µm) sample (0.5 g) was mixed with 40 mL of a solvent containing 60% ethanol in 0.5% formic acid, and further constantly stirred at room temperature for 1 h. The mixture was centrifuged (6000× g, 20 min) and the supernatant was then used for HPLC analysis, total polyphenols, and in vitro antioxidant activity determination. Different phenolic components were determined using an HPLC system (Agilent 1220, Agilent Technology, USA), with a binary pump and UV-Vis detector (Agilent Technology, USA). Separation was performed on an Agilent TC-C18 column (5 μm, 4.6 mm × 250 mm) at 25 °C and a wavelength of 280 nm was used. The following mobile phases were used: 0.5% acetic acid (A) and 100% acetonitrile (B) at a flow rate of 0.8 mL/min. The gradient elution started with 14% B, between 6 min and 30 min, linearly increased to 25% B, and then to 50% B at 40 min. The identification of the compounds was confirmed by a comparison of retention times utilizing the standard solutions and standard calibration curves of different phenolics.

CE analyses were run on an Agilent 7100 Capillary Electrophoresis System with a UV-Vis detector (Agilent Technologies, Waldbronn, Germany). A total of 50 μm fused silica capillaries (375 μm outer diameter) were purchased from CM Scientific Ryefield (Dublin, Ireland) and were cut to a total and effective length of 48.5 and 40.0 cm, respectively. UV detection was carried out at 210 nm and the separations were all carried out in positive polarity mode. The temperature was set at 22 °C. Hydrodynamic injection of the sample was performed in two steps, applying first 50 mbar pressure for 5 s to the sample injection vial, then 50 mbar for 5 s to a buffer-filled vial, for plug injection.
Each new capillary was rinsed with 1 M NaOH, 0.1 M NaOH, and water for 5 min each. Daily, the capillary was rinsed with 0.1 M NaOH and water for 2 min each before use. Between the runs, the conditioning was made as follows: MeOH (1 min), 0.1 M NaOH (1 min), water (1 min), and BGE (3 min).
The optimized CE conditions corresponded to the following, with the MODR in brackets: capillary total length, 48.5 cm (effective length, 40.0 cm); temperature, 22 °C; voltage, 25 kV (23–29 kV); BGE: 23 mM (21–26 mM) phosphate-borate buffer pH 9.70 (9.50–9.77), 65.0 mM SDS, 1.00% v/v (0.25–1.29% v/v) n-butanol, 20 mM (21–26 mM) DMβCD.

Determination of EDCs was obtained through a HPLC system coupled to an UV–Vis detector (Agilent Technologies) and a reverse-phase column Agilent Eclipse XDE-C18 150 × 4.6 mm, 5 μ. A reverse-phase column Agilent Eclipse XDE-C18 150 × 4.6 mm, 5 μ, was used for the chromatographic measurements. The final reaction mixture was performed at 25 °C in 1 mL vial containing 10 mg L⁻¹ of each analyte, 10 % (v/v) buffer McIlvain pH 5 and 100 U L⁻¹ (50.3 U mg⁻¹) of laccase; then the solution was vortex-mixed briefly for homogenizing and sheltering from light. The enzymatic treatments were measured by triplicate. An injection volume of 20 μL and 1 mL min⁻¹ gradient elution by means of (A) acetonitrile (ACN) and (B) 10 mM phosphate buffer (pH 3.5) were applied. The gradient program was set as follows: 0–11 min, 25 % (A); 11–23 min, 95 % (A) and 23–30 min, 25 % (A). BPA, NP, TCS and EE2 were detected at three wavelengths, 206, 290 and 275 nm. The chromatographic analysis was carried out up to 12 h and the extent of the reaction was estimated by the decrease of the corresponding analyte peak analyzed by HPLC-UV chromatography technique and quantified using a calibration curve.