Waters 2695 hplc

To analyze if there were changes of BPA concentration after mixing with TiO₂-NPs, different concentrations of BPA solutions (5 and 10 mg/L) and combined solutions (BPA 5 mg/L + TiO₂ 10 mg/L, BPA 5 mg/L + TiO₂ 50 mg/L, BPA 10 mg/L + TiO₂ 10 mg/L, and BPA 10 mg/L + TiO₂ 50 mg/L) were prepared. Then the combined solutions were added to 50-mL plastic centrifuge tubes (in a preliminary experiment, the plastic centrifuge tubes were proven to have no effect on the BPA concentration). Subsequently, the tubes were centrifuged for 10 min at 14,000 rpm using a high-speed centrifuge (HIMAC CR 22G II, Hitachi, Ltd., Chiyoda-ku, Japan), and the supernatant was collected and centrifuged again at 14,000 rpm. Before injection, the BPA solutions and the combined solutions were passed through 0.22-μm filter membranes, respectively. The samples were analyzed by high-performance liquid chromatography (Waters 2695 HPLC, Waters Corp., Milford, MA, USA) with a photodiode array detector (Waters 2998 PDA) for BPA levels. The samples were separated on a CAPCELL PAK C₁₈ column (4.6 × 150 mm, 5 μm, Waters Corp.) using a mobile phase of 60% acetonitrile and 40% water at a flow rate of 1.0 mL/min. The injection volume was 10 μL, and the column temperature was 20°C. The pH values for all of the samples before and after the adsorption experiments were similar and were approximately neutral.

The TBARS assay was performed according to a previously method described by Papastergiadis et al. (30 (link)) with slight adjustments. Briefly, 1 g HPBF was mixed with a total of 5 mL of 7.5% TCA (w/v) with 0.02% (w/v) of EDTA. The mixed solution was homogenized with a refrigerated centrifuge (Hunan Herexi Instrument and Equipment Co., Ltd., Changsha, China) for 10 min at 8000 rpm. The homogenate was treated with 5 mL TBA reagent (20 mM) at 90°C for 30 min, and then filtered through a 0.22 Millipore membrane filter (MREDA Technology Inc., Beijing, China) after cooled using running water. Ten μL mixture solution was separated by Waters 2695 HPLC on a Waters SunFireTM C18 column (4.6 × 250 mm, 5 μm) using a mobile phase of 10 mM ammonium acetate and methanol (7:3, v/v). The column temperature was 30°C. The detection wavelength was 532 nm using Waters 2996 photodiode array detector (PAD). A standard curve made up of diluted 1,1,3,3-tetraethoxy propane solutions was used to calculate the amount of TBARS. The results were given in milligrams of MDA equivalents per kilogram of the sample (mg MAD/kg).

The ability of LAC_2.9 to transform guaiacylglycerol-β-guaiacyl ether and veratrylglycerol-β-guaiacyl ether was performed essentially as described by Brown et al. (2012 (link)). Briefly, 1 mM of lignin model compound (LMC) was incubated with 0.2 μM LAC_2.9 in 20 mM sodium phosphate, pH 6 containing 1 mM CuSO₄. The reactions were incubated at 60 °C with stirring and quenched after 30 min or 6 h by adding acetic acid to 0.5% final concentration. The quenched reaction was centrifuged at 16,000×g for 5 min, and the cleared solution was analyzed by reverse-phase HPLC. Samples were analyzed using a Waters 2695 HPLC (Waters, Milford, MA, USA) equipped with a Luna^® 5 µm C18(2) column 250 × 4.6 mm (Phenomenex, Torrance, CA, USA) and a UV detector. The column was operated at 0.7 ml min⁻¹ and the sample was eluted using a 16.8 ml linear gradient of 1% formic acid in H₂O to 100% methanol.

Around 5 ml of the above sample was used for stability analysis. Then, 10 ml of absolute ethanol was mixed with 10 ml of 60% KOH, then shaken at room temperature for 3 h, and extracted with cyclohexane:n-hexane:ethyl acetate = 1:2:2. The extraction was repeated 3 times. The extracts were combined and rotary evaporated to near dryness in a 30°C water bath, then dissolved to 10 ml using a 0.1% BHT absolute ethanol for HPLC analysis. A standard curve was plotted using 97% lutein to characterize the content changes of lutein during the degradation process.
The change in lutein content was determined by Waters 2,695 HPLC (Waters Technology Co., Ltd., USA). The chromatographic column model was Venusil XBP C30 (5 μm, 250 mm × 4.6 mm). Phase A (methanol:water = 88:12), phase B (methyl tert-butyl ether). At 0–18 min, phase A changed from 100% to 10%; at 18.1 min, phase A changed from 10% to 100%, and kept it for 10 min. The flow rate was 1.0 ml/min, and the injection volume was 50 μl. The degradation rate was calculated by Equation (7), and the degradation kinetics was determined by Equations (8–10).
Kinetic equations (8–10) (37 (link))
where, C₀ represents the initial content of lutein before stability analysis, and C_t represents lutein content at time t during stability analysis. k₀, k₁, and k₂ represent the kinetic constants.

Ascorbic acid was quantified with paired ion reverse phase HPLC coupled with electrochemical detection (ECD), as previously described (Frei et al., 1989 (link)). In short, cells were harvested with trypsin, and the cell pellet was extracted with 10% perchloric acid with 1 mM diethylenetriamine pentaacetic acid (DTPA). The supernatant was diluted 10× with mobile phase and adjusted to pH 5.0 with 2.58 M K₂PO₄ (pH 9.8). Samples were analyzed with a Waters 2695 HPLC equipped with a Supelcosil LC-8 column (25 cm × 4.6 mm i.d., Supelco, St. Louis, MO, United States) and a LC-18 guard column (2 cm × 4.6 mm i.d., Supelco). The eluant (40 mM sodium acetate, 7.5% vol/vol methanol, 0.54 mM DTPA, 1.5 mM dodecyl-triethylammonium phosphate in purified Milli-Q water, taken to pH 4.75 with glacial acetic acid) was delivered at 1.0 mL/min. Analysis was performed on a LC 4B amperometric electrochemical detector equipped with a glassy-carbon working electrode and a Ag/AgCl reference electrode (Bioanalytical Systems, West Lafayette, IN, United States). Ascorbic acid was analyzed with an applied potential of +0.5 V with a sensitivity of 50 nA, and eluted as a single peak at 5.8 min.

We used a Waters 2695 HPLC (Alliance) system with a photodiode array detector equipped with a Lichrosorb RP-18 reversed phase analytical column (C18 35 μm, 4.6 × 50 mm PIN 186003034). Analysis was achieved with stepped linear gradient of solvent A (0.08% TFA in H₂O) and in solvent B (0.08% TFA in 100% acetonitrile) for 30 min with a flow rate 3 mL/min. The conjugation of OM with (KG)₅-MOG_35-55 peptide was evaluated by HPLC. The (KG)₅-MOG_35-55 HPLC peak disappeared within six hours indicating completion of conjugation to OM.

HPLC analysis was performed on a Waters 2695 HPLC equipped with a Waters 2996 photodiode array detector (Waters). 50 μl extract was injected onto a 250 × 4.6 mm Luna 5 μm phenyl-hexyl column (Phenomenex) and separated on a 35 min linear gradient from 5 to 100% acetonitrile with 0.1% formic acid at a flow rate of 1 ml/min. Data were collected and analyzed using Waters software and plotted using Graphpad Prism 9 (GraphPad Software).