Co 4060 column oven

The fluorescence intensity of SOSG (Thermo Fisher Scientific, Waltham, MA, USA) oxidized by ¹O₂ was determined from the fluorescence of the HPLC peaks. The photocatalyst (2 µM) and SOSG (10 µM) were added to 50% CH₃CN solution in 10 mM MES buffer (pH 7.4) in a 1.5 mL tube. The solution was irradiated with blue light (RELYON, Twin LED light, 455 nm) for 30 s on ice. After irradiation, the solution was diluted 2.6-fold with 0.1% aqueous formic acid and analyzed using HPLC. Analytical HPLC was carried out on a JASCO PU-4580 HPLC Pump, JASCO LG-4580 Quaternary Gradient Unit (Tokyo, Japan), and JASCO DG-4580 Degassing Unit with a JASCO MD-2018 Plus Photodiode Array Detector, JASCO CO-4060 Column Oven, JASCO As-455 HPLC Autosampler, and JASCO LC-NetII/ADC Interface Box using a C18 reverse phase column (Inertsil ODS-4, 150 × 4.6 mm, 5 μm (GL Science, Inc., Tokyo, Japan)). The HPLC conditions were as follows: mobile phase A, 0.1% formic acid in H₂O, mobile phase B, 0.1% formic acid in CH₃CN. 0−5 min, 5% B; 5−27 min, 5−100% B; 27−32 min, 100% B. The fluorescence of the separated peaks was detected using HPLC (Ex 504 nm/Em 525 nm).

The ethanol extract (10 μl) of each sample was subjected to three-dimensional high-performance-liquid chromatography (HPLC) analysis to detect the chemical constituents. The HPLC system consisted of a PU-4180 pump, a MD-4015 multiwavelength detector (200–400 nm), and a CO-4060 column oven (JASCO, Tokyo, Japan). The column was a Mightysil RP-18GP S (150 × 4.6 mm i.d.; Kanto Chemical Co., Inc., Tokyo, Japan). The solvents were (A) water/acetonitrile (17:3) and (B) water/acetonitrile (3:2). A linear gradient of 100% A and 0% B changing over 45 min to 0% A and 100% B was applied, and then 0% A and 100% B were continued for 15 min. The flow rate and the column temperature were 1.0 ml per min and 40°C, respectively. Each component was identified by comparing the peak retention times and wavelengths with those of authentic compounds.

A RP-HPLC system JASCO (Tokyo, Japan), equipped with AS-4150 RHPLC Autosampler, PU-4180 RHPLC Pump, LC-NetII/ADC Interface Box, CO-4060 Column Oven, MD-4010 Photo Diode Array Detector, and ChromNAV software, version 2.0 (JASCO, Tokyo, Japan) was used. For the quantification of the VES-GEM conjugate, a Zorbax Eclipse XDB-C18 column (5 μm, 4.6 mm × 250 mm, Agilent Technologies, Santa Clara, CA, USA) at 30 °C and methanol as the mobile phase, with a flow rate 1 mL/min, was used. The detection wavelength was set at 248 nm [39 (link)]. All samples were filtered before injection through a PTFE hydrophilic Scharlau syringe filter (13 mm, 0.22 µm). The injection volume was 20 µL. For the VES-GEM calibration curve, a stock solution of VES-GEM in ethanol (50 ppm, 10 mL) was progressively diluted to standard solution concentrations of 40 ppm, 30 ppm, 20 ppm, 10 ppm, 5 ppm, and 1 ppm. The calibration curve of the quantified VES-GEM concentration, as a function of peak area (absorbance detected) (Figure S1), was drawn and showed good linearity within a concentration range of 1–50 ppm: linear regression equation y = 20779x + 2003.5, R = 0.9995, LOD = 0.15 ppm, LOQ = 0.236 ppm. The retention time was t = 8.0 min (Figures S2 and S3).