Prominence series uflc system

Gradient HPLC analysis was carried out using the Shimadzu^® Prominence series UFLC system equipped with a CBM-20A controller bus module, an LC-20AD liquid chromatograph, a CTO-20A column oven, a SPD-20A UV–Visible detector allowing absorption measurement at two chosen wavelengths and a RF-20A fluorescence detector allowing fluorescence measurements at specific excitation and emission wavelengths. For all measurements, the UV–visible detectors were set respectively at 295 and 350 nm. Sample aliquots of 20 µL were injected in a 95 % solvent A—5 % solvent B (A = Milli-Q water/TFA 99.9:0.1 v/v; B = CH₃CN/Milli-Q water/TFA 90:9.9:0.1 v/v/v) into a Jupiter C4 column (150 × 4.60 mm, 5 µm, 300 Å, Phenomenex^®) at a flow rate of 1 mL min⁻¹ over 5 min. In a second step, samples were eluted by a gradient from 5 to 90 % of solvent B in solvent A over 15 min. Finally, the concentration of solvent B was maintained over 5 min. Then, the concentration of solvent B was decreased to 5 % over a period of 5 min to re-equilibrate the system, followed by additional 5 min at this final concentration. Before each sample measurement, a baseline was recorded following the same conditions by loading Milli-Q water onto the injection loop.

Gradient HPLC analysis was performed using the Shimadzu Prominence series UFLC system with a LC-20 AD liquid chromatograph, a CBM-20A controller bus module, a CTO-20A column oven, and an SPD-20A UV-visible detector. UV-visible absorption was measured at 295 nm. Separation was performed at a flow rate of 1 mL min^-1 using C4 reverse phase HPLC column (Jupiter^®, 5 µm, 300 A, 150 x 4.6 mm). The gradient initial solution was 95% solvent A - 5% solvent B (A = H₂O / ACN / TFA: 98.9 / 1 / 0.1% v, B = ACN / H₂O / TFA: 89.9 / 10 / 0.1% v) over 5 min. During the next step, samples were eluted over 15 mins by a gradient developed from 5 to 90% of solvent B in solvent A. The concentration of solvent B was maintained over 5 min and then the concentration of solvent B was decreased to 5% over a period of 5 min to re-equilibrate the system, followed by an additional 5 min at this final concentration.